Citation
Reengineering the TMJ Disc: An Evaluation of Scaffold Design, Cell Incorporation, and Stimulated Functionalization

Material Information

Title:
Reengineering the TMJ Disc: An Evaluation of Scaffold Design, Cell Incorporation, and Stimulated Functionalization
Creator:
Juran, Cassandra
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
MCFETRIDGE,PETER S
Committee Co-Chair:
DOBSON,JON P
Committee Members:
BANKS,SCOTT ARTHUR
HAHN,DAVID WORTHINGTON
DOLWICK,MELVIN F
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Cells ( jstor )
Cultured cells ( jstor )
Dental research ( jstor )
Lasers ( jstor )
Scaffolds ( jstor )
Seeding ( jstor )
Shear modulus ( jstor )
Shear stress ( jstor )
Temporomandibular joint ( jstor )
Tissue engineering ( jstor )
biomaterial
bioreactor
fibrocartilage
mechanotransduction
temporomandibular

Notes

General Note:
Functioning primarily as a stress distribution bumper within the joint, the temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe Temporomandibular Disorders has been the primary driving force for the introduction and development of TMJ disc Tissue Engineering as an approach to alleviate this priority clinical issue. A great deal of promise has been shown with the application of tissue engineering principles to regenerate living tissues, that would ideally accommodate typical loads of the joint and regain physiologic functionality. The branches of study investigated thus far have primarily focused on synthetic scaffold material, characterizing the native anatomy and physiology, cellular culture technique, and decellularization of TMJ discs. Cell survival and integration into the scaffold has proven problematic largely due to limited porosity, resulting in poor transport conditions that limit cell migration into the scaffold. The purpose of my thesis is to utilize a native porcine TMJ disc (pTMJ) as a xenogeneic ex vivo scaffold, and further modify the discs structure to enhance reseeding and transport conditions to create a mechanically and biologically functional disc. To then evaluate different physiologic mechanical loading conditions influence on the remodeling, cell phenotype, and mechanical viability of the disc.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Juran, Cassandra. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2016

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http://jdr.sagepub.com/Journal of Dental Research http://jdr.sagepub.com/content/92/2/193 The online version of this article can be found at: DOI: 10.1177/0022034512468749 2013 92: 193 originally published online 19 November 2012 J DENT RES C.M. Juran, M.F. Dolwick and P.S. McFetridgeShear Mechanics of the TMJ Disc : Relationship to Common Clinical Observations Published by: http://www.sagepublications.com On behalf of: International and American Associations for Dental Research can be found at: Journal of Dental Research Additional services and information for http://jdr.sagepub.com/cgi/alerts Email Alerts: http://jdr.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? Nov 19, 2012 OnlineFirst Version of Record Jan 15, 2013 Version of Record >> at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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193 DOI: 10.1177/0022034512468749 Received May 8, 2012; Last revision October 19, 2012; Accepted October 23, 2012 A supplemental appendix to this article is published elec tronically only at http://jdr.sagepub.com/supplemental. International & American Associations for Dental ResearchC.M. Juran, M.F. Dolwick, and P.S. McFetridge*J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, JG-56, Biomedical Sciences Building, Gainesville, FL 32611-6131, USA; *cor responding author, pmcfetridge@bme.ufl.edu J Dent Res 92(2):193-198, 2013ABSTRACTThe temporomandibular joint (TMJ) is a complex hinge and gliding joint that induces significant shear loads onto the fibrocartilage TMJ disc during jaw motion. The purpose of this study was to assess regional variation in the discs shear loading characteristics under physiologically relevant loads and to associate those mechanical findings with common clinical observations of disc fatigue and damage. Porcine TMJ discs were compressed between an axially translating bottom platen and a 2.5-cm-diameter indenter within a hydrated testing chamber. Discs were cyclically sheared at 0.5, 1, or 5 Hz to 1, 3, or 5% shear strain. Within the anterior and intermediate regions of the disc when sheared in the anteroposterior direction, both shear and compressive moduli experienced a significant decrease from instantaneous to steady state, while the posterior regions compressive modulus decreased approximately 5%, and no significant loss of shear modulus was noted. All regions retained their shear modulus within 0.5% of instantaneous values when shear was applied in the mediolateral direction. The results of the discs regional shear mechanics suggest an observable and predictable link with the common clinical observation that the posterior region of the disc is most often the zone in which fatigue occurs, which may lead to disc damage and perforation.KEY WORDS: tissue engineering, temporomandibular disc, joint disease, jaw biomechanics, extracellular matrix (ECM), biomechanics.INTRODUCTIONThe temporomandibular joint (TMJ) regulates movements of the mandible with respect to the temporal bone of the skull. The fibrocartilagenous TMJ disc has, in the past decade, been the subject of more extensive mechanical evaluations. The majority of published biomechanical analyses have focused on the TMJ discs compressive properties (Tanaka et al., 2003a,e; Allen and Athanasiou, 2005; Lumpkins and McFetridge, 2009) and have found that the disc possesses significant regional variations (Lumpkins and McFetridge, 2009; Kuo et al., 2010). While compressive properties are important during joint loading, the TMJ has primarily a gliding joint function that induces com pressive as well as significant shear forces. Previous studies have evaluated shear under static shear loading conditions and have shown regional variations of the TMJ disc properties (Lai et al., 1998). Current investigations of the TMJ under dynamic shear conditions have focused on the central region of the discs intermediate zone (Tanaka et al., 2003b, 2004; Koolstra et al., 2007). The relationship between repetitive shear and compressive loading of the intervertebral disc and disc damage has been alluded to by Callaghan and Iatridis and colleagues, who suggested that disc herniation and damage may be more linked to repeated flexion extension and shear motions than to applied joint compression (Callaghan and McGill, 2001; Iatridis and ap Gwynn, 2004). In these investigations, the regional shear and compressive characteristics of the porcine TMJ disc and the interconnectivity and dependency of these characteristics on one another and variables relevant to physiologic function have been evaluated. We hypothesized that not only is the shear modulus (G) of the TMJ disc dependent on frequency, shear, and compressive strain, but also that the compressive modulus of elasticity (E) is dependent on the appli cation of cyclic shear loads (Lumpkins et al., 2008). Additionally, we hypoth esized that the interdependence of the compressive and shear elastic moduli will reveal material trends consistent with damage seen clinically in patients with temporomandibular disorders.MATERIALS & METHODSSpecimen PreparationPartially dissected fresh porcine TMJs were isolated from male animals ages 6 to 9 mos, purchased with IACUC approval (IACUC Protocol # 201207534) from Animal Technologies Inc. (Tyler, TX, USA). Dissection was conducted as previously described (Lumpkins et al., 2008; Appendix 1). After dissection, discs were stored in 0.15 M phosphate-buffered saline (PBS, pH 7.4) at 4C until use. All samples were stored for fewer than 12 hrs before being tested (Lumpkins et al., 2008). Immediately before mechanical testing, a circular stainless steel punch with a 6-mm inner diameter was used to extract Shear Mechanics of the TMJ Disc: Relationship to Common Clinical Observations at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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194 Juran et al. J Dent Res 92(2) 20133 samples from each disc: anterior, intermediate, and posterior samples (Fig. 1B). Anterior-to-posterior (A-P) and medial-tolateral (M-L) directions were labeled by means of a waterresistant marker on each disc sample (Fig. 1B, insert), to orient the sample correctly for testing within the hydrated testing chamber.Sample GroupsThe TMJ discs were divided into 3 testing groupsanterior, intermediate, and posterioreach undergoing 27 testing proce dures [frequency (F) variation (0.5 Hz, 1 Hz, and 5 Hz), com pressive strain () variation (5%, 10%, and 15%), and shear strain () variation (1%, 3%, and 5%)], with 9 specimens for each testing procedure. The order of the testing procedure was randomized for each sample group to minimize sample error (see Appendix 7, Appendix Fig. 3).Biomechanical Testing Cyclic Shear LoadingA Biomomentum Mach1 Micromechanical System (Biomomentum Inc., Laval, Quebec, Canada) was used for all mechanical testing. The Mach1 is a multi-axial modular mechanical testing rig which can measure or impart compressive and shear loading. The testing chamber was filled with 0.15 M PBS (pH 7.4) to maintain hydra tion, similar to the in vivo environment (Piette, 1993) and to maintain consistency with previous studies on TMJ tissue mechanics (Tanaka et al., 2003c; Allen and Athanasiou, 2006). An axially translating bottom plate and an axially stationary vertically translating 2.5-cm-diameter indenter were used to generate the shear and compressive loads. Prior to being tested, samples were allowed to equilibrate (unloaded) in PBS for 5 min at 37 1C. Sample height was determined by a pre-programmed function of the Mach1 called find contact (see Appendix 2). With the strain profile illustrated in Fig. 1C, compressive and shear strain defor mations (indenter displacement, L position; and axially translat ing bottom plate, x position) were applied to the tissue and assessed according to the calculations = L / L0 and = x / L0, where L is the decrease in sample thickness and x is the axial displacement in the shear direction. Both parameters relative to the initial thickness L0 were needed to produce the desired strain range (see Appendix 6). The Mach1 measures resulting force (FX and FZ) experience by the indenter during the compressive and shear load ing. The resulting compressive stress on each sample is defined by = FZ/A, where FZ is the compressive force and A is the crosssectional area of the sample, and the resulting shear stress is = Fx/A, where FX is the resulting axial force and A is again the cross-sectional area of the sample (Lumpkins et al., 2008). Samples were sheared under sinusoidal strain defined by = sin(t), where and are the respective strain amplitude and frequency, and t is time. We analyzed results of the cyclic loading tests by calculating the hysteresis, peak stresses, and the instantaneous and steady-state compressive (Eint,Ess) and shear moduli (Gint,Gss). The general calculations for the compressive and shear moduli are E = / and G = / respectively.Statistical AnalysisEach set of test group data was calculated based on the testing of 9 samples (n = 9). We calculated standard deviations and used one-way analysis of variance (ANOVA) testing to determine statistical significance between and among test groups for bio mechanics. Significance was established by the Tukey-Kramer test (p = 0.05, n = 9). Figure 1. Joint anatomy and simplified free-body diagram, regional sampling, and testing method. (A) The gross anatomy of the TMJ and the placement of the disc within the glenoid fossa. The insert illustrates the position of the TMJ disc when the mandibular condyle slides forward with respect to the articular eminence when the jaw opens. (B) The sampling regions of the TMJ disc and the directional notation used to orient the sample properly for testing (gray line, anteroposterior direction; black line, mediolateral direction). A representative testing strain profile is shown in (C) (description in text body), and (D) depicts the sequential testing procedure used to generate the strain profile of (C) (dashed arrows indicate active loading, and solid arrows indicate sustained loading). at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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J Dent Res 92(2) 2013 Shear Mechanics of the TMJ Disc Relative to Clinical Observations 195 RESULTSThe average thicknesses reported for the tested regions were 3.66 0.24, 2.00 0.29, and 3.88 1.45 mm for the anterior, intermediate, and posterior regions, respectively. Shear modulus was evaluated for the 1st strain cycle ( Gint) and for the 10th strain cycle ( Gss) to show the changes exerted by frequency and compressive and shear strain on the material properties over the load application (Fig. 2). When shear loads were displaced in the A-P direction, the stiffness of all regions decreased from Gint to Gss, with the most noteworthy change occurring with fre quency increase (Fig. 2A). Change in Gint, evaluated solely as a function of frequency increase from 0.5 Hz to 5 Hz, was, on average, an 8.6% increase for all and conditions evaluated. When compressive strain was increased and the other test condi tions were maintained, the disc experienced a similar increase in shear modulus (Fig. 2B); however, when the shear strain was increased, the shear modulus decreased, becoming more elastic (Fig. 2C). Most noticeably, for the posterior region tested at the high frequency (5 Hz) and high compressive strain (15%) (data not represented in Fig. 2C), Gint = 1.556 0.327 MPa and Gss = 0.797 0.315 MPa at = 1% and Gint = 1.078 0.158MPa and Gss = 0.686 0.072MPa at = 5%, an average decrease of 22.5% from the instantaneous to the steady-state condition. Similar to the shear modulus change with (Fig. 2C), the compressive modulus also became more elastic with repeated shear in all regions of the disc (Fig. 3). The cycle-dependent compressive modulus of the posterior region shows that, with each shear cycle application, the compressive modulus increased by nearly 5% of the Eint value before continuing the decreasing trend. The average magnitude of the E (peak height) when sheared in the A-P direction was 1.5 0.0 kPa, and in the M-L direction was 0.40 0.03 kPa in the posterior region, and became more regular as the compressive modulus reached a steady-state condition (cycles 6 through 10). This phenomenon was also seen in the anterior region, but only when shear strain was applied in the M-L direction, and the magnitude was less than 25% of the posterior regions E (table in Fig. 3). The shear modulus characteristics are described in Fig. 4. Only the posterior region statistically maintained its shear modulus from Gint to Gss when sheared in the A-P direction. All regions when sheared in the M-L direction maintained within 20% of their Gint stiffness over the strain application cycle.DISCUSSIONThe ability of the TMJ disc to act as a buffer between the articu lating skeletal structures of the TMJ is fundamental to jaw movement; thus, it is paramount to understand the mechanical response of a healthy TMJ disc when exposed to loading similar to that experienced in vivo. The current investigation focused on the mechanical characteristics of the disc during cyclic shear Figure 2. Changes in shear modulus with frequency compressive strain, and shear strain. Mean values of the instantaneous (1) and steady-state (2) shear moduli as a function of frequency, with = 1% and = 10% (A), compressive displacement, F = 1 Hz, and = 1% (B), or shear displacement, = 10% and F = 1 Hz (C). Samples were shear-strained in the anterior to posterior direction for all data presented. Increases in frequency and compressive strain caused shear stiffening, and the application of greater shear strain induced the shear modulus to become more elastic in the anterior and intermediate regions of the disc. Error bars represent standard deviations. at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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196 Juran et al. J Dent Res 92(2) 2013testing, when the magnitude of shear strain (), compressive strain (), and frequency (F) are varied under broadly physiolog ical parameters (see Appendix 5). Previous investigations by Tanaka and co-workers have shown that the disc experiences shear stiffening, or increase in elastic modulus, as or F is increased, and, conversely, shear softening with increasing (Tanaka et al., 2003b, 2004). Fig. 2 depicts the dependency of the disc regions on frequency, com pressive strain, and shear strain. Shear stiffening was hypothe sized to be a combination of 2 effects: the first due to interstitial fluid or bulk matrix outflow as pressurized fluids are squeezed from the point of strain application; and the second due to the frequency of application being increased, which results in a lag in fluid resorption between the successive strain applica tions. These conclusions were further supported by our results and can be expanded to be a universal trend for the regions tested. The shear softening with increased shear strain, however, was observed only in the anterior and intermediate regions of the disc. It has previously been hypothesized that observed shear softening is due to the proteoglycan [glycosaminoglycan (GAG)] component and water matrix within the disc possessing non-Newtonian characteristics. GAGs are highly hydrophilic sugar chains that act to maintain the discs resistance to pres sure. The lack of significant shear softening in the posterior region, then, is in agreement with this hypothesis, since the posterior band of the disc contains a lower GAG content than the anterior or intermediate zones (Nakano and Scott, 1996; Almarza et al., 2006) (Fig. 4). A difference between our experi mental design and that of Tanaka et al. is that the latters work evaluated the shear properties of the porcine disc at porcine core temperature (39C), and our evaluation was done at human body temperature. This temperature difference may account for the Figure 4. Mean regional values of the instantaneous and steady-state shear moduli of each disc region when strained in the anterior-toposterior direction and the medial-to-lateral direction. The anterior and central regions experienced a significant decrease in shear modulus when strained in the anterior-posterior (A-P) direction. This increased elasticity was not seen in the posterior region (* p < 0.05; ** p < 0.01). Figure 3. Changes in compressive modulus when cyclic shear strain was applied in the anterior-to-posterior or medial-to-lateral direction. Representative zonal compressive modulus evolutions over the load application are shown in (A), (B), and (C). Mean values of the compressive modulus of each region of the TMJ disc are shown in the table. The anterior and intermediate regions had less reduction of their compressive modulus, retaining their compressive modulus even when repeatedly sheared. The posterior region became more elastic with application of shear strain. All regions had significant reduction in initial to steady-state modulus when sheared in the medial-lateral direction ( p < 0.05). Anteriorposterior shear strain application data are represented by the black curves, and medial-lateral shear strain application data are represented by the gray curves. at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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J Dent Res 92(2) 2013 Shear Mechanics of the TMJ Disc Relative to Clinical Observations 197 variances in our mechanical results, since higher temperatures reduce stiffness and strength of the disc, since many ECM com ponents (collagen and proteoglycans) are temperature-sensitive (Detamore and Athanasiou, 2003). Application of cyclic shear in the A-P direction decreased the compressive modulus until a steady state was reached; however, in the posterior region, significant bumps were observed in the mechanical profile (Fig. 3C). We hypothesized that the increase in compressive stiffness associated with these bumps represents a material property that acts as a breaking system (force). Compression causes outflow of interstitial fluid or a shifting of the bulk matrix from the point of strain application. As the fluid matrix shifts, the periphery swells to maintain the tissue volume and causes increased hoop stresses and hydraulic pressures that inhibit the force dispersion and act to recoil the fibers back toward their undistorted orientation. Compressive properties of the disc are strongly dependent on interstitial fluid flow (Allen and Athanasiou, 2006), and with fewer paths for tissue fluid to escape when the disc is under shear, E will respond in intermit tent increased stiffness. A possible limitation of these investiga tions is that we evaluated disc punches that did not necessarily retain the boundary conditions of the whole disc; however, the ECM fiber alignment superstructure remained intact. This trend was observed only in the anterior and posterior zones, likely due to fiber arrangement, where collagen fibers were oriented in a mediolateral arc that increased hoop stresses and hydrostatic forces (Detamore et al., 2005; Almarza and Athanasiou, 2006). By comparison, the intermediate region, where the collagen fibers were aligned in the antero-posterior direction, was shown to culminate in channels that shuttled interstitial fluid quickly within the region. The mechanical consequence is that the inter mediate zone was more elastic than the periphery of the disc (Lumpkins and McFetridge, 2009). Perforation is often associated with disc derangement or osteoarthritis; however, tearing of the posterior-lateral region is also seen in asymptomatic discs (Kuribayashi et al., 2008; Liu et al., 2010). The results of the current study showed that the steady-state shear modulus was stiffest in the discs posterior region, indicating that this region is different structurally and/or in composition. These mechanical results are in agreement with clinical observations of regional disc damage. It is clinically accepted that TMJ disc fatigue occurs most frequently in the discs lateral-posterior region (Kuribayashi et al., 2008). We hypothesized that this retained stiffness with repeated shear strain applications limits the discs ability to distribute (disseminate) loads away from the impact point, leading to greater residual localized stress summation. It was further hypothesized that these localized stresses result in material fatigue and even tual failure, seen clinically as disc perforations. Shear strain in the M-L direction is less prominent in healthy TMJ disc loading (Fushima et al., 1995; Athanasiou et al ., 2009). However, shear strain is associated with the nonpathological tooth-grinding and jaw-clenching that are seen in bruxism, which can potentially lead to TMDs (Kuboki et al., 1996; Nagahara et al., 1999; Gallo et al., 2000). Sometimes simplified as hyperactivity of the lateral pterygoid, clenching and bruxing mechanics are more completely described as para functional activity of 1 or more of the major masticatory muscles (masseter, medial pterygoid, temporalis, and lateral pterygoid) (Nagahara et al., 1999; Hirose et al., 2006). The mechanical testing of the discs elastic moduli when sheared in the M-L direction under high compressive load is meaningful for the assessment of any potential relationship between bruxing and TMJ disc perforation. Similar to the shear modulus characteris tics of the posterior region under A-P shear, all regions of the disc have an observable trend of retaining their shear modulus from Gint to Gss when load is applied with M-L loading (Fig. 4). In conclusion, these results confirm that the mechanical char acteristics of the TMJ disc are highly dependent on the ECM microenvironment and its regional composition. The posterior region of the disc, which is the most commonly observed zone in which the disc shows fatigue, has been shown to maintain its stiffness when compressed or sheared cyclically. While there is no direct association between theoretical or experimental mod els and the clinical experience, these results are in agreement with mathematical modeling results showing that large stresses developed in the posterior region of the disc and retrodiscal tissue during prolonged clenching, and higher still in these regions when antero-lateral internal derangement is included (Hirose et al., 2006; Nishio et al., 2009). The hypothesis that there is a relationship between the discs regional mechanical properties and common clinical observations of TMJ disc damage is sup ported by the data collected in these works. These results sup port further investigation of fluid movement within the disc scaffold and greater development of a physiologically represen tative testing regime.ACKNOWLEDGMENTSWe gratefully acknowledge the National Institute of Dental and Craniofacial Research at the US National Institutes of Health (NIH; 1R21DE022449) and the J. Crayton Pruitt Family Department of Biomedical Engineering at the University of Florida for funding. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. en-GBREFERENCESAllen K, Athanasiou K (2005). A surface-regional and freeze-thaw charac terization of the porcine temporomandibular joint disc. Ann Biomed Eng 33:951-962. Allen K, Athanasiou K (2006). Viscoelastic characterization of the porcine temporomandibular joint disc under unconfined compression. J Biomech 39:312-322. Almarza A, Athanasiou K (2006). Effects of hydrostatic pressure on TMJ disc cells. Tissue Eng 12:1285-1294. Almarza A, Bean A, Baggett L, Athanasiou K (2006). Biochemical analysis of the porcine temporomandibular joint disc. Br J Oral Maxillofac Surg 44:124-128. Athanasiou KA, Almarza AA, Detamore MS, Kalpakci KN (2009). Tissue engineering of temporomandibular joint cartilage. San Rafael, CA: Morgan & Claypool Publishers. Callaghan J, McGill S (2001). Intervertebral disc herniation: studies on a porcine model exposed to highly repetitive flexion/extension motion with compressive force. Clin Biomech (Bristol, Avon) 16:28-37. Detamore M, Athanasiou K (2003). Tensile properties of the porcine tem poromandibular joint disc. J Biomech Eng 125:558-565. Detamore M, Orfanos J, Almarza A, French M, Wong M, Athanasiou K (2005). Quantitative analysis and comparative regional investigation of at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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198 Juran et al. J Dent Res 92(2) 2013the extracellular matrix of the porcine temporomandibular joint disc. Matrix Biol 24:45-57. Fushima K, Gallo L, Krebs M, Palla S (1995). Medial and lateral TMJ space variations during mastication. J Dent Res 74(Spec Iss):588, abstract #1504. Gallo L, Nickel J, Iwasaki L, Palla S (2000). Stress-field translation in the healthy human temporomandibular joint. J Dent Res 79:1740-1746. Hirose M, Tanaka E, Tanaka M, Fujita R, Kuroda Y, Yamano E, et al. (2006). Three-dimensional finite-element model of the human temporoman dibular joint disc during prolonged clenching. Eur J Oral Sci 114:441448. Iatridis J, ap Gwynn I (2004). Mechanisms for mechanical damage in the intervertebral disc annulus fibrosus. J Biomech 37:1165-1175. Koolstra JH, Tanaka E, Van Eijden TM (2007). Viscoelastic material model for the temporomandibular joint disc derived from dynamic shear tests or strain-relaxation tests. J Biomech 40:2330-2334. Kuboki T, Azuma Y, Orsini M, Takenami Y, Yamashita A (1996). Effects of sustained unilateral molar clenching on the temporomandibular joint space. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 82:616624. Kuo J, Zhang L, Bacro T, Yao H (2010). The region-dependent biphasic viscoelastic properties of human temporomandibular joint discs under confined compression. J Biomech 43:1316-1321. Kuribayashi A, Okochi K, Kobayashi K, Kurabayashi T (2008). MRI find ings of temporomandibular joints with disk perforation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 106:419-425. Lai W, Bowley J, Burch J (1998). Evaluation of shear stress of the human temporomandibular joint disc. J Orofac Pain 12:153-159. Liu XM, Zhang SY, Yang C, Chen MJ, Y Cai X, Haddad MS, et al. (2010). Correlation between disc displacements and locations of disc perfora tion in the temporomandibular joint. Dentomaxillofac Radiol 39:149156. Lumpkins S, McFetridge P (2009). Regional variations in the viscoelas tic compressive properties of the temporomandibular joint disc and implications toward tissue engineering. J Biomed Mater Res A 90:784-791. Lumpkins S, Pierre N, McFetridge P (2008). A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc. Acta Biomater 4:808-816. Nagahara K, Murata S, Nakamura S, Tsuchiya T (1999). Displacement and stress distribution in the temporomandibular joint during clenching. Angle Orthod 69:372-379. Nakano T, Scott P (1996). Changes in the chemical composition of the bovine temporomandibular joint disc with age. Arch Oral Biol 41:845853. Nishio C, Tanimoto K, Hirose M, Horiuchi S, Kuroda S, Tanne K, et al. (2009). Stress analysis in the mandibular condyle during prolonged clenching: a theoretical approach with the finite element method. Proc Inst Mech Eng H 223:739-748. Piette E (1993). Anatomy of the human temporomandibular joint. An updated comprehensive review. Acta Stomatol Belg 90:103-127. Tanaka E, del Pozo R, Tanaka M, Aoyama J, Hanaoka K, Nakajima A, et al. (2003a). Strain-rate effect on the biomechanical response of bovine temporomandibular joint disk under compression. J Biomed Mater Res A 67:761-765. Tanaka E, Hanaoka K, van Eijden T, Tanaka M, Watanabe M, Nishi M, et al. (2003b). Dynamic shear properties of the temporomandibular joint disc. J Dent Res 82:228-231. Tanaka E, Kawai N, van Eijden T, Watanabe M, Hanaoka K, Nishi M, et al. (2003c). Impulsive compression influences the viscous behavior of porcine temporomandibular joint disc. Eur J Oral Sci 111:353-358. Tanaka E, Kikuzaki M, Hanaoka K, Tanaka M, Sasaki A, Kawai N, et al. (2003d). Dynamic compressive properties of porcine temporomandibular joint disc. EurJ Oral Sci 111:434-439. Tanaka E, Kawai N, Hanaoka K, van Eijden T, Sasaki A, Aoyama J, et al. (2004). Shear properties of the temporomandibular joint disc in relation to compressive and shear strain. J Dent Res 83:476-479. at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research


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1 REENGINEERING THE TMJ DISC: AN EVALUATION OF SCAFFOLD DESIGN, CELL INCORPORATION, AND STIMULATED FUNCTIONALIZATION By CASSANDRA MARIE JURAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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2 2014 Cassandra Marie Juran

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3 To those in my life who have inspired and challenged my imagination

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4 ACKNOWLEDGMENTS In addition to representing the culmination of over three years of graduate work, this thesis serves as a testament to the people who contributed to my education over the first twenty eight years of my life. I would first like to thank my mentor, Dr. Peter McFetridge, for offering me the opportunity to work on this innovative project in the fall of 2010. I thank him for his continuous guidance and resear ch advisement over these years, for always leaving his door open. I also wish to thank the other members of my supervisory committee for their useful suggestions and critique of both my proposal and dissertation: Dr Jon Dobson, Dr. Scott Banks, Dr. David W. Hahn an d a special thanks to my clinical committee member Dr. M. Franklin Dolwick whose advice and support was invaluable. I also thank my committee members for providing advice on venturing out into the unknown after graduation. I want to acknowledg e all of my lab mates, research assistants, and other colleagues at the University of Florida for their cooperation. I especially thank the past and present students of the McFetridge lab for their constructive criticism, collaboration, and for moral and i ntellectual support, for without them this work could not have been completed. I individually thank the following students for direct advisement of this project: Dr. Salma Amensag, Dr. Joe Uzarski, Dr. Marc Moore, Dr. Zehra Tosun, Claudia Siverino, Andrea Matuska, Leslie Goldberg, Mediha Gurel, and Aurore Van de Walle. I owe a great deal of gratitude to my parents, Franz and Catherine, who always supported me, challenged me, and reminded me that I was unique and that my way of approaching and investigati ng questions was as well My brother, Franki e, has always tested my patience and my ingenuity with his ability to get us into trouble and I owe a

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5 great deal of my ability to adapt my thinking process to new and unexpected hurdles to him Lastly, this w ork would not be possible without our sources of funding. I would like to thank the National Institute of Health for providing the financial support (NIH R21 DE022449 ).

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATI ONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 THE TEMPOROMANDIBULAR JOINT DISC: REGENERATIVE MEDICINE AND TISSUE ENGINEERING ................................ ................................ ................ 17 The Temporomandi bular Joint: Anatomy, Dysfunction, Treatments, and Disc Characterization ................................ ................................ ................................ .. 17 Tissue Engineering Efforts ................................ ................................ ...................... 19 Ideal Design and History of TMJ Disc Tissue Engineering ............................... 19 Tissue Characterization ................................ ................................ .................... 22 Cell s ................................ ................................ ................................ ........... 22 Biochemical composition ................................ ................................ ............ 22 Biomechanical characterization ................................ ................................ 23 Hurdles to Tissue Engineering using Whole Disc Tissue Xenogeneic Graft ........... 26 Key Investigations ................................ ................................ ................................ ... 27 Research Design ................................ ................................ ................................ .... 27 Significance ................................ ................................ ................................ ............ 28 Innovation ................................ ................................ ................................ ............... 29 2 GENERAL MATERIALS AND METHODS ................................ .............................. 36 Experimental Methods ................................ ................................ ............................ 36 Porcine TMJ Disc: Procurement and Dissection ................................ .............. 36 Decellularization of the Porcine TMJ Disc ................................ ........................ 36 Lyophilization of Fibrocartilage Disc Scaffold ................................ ................... 37 ................................ .... 37 Analytical Methods ................................ ................................ ................................ .. 38 Biochemical Analysis ................................ ................................ ........................ 38 Glycosaminoglycans content analysis ................................ ....................... 38 Total collagen content analysis: modified hydroxyproline assay ................ 38 Cellular Integration Analysis ................................ ................................ ............. 39 Fluorescent stains ................................ ................................ ...................... 39 Fluorescent imaging and analysis ................................ .............................. 40 Histology ................................ ................................ ................................ ........... 40 Tissue processing ................................ ................................ ...................... 40 Hematoxylin & eosin stain ................................ ................................ .......... 40

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7 ................................ ................................ ........... 41 Color imaging ................................ ................................ ............................. 41 Cellular proliferation and metabolic activity ................................ ................ 41 Mechanical Analysis: Cyclic Compressive Analysis ................................ ......... 42 Imaging ................................ ................................ ................................ ............. 43 Scanning electron microscopy (SEM) ................................ ........................ 43 Transmission electron microscopy (TEM) ................................ .................. 44 3 SHEAR MECHANICS OF THE TMJ DISC: RELATIONSHIP TO COMMO N CLINICAL OBSERVATION ................................ ................................ ..................... 49 Introduction ................................ ................................ ................................ ............. 49 Materials and Methods ................................ ................................ ............................ 50 Specimen Preparation ................................ ................................ ...................... 50 Sample Groups ................................ ................................ ................................ 50 Disc Geometry and Sample Height ................................ ................................ .. 51 Physiologic Load Choice ................................ ................................ .................. 51 Profile Application ................................ ................................ ............................. 52 Testing Order Evaluation ................................ ................................ .................. 52 Biomechanical Testing Cyclic Shear Loading ................................ ................ 53 Recovery Time ................................ ................................ ................................ 54 Slippage Evaluation ................................ ................................ .......................... 55 Statistical Analysis ................................ ................................ ............................ 55 Results ................................ ................................ ................................ .................... 55 Discussion ................................ ................................ ................................ .............. 57 Conclusions ................................ ................................ ................................ ............ 60 4 MECHANOBIOLOGICAL ASSESMENT OF TMJ DISC SURFACES: A NANOINDENTATION AND TEM STUDY ................................ ............................... 68 Introduction ................................ ................................ ................................ ............. 68 Materials and Methods ................................ ................................ ............................ 71 Depth Dependent Nanoindenter Micromechanical Testing .............................. 71 Statistical Analysis ................................ ................................ ............................ 72 Results ................................ ................................ ................................ .................... 72 Discussion ................................ ................................ ................................ .............. 74 Conclusions ................................ ................................ ................................ ............ 7 7 5 LASER MICRO PA TTERNING SCAFFOLD DEVELOPMENT AND BIOMEDICAL ASSESMENT ................................ ................................ ................... 83 Introduction ................................ ................................ ................................ ............. 83 Materials and Methods ................................ ................................ ............................ 86 Experimental Methods ................................ ................................ ...................... 86 Laser micro patterning ................................ ................................ ............... 86 Hydration ................................ ................................ ................................ .... 86 Analytical Methods ................................ ................................ ........................... 87

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8 Biochemical quantification ................................ ................................ .......... 87 Permeability coefficient analysis ................................ ................................ 87 COMSOL computational fluid modeling ................................ ..................... 87 Mechanical evaluation ................................ ................................ ............... 88 Statistical Analysis ................................ ................................ ............................ 89 Results ................................ ................................ ................................ .................... 89 Scaffold Optimization for LMP ................................ ................................ .......... 89 Laser Micro Patterning (LMP) Analysis ................................ ............................ 90 Discussion ................................ ................................ ................................ .............. 92 Conclusions ................................ ................................ ................................ ............ 96 6 MESENCHYMAL STEM CELL CHOICE, VALIDATION, AND NOVEL SEEDING METHODOLOGY ................................ ................................ ................................ 105 Introduction ................................ ................................ ................................ ........... 105 Methods ................................ ................................ ................................ ................ 107 Human Mesenchymal Stem Cell (h MSC) Isolation and Differentiation ......... 108 Immunohistochemistry of MSCs ................................ ................................ ..... 109 Hydration Seeding Computation and Live/DEAD Viability Evaluation ............ 109 Hydration Seeding Empirical Evaluation ................................ ........................ 112 Feedback Optimization ................................ ................................ ................... 112 7 day culture ................................ ................................ ............................ 113 Evaluations ................................ ................................ .............................. 113 Results ................................ ................................ ................................ .................. 114 MSC Isolation and Evaluation ................................ ................................ ........ 114 Computational Hydration Seeding and Live/DEAD Viability Evaluation ......... 114 Computation model of hydration seeding by standard culture media ....... 114 Computation model of hydration seeding by dextran modified culture media ................................ ................................ ................................ .... 115 Hydration Seeding and 7 Day Culture Experimental Assessment .................. 115 Discussion ................................ ................................ ................................ ............ 116 Conclusions ................................ ................................ ................................ .......... 120 7 CELLULAR ADHESION AND CYTOCOMPATABILITY PROOF .......................... 131 Introduction ................................ ................................ ................................ ........... 131 Methods and Ma terials ................................ ................................ .......................... 132 Scaffold Culture ................................ ................................ .............................. 132 Biomechanical Analysis ................................ ................................ .................. 132 Hydraulic Permeability ................................ ................................ .................... 132 Cellular Incorporation ................................ ................................ ..................... 133 Stat istical Analysis ................................ ................................ .......................... 134 Results ................................ ................................ ................................ .................. 134 Mechanical Evaluation ................................ ................................ ................... 134 Cellular Adhesion and Regional Seeding Efficacy ................................ .......... 134 Cellular Integration and Early Remodeling ................................ ..................... 135 Discussion ................................ ................................ ................................ ............ 136

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9 Conclusions ................................ ................................ ................................ .......... 140 8 PROJECT CONCLUSIONS, CLINICAL IMPACT, AND FUTURE DIRECTION .... 149 Project Conclusions ................................ ................................ .............................. 149 Project Clinical Impact ................................ ................................ .......................... 149 Project Summary ................................ ................................ ................................ .. 150 Project Future Directions ................................ ................................ ...................... 153 LIST OF REFERENCES ................................ ................................ ............................. 158 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 169

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10 LIST OF TABLES Table page 5 1 Biochemical evaluation of the native TMJ disc and acellular TMJ disc scaffolds ................................ ................................ ................................ ........... 101 6 1 Definition of COMSOL Multiphysics computational modeling independent variables for the TMJ disc poroelastic matrix material properties. .................... 123 6 2 Definition of COMSOL Multiphysics computational modeling independent variables for the TMJ disc fluid material and particles properties. .................... 124 7 1 Hydraulic Conductivity and Theoretical Per meability of the nati ve and worked TMJ disc scaffold ................................ ................................ .............................. 142

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11 LIST OF FIGURES Figure page 1 1 Temporomandibular Joint Anatomy ................................ ................................ .... 30 1 2 Regional cell population of the Temporomandibular Joint Disc .......................... 31 1 3 Proteoglycan regional content of the porcine TMJ disc ................................ ...... 32 1 4 Anatomy and tensile mechanical properties of the TMJ disc attachments ......... 33 1 5 Extracellular matrix fibril alignment of the TMJ disc ................................ ............ 34 1 6 Viscoelastic compressive biomechanics of the porcine TMJ disc ....................... 35 2 1 Porcine Temporomandibular Dissection ................................ ............................. 45 2 2 Preliminary processing the native TMJ disc for laser ablation micro patterning 46 2 3 region mesenchymal stem cells are derived from for these experiments ........... 47 2 4 Compressive mechanical testing regime ................................ ............................ 48 3 1 Joint anatomy and simplified free body diagram, regional sampling, and testing method ................................ ................................ ................................ .... 61 3 2 Change in Shear Modulus with frequency, compressive strain, and shear strain ................................ ................................ ................................ ................... 62 3 3 Change in compressive modulus when cyclic shear strain is applied in the anterior to posterior or medial to lateral direction ................................ ................ 63 3 4 Mean regional values of the instantaneous and steady state shear modulus of each disc region when strained in the anterior to posterior direction and the medial to lateral direction. ................................ ................................ ............. 64 3 5 Recovery time evaluation ................................ ................................ ................... 65 3 6 Representative Stress Strain plot depicting axial slippage at lower compressive strain values ................................ ................................ .................. 66 3 7 Random testing order strain profile application ................................ ................... 67 4 1 Joint and Disc Anatomy with Depth Dependent Regional Zone Highlighted. Schematic Joint Anatomy with Disc Placement and orientation elucidated and SEM micrographs of the TMJ disc ................................ ............................... 78

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12 4 2 Representative surface dependent step creep response and TEM correlations ................................ ................................ ................................ ......... 79 4 3 Cyclic ultra micro indentation mechanical characterization ................................ 80 4 4 TEM of the superior disc surface ................................ ................................ ........ 81 4 5 Apoptotic cell zone at inferior surface periphery ................................ ................. 82 5 1 TMJ disc scaffold preparation and optimization for laser micro patterning. Mechanical consequence of lyophilization ................................ .......................... 98 5 2 Mechanical consequence of decellularization and lyophilization on fibrocartilage TMJ disc ................................ ................................ ........................ 99 5 3 Laser Micro Patterning schematic and final printed TMJ patterns with dimensional data ................................ ................................ .............................. 100 5 4 Rehydration and permeability experiment and COMSOL MultiPhysics modeling fluid mechanics within the LMP pore ................................ ................. 102 5 5 Whole scaffold evaluation of LMP mechanical consequence. Representative and modulus values ................................ ................................ .......................... 103 5 6 Nano ind entation evaluation of thermal damage to ECM scaffold .................... 104 6 1 and differentiation ................................ ................................ ................................ .... 122 6 2 velocity and cell scale particle tracing modeling ................................ ............... 125 6 3 COMSOL Multiphysics Modeling of the Moment Field associated with hydration seeding ................................ ................................ ............................. 126 6 4 Cut away views of COMSOL Multiphysics Modeling tracing cell scale particles during initial (5min) rehydration seeding ................................ ............. 127 6 5 H&E histology of the hydration seeded and control pipet seeded constructs at 2 hours of culture ................................ ................................ .............................. 128 6 6 DNA quantification and cellular metabolism of the scaffold and regional variation of DNA quantification over 24 hours of culture ................................ ... 129 6 7 Variation in DNA quantification and cellular metabolism of the hydration seeded and pipet seeded scaffolds cultured over 7 days ................................ 130 7 1 Mechanical Effects of the scaffold processing technique ................................ 142

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13 7 2 Cell Adhesion over initial 24 hours ................................ ................................ ... 144 7 3 DNA quantification and cell seeding density as a function of thickness ............ 145 7 4 Cellular Adhesion to the surface layer and through the thickness of the TMJ disc scaffold ................................ ................................ ................................ ...... 14 6 7 5 Cellular Proliferation and Metabolism and mechanical characterization over culture period ................................ ................................ ................................ .... 147 7 6 Histological evaluation of culture at day 21 ................................ ...................... 148 8 1 Experimental design and preliminary results comparing cyclic compression and combined cyclic tension/compression mechanical stimulation to promote wound healing mechanisms of the MSCs and functionalize the LMP TMJ ....... 157

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14 LIST OF ABBREVIATIONS ECM E XTRACELLULAR M ATRIX GAG G LYCOSAMINOGLYCAN ID I NTERNAL D ERANGEMENT LMP L ASER M ICRO P ATTERNED P TMJ P ORCINE T EMPOROMANDIBULAR J OINT SDS S ODIUM D ODECYL S ULFATE TMD T EMPOROMANDIBULAR D ISORDER TMJ T EMPOROMANDIBULAR J OINT

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REENGINEERING THE TMJ DISC: AN EVALUATION OF SCAFFOLD DESIGN, CELL INCORPORATION, AND STIMULATED FUNCTIONALIZATION By Cassan dra Marie Juran May 2014 Chair: Peter S. McFetridge Major: Biomedical Engineering Functioning primarily as a stress distribution bumper within the joint, the temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe Temporomandibular Disorders has been the primary driving force for the in troduction and development of TMJ disc Tissue Engineering as an approach to alleviate this priority clinical issue. A great deal of promise has been shown with the application of tissue lly accommodate typical loads of the joint and regain physiologic functionality. The branches of study investigated thus far have primarily focused on synthetic scaffold material, characterizing the native anatomy and physiology, cellular culture techniqu e, and decellularization of TMJ discs. Cell survival and integration into the scaffold has proven problematic largely due to limited porosity, resulting in poor transport conditions that limit cell migration into the scaffold.

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16 The purpose of my thesis is to utilize a native porcine TMJ disc (pTMJ) as a xenogeneic ex vivo scaffold, and further modify the discs structure to enhance reseeding and transport conditions to create a mechanically and biologically functional disc. To then evaluate different physio logic mechanical loading conditions influence on the remodeling, cell phenotype, and mechanical viability of the disc.

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17 CHAPTER 1 THE TEMPOROMANDIBULAR JOINT DISC: REGENERATIVE MEDICINE AND TISSUE ENGINEERING The Tem poromandibular Joint: Anatomy, Dysfunction, Treatments, and D i sc C haracterization The Temporomandibular Joint (TMJ) is a physiologically dynamic diarthroidal joint that joins the mandible, or lower jaw, to the temporal bone of the skull [1] (Figure 1 1). The primary components of TMJ articulation are the condyle, articular fossa, articular eminence, and the small fibrocartilage disc which acts as a bumper distributing and disseminating loading throughout joint motion. The vast complexity of the TMJ yields itself to a wide variety of pathologies, collectively termed Temporomandibular Disorders (TMD). Epidemiological studies have reported that as many as 20 25% of the population experience symptoms of TMDs, which can range from painless cli cking and locking of the jaw to debilitating pain and dysfunction. Of these conditions, a large percentage (about 70%) of patients seeking treatment suffer from an internal derangement of the fibrocartilagenous disc such that the disc exhibits an abnormal relationship to the TMJ skeletal structure [2 7] Treatments for TMDs in the early stages include bite alteration by means of mo uth guards or skeletal splinting. Conservative intermediate therapies include injections of Botulism toxin type A (Botox) and local anesthetics to relieve pain and hyaluronic acid and steroids to encourage joint recovery by natural pathways. These therapi es are not approved by the Food and Drug Administration and their success is arguable [8, 9] More aggressive therapies for intermediate stage dysfunction and early degeneration involve surgical procedures. Arthroscopy is a procedure in which a miniature telescoping instrument is used to look inside your joint to diagnose joint problems [10 12] Arthrocentesis is a process in which

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18 two needles or the arthroscope and a needle are placed into t he joint, and the joint is flushed with a sterile saline solution or a lactated Ringers solution. The purpose of this procedure is to remove tissue breakdown products and reduce inflammation [13 16] Some surgeons will not only look inside and/or wash out the joint, but also perform surgical procedures like removing scar tissue, smoothing the bo ne and even attempt repositioning the disc. Once the disorder progresses to a point that the internal structures have become damaged the only surgical treatment to address the dysfunction is arthrotomy, open joint TMJ surgery. Arthrotomy surgeries in clude discoplasty or repositioning of the disc, and discectomy, total removal of the TMJ disc. Sometimes in addition to discal surgical treatment manipulation of the skeletal structures is needed to restore function, a procedure called arthroplasty. In t he most severe TMD cases the removal of damaged or degenerative disc tissue and bone is so severe that an implanted metal joints are [17 21] These treatments are extremely invasive and destructive to the surrounding tissue causing additional problems to joint as time progresses. Current treatments for TMDs are limited to managing symptoms rather than full recovery, and as a whole cost an estimated 32 billion dollars annually in the United States [4]. The debilitating nature and limited treatment options of severe TMDs have been the primary driving force for the development of TMJ disc Tissue Engineering as an approach to al leviate this priority clinical issue. Within this field, investigations have

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19 primarily focused on characterizing the native anatomy and physiology, cellular culture techniques, synthetic scaffold materials and decellularization of TMJ discs. Tissue Engineering Efforts Ideal Design and History of TMJ Disc Tissue Engineering The aim of using the tissue engineering approach is to replace damaged or diseased TMJ discs before degeneration of the surrounding architecture progresses to a point which total j oint replacement becomes a necessity. An ideal newly implanted tissue engineered disc would ideally accommodate all joint loading and regain biological function. This disc would be composed of a matrix able to recapitulate the energy distributive ability required for proper function of the complexly loaded joint. The graft would also support cellular integration and nutrient/waste diffusion to accommodate early remodeling of the predominantly avascular neo tissue. The use of alloplastic materials to repl ace the disc and the skeletal components condyle head in 1891 [22] The first recorded use of a metal implant in the TMJ was by Castigliano and Gross and Kleitch in 1951 using vitallium a cobalt chromium alloy to replace the entirety of the patients mandible [23 ] However the most infamous alloplastic replacements of the TMJ components was the 1970s to 1990s Vitek Proplast Teflon intra articular disc replacements which suffered from catastrophic failure due to material wear and ensuing surrounding tissue dama ge and degeneration [20, 24 27] The incredible dysfunction of the Vitek and several similar implants caused the FDA to issue a class I recall of several TMJ alloplastic implants [2] However, today many alloplastic total joint replacement devices (TMJ Concepts, TMJ Medical, etc.) are on the market and produce successful clinical results after recovery from surgery.

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20 Unfortunately all these devices are implanted by traumatic open joint surgery and removal of large sections of the TMJ skeletal structures, replacing them with mechanical components [1 9, 28, 29] These therapies are still controversial in part because of fear from past catastrophes of TMJ implant devices and in part because of the severity of the surgical procedure, the post operative recovery, physical therapy, and often secondary surgeries to correct damage cause by the implant over time. Autogeneous tissue implants, or tissues grafts originating from the patient him/herself, used to ease the sym ptoms of TMDs are recorded being employed as early as 1860 when Verneuil employed the use of the temporalis flap extended into the disc space after removing the disc [30] Murphy reported the use of autogen e ous fat for interposition after lysis of TMJ ankylosis [31] Risdon applied free muscle to the problem in 1934 [32] It was not until the 1970s that the use of autogeneous articular cartilage derived from the hip was explored by Perko [33] All these autogeneous materials suffered from difficulties with stabilization, excessive elasticity and lack of mechanical rigidity to withstand joint loading, and susceptibility to perforation or fracture due to thinness. Georgiade investigated the use of dermis as a disc replacement and demonstrated some promising patient recovery [34] This material was further validated in the investigations of Stewart who showed that the implant survived as an effective interpositional scar which in some instances showed fibrocartilagen ous histologic characteristics. The major limitation to the use of this material was the formation of cysts and eventual ankylosis likely due to the thinness of the dermal graft. However, the histologic compatibility seen has inspired investigations into the use of other connective tissues as disc replacement grafts. The major problem using jaw ligaments

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21 as TMJ disc homologs is the ligaments complex involvement in the function of the TMJ. As such grafting attempts have shown poor results often resulting in altered mastication cycle several months after graft placement. Allogeneic transplant technology is the transplant of cells, tissues, or organs, to a recipient from a genetically non identical donor of the same species The use of this tissue source g ained predominance during WWII to treat injured soldiers [35] The techniques and immune regulatory drugs developed to improve the outcomes of these treatments aided in the development of the field of organ transplantation. Allograft therapies for the TMJ disc in humans have not be en published in the literature, however several animal studies have been conducted. Kurita, in the late 1990s, demonstrated the short term effects of disc allograft in osteoarthritic sheep [36] results suggest that implanted fresh disc allografts can pre vent fibrous ankylosis and repair the joints fibrously in the early stages after grafting. More recently in 2003 and 2006, Sato demonstrated the viability of s tored tendon allograft for TMJ disc replacement following discectomy in rabbits [37] The use of xenogeneic tissue implants as therapies to treat TMJ disorders have several advantages over alloplastic, allogeneic, and autogenic grafts. Xenogeneic tissue is readily available, immune regulation for pig heart valves and skin transplants already in use clinically, and all mammalian ECM proteins are fundamentally identical. For the TMJ disc xenograft porcine disc tissue has demonstrates comparable size, structure, and cell populat ions [38, 39] Additionally pig jaws function similarly to humans during mastication and vocalization. The phases of mastication in a pig are the same as in a human or higher primate but occur at about double the speed. The

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22 morphologic dev elopment and compositional make up of the porcine TMJ is statistically similar to the human TMJ disc and the porcine jaw structure, chewing, and tooth development models have been used as homologs for humans in dental teaching programs for decades [38] Tissue Characterization Initial findings assessing the suitability of the porcine xenograft TMJ as a scaffold were focused on disc cells, biochemical com position, and biomechanical properties [40] Cells Early investigations into t he cells of the human TMJ disc reported that the ll populations of rounder cells Studies of cell communities of the bovine TMJ disc showed that both fibroblast and regional populations of chdrocytes within the disc [41] While primate studies indicated the cellular populations were predominantly chondrocyte like cells described as roundish and surrounded by lacunae [42] An immunohistochemistry and tra nsmission electron microscopy (TEM) investigation into the cell type and distribution within the porcine TMJ disc revealed that fibroblast like cells were present throughout the disc with greatest density at the anterior and posterior bands and an abundanc e of chondrocyte like cells in the center of the TMJ disc (Figure 1 2) [43] The correlating conclusion of all these cell investigation is that the TMJ disc contains a non homogeneous distribution of cell subpopulations predominantly a universal fibroblast like population and an intermediate zone specific chondr ocyte like population. Biochemical composition Compositionally the TMJ disc is predominantly collagen (total collagen content 68.2% dry weight) [44] with regional variation of type and matrix structure. The disc is

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23 predominantly made up of the structural protein co llage type I. Type I is found throughout the disc with almost exclusive presence in the anterior and posterior bands and at high concentration in the intermediate zone. The compositional differences between the peripheral regions and the intermediate dis c are that the former possesses thick (10 intermediate zone collagen type I is discontinuous and highly undulated [45] Within the intermittent collagen typ e I are clusters of collagen type II (hyaline/articular cartilage major structural component) and elastin. In addition to structural proteins of the TMJ disc are a variety of glycosaminoglycans (GAGs). By far the most abundant GAG in the TMJ disc is chond roitin sulfate, accounting for 74% total GAG content [45 47] Chondroitin 4 sulfate is found in highest concentration in the intermediate zone which corresponds well to the higher density of chondrocyte like cell populat ions mentioned previously [43] Chondroitin 6 sulfate lik e chondroitin 4 sulfate is localized in the intermediate zone but also found profusely in the anterior band of the medial region. This compositional trend is followed by Dermatan sulfate PG and keratin sulfate. Hyaluronic acid, a GAG associated with arti culating cartilage, is found in low concentration (less than 1% dry weight) throughout the disc. Proteoglycan assessment reveal that a large proportion of the GAG are adhered isc are aggrecan and decorin with regional variations illustrated in Figure 1 3 [46 48] Biomechanical characterization The structural makeup of the TMJ disc supports the mechanical robustness needed for TMJ articulation. The TMJ disc experiences tension, compression, shear,

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24 and surface rotation and drag loading during joint articulation. The mechanical demands of the disc have been studied over the past decade and a half with quantification of the mechanical characteristics being well defined. Each loading modality has bee n examined independently by uniaxial testing and more recent work testing [49 51] The TMJ disc is oriented and held in place by discal attachments (Figure 1 4) [52] Evaluation of these attachments found anisotropy of structural ECM fibrils demonstrated in 5 of the 6 evaluated attachments, with the exception of the anterior in ferior attachment. Anteroposteriorly, the lateral attachment had the highest tensile modulus value (8.3 MPa) and the anterior superior was least stiff (1.4 MPa). Mediolaterally, the posterior superior attachment was stiffest (16.3 MPa) and the medial was l east stiff (1.4 MPa). The greatest strain was observed in the lateral attachment in the mediolateral direction and the posterior superior attachment in the anteroposterior direction. With greatest strains in the most commonly observed directions of disc di splacement, these works hypothesized that compromise in the posterior and lateral attachments contributes to partial lateral and anterior disc displacement [53, 54] The attachments restrain the TMJ disc such that the disc is held in tension over the condyle of the mandible, creating a trampoline like reactive structural arrangement. Thus the tensile characteristics of the disc are of critical importance as a target parameter for tissue engineering a disc replacement, as 70% of TMDs are statistically correlated to disc displacement or failure of the tensile ability of the disc [55] The regional properties of the TMJ disc under tensile loading have a strong relationship to

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25 the ECM architecture. Qualitative observations from several published works have stated that fibril orientation has directional specificity in the d isc, oriented anterior to posterior in the intermediate zone and with ring like orientation along the periphery (Figure 1 5) [47, 56 59] The structure function relationship between tensile mechanical ability an d ECM architecture is apparent when the tensile loading is applied in the mediolateral direction. This directional loading shows that the regions with medial to lateral fibril orientation (the anterior and posterior bands) have higher modulus values than the intermediate zone which possesses anterior to posterior fibril alignment [47] The differences in the structure function relationship to tensile properties associated with this loading orientation are less overt. The higher modulus values for the medial (14.3MPa) and central (18.5MPa) regions as compared to the lateral (10.6MPa) region are attributed to differences in the ECM fiber diameters. The lateral region is composed of smaller diameter fibers offering less resistance to tensile stretching and un crimping of the undulating protein fibrils. The majorit y of published biomechanical analyses have focused on the TMJ discs compressive properties [60 63] and have found that the disc possesses significant zonal specificity [61, 64] The highly incongruent skeletal structure of the TMJ impart regional compressive characteristics which support the energy distributive function of the d isc. Published evaluations of dynamic mechanical regional variations of the TMJ disc under unconfined compression agree that the compressive modulus is highly dependent on strain amplitude and frequen cy at which loading is imparted However, in the liter ature is an evident disconnect between the studies conducted. The animal model and experimental methods are particularly divergent throughout previous reports.

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26 To clarify and develop relevant target parameters for future tissue engineering the McFetridge research group tested regional dynamic compressive evaluations on porcine TMJ discs and fit the results to a viscoelastic model (Figure 1 6 ) [61] Variability of the intermediate regions (central, medial, and lateral) were assessed using a uniaxial mechanical testing paradigm. Results indicated the compressive modulus has a greater dependency on strain applied than the frequency at w hich the compression is imparted. Also, all regions tested display a decrease in modulus value from instantaneous to steady state. A finding typical of viscoelastic materials. Hurdles to Tissue Engineering using Whole Disc Tissue Xenogeneic Graft The use of whole tissue xenogeneic grafts for the TMJ disc present several key problems. First the published literature does not adequately describe several key loading modalities of the TMJ. As stated previously the TMJ disc experiences tension, compression and shear loading. Tensile and compressive characterization has been conducted thoroughly and by several research groups [52, 57 59] characteristics however have undergone little investigation. Also, these investigations looked at un iaxial shear loading, which is not physiologically representative as shear loading is accompanied by compressive loading. Another characterization investigation key to tissue engineering efforts which is insufficiently described in the literature is the d isc surface structure function relationship. The disc is a diarthroidal joint, and as such each surface of the disc is exposed to different articulating skeletal architecture and chemical environment which effect the immediate surface zones properties. T his quality of the disc is imperative for functional implant of the engineered disc replacement. Another major restriction to the use of whole tissue xenogeneic engineering, in vivo or in vitro, is the tissue possesses subcellular microporosity. The impli cation being

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27 that cells from the joint cannot penetrate the grafts porosity or obtain nutrients during remodeling. Computational studies of diffusion of small molecules through a matrix of rom a nutrient source. This finding is corroborated by experimental studies conducted by the McFetridge et al., 2011 which showed cellular infiltration onto a vascular graft was [130] Key Investigation s This dissertation describes five key investigation conducted to establish 1) important disc characteristics not thoroughly described in the literature to be used as target properties for disc engineering efforts and, 2) describe and evaluate a novel lase r ablated path of infiltration to support cellular integration and subsequent nutrient diffusion during tissue remodeling of the xenogeneic graft. Study 1: Define shear mechanical characteristics of the native porcine disc for physiologically representati ve loading conditions. Study 2: Investigate the structure/function relationship of the immediate disc surfaces using Ultra nanoindentation and TEM imaging. Study 3: Incorporate laser ablated microporosity to enhance permeability, hydration, and transport conditions while minimizing mechanical degradation due to volume loss and microenvironment disruption. Study 4: novel hydration seeding modality for cellular integration and viability. S tudy 5 : Test the hypothesis that physiologic mechanical stimulation encourages fibrochondrocytes to remodel the LMP TMJ scaffold toward its native properties Research Design This dissertation describes the progressive development of a functional ex vivo acellular bioscaffold derived from porcine TMJ discs into tissue engineered cellularized

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28 laser drilled TMJ disc implant. In addition to developing functional TMJ disc tissue I have aimed to determine parameters that can be used to define critical modaliti es that are expandable to engineering other articular cartilage deficits. The overall concept of the experimental design is to expand current investigations to fully develop the tissue engineered construct. My first goal is to develop a freeze dried ace llular disc scaffold that enhances laser interactions and advantageously act to restore native disc structure and biomechanics lost during SDS decellularization. The second phase of the experimental design is to determine what laser drilled porosity condit ions are optimal for cell to ECM interaction and physiologic function. To assess the scaffolds ability to support cell culture after seeding two culture techniques will be evaluated; static, and cyclic dynamic loading. The proposed porcine TMJ disc (pTM J) scaffold seeks not to manage symptoms alone of TMDs but to alleviate them by replacing the damaged or diseased TMJ disc. Tissue engineering a functional TMJ disc is a promising technology which may serve to alleviate pain associated with TMDs, slow pro gression of TMJ tissue resorption due to improper load distribution, and return normal jaw function to the previously impaired joint. Significance The significance of this work is twofold. First, create a viable TMJ disc replacement for those afflicted with severe TMDs, and second further our understanding of Temporomandibular Joint disc physiology and function. These investigations were significant because: Utilized a freeze drying technique to reinstate native TMJ structure and mechanics after decell ularization. A property that is often lost with aggressive ex vivo tissue decellularization protocols.

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29 Utilization of hydration physics to seed cells throughout the tissue scaffold by capillary action of the cell rich fluid uptake by the freeze dried scaff old. As the disease often effects the whole disc structure, including ligaments that maintain the discs physical location, this approach allows the ligaments associated with the scaffold to be included as part of the entire structure, thus significantly im proving the potential of the surgical procedure. In addition to treatment of TMDs the technology developed through the work of this dissertation can be expanded to other diseased or damaged articular cartilage tissues. The TMJ disc model system has very similar biochemical characteristics to the vertebral discs of the spine. Damage to these discs is a degenerative condition that plagues nearly 70% of the United States population at some period of their life [7] The porcine TMJ (pTMJ) scaffold aims to return normal jaw function to a previously impaired joint, an d eliminate the need for costly recurrent treatment. Innovation These investigations explore the utilization of laser micro fabrication to create artificial porosity for in the field of tissue engineering, specifically to construct a TMJ disc scaffold. Th ese works present the use of stem cells as a cell source for in vitro TMJ tissue remodeling and investigate the use of mechanical stimulation as a differentiation and functionalization mechanism in the TMJ disc. An engineered disc may serve to alleviate p ain associated with TMJ disorders, and slow progression of TMJ tissue resorption due to improper load distribution.

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30 Figure 1 1. Temporomandibular Joint Anatomy. A) and B) illustrates the articulation of the mandibular condyle within the infratemporal fossa while C) is an MRI scan which shows the total joint anatomy. D) is an expanded illustration of the major skeletal connections of the TMJ and the articulating disc. Photos courtesy of Dr. M Franklin Dolwick. Martini, F. & Ober, W. Visual Anatomy & Physiology. Benjamin Cummings.

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31 Figure 1 2. Regional cell population of the Temporomandibular Joint Disc. TEM images represent the morphological differences between the fibroblast like and chondrocyte like populations with differences in the populations regional distributions represented in the bar graphs adjacent. Detamore et al ., Cell Type and Distributio n in the TMJ Disc. J Oral Maxillo Surg 2006

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32 Figure 1 3. Proteoglycan regional content of the porcine TMJ disc. The major proteoglycan concentrations of the TMJ disc are tabulated for median abundance per regional segment. Allen et al., Tissue Engi neering of the TMJ Disc: A Review. Tissue Engineering, 2007

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33 Figure 1 4. Anatomy and tensile mechanical properties of the TMJ disc attachments. Structural views are shown in (A) sagittal and (B) coronal views of the attachments, including the medial (MA) and lateral (LA) attachments, and the posterior (posterior superior, PS and posterior inferior, PI) and anterior (anterior superior, AS and ant erior inferior, AI) attachments. (C and D) illustrate the loading direction evaluated and correspond to the bar graphs below. Murphy et al. Tensile Characterization of Porcine Temporomandibular Joint Disc Attachments J Dent Res 2013

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34 Figure 1 5. Ext racellular matrix fibril alignment of the TMJ disc. Bright field images of the anterior (A) posterior (E) medial (B) lateral (D) and central (C) zones of the disc. The fibrils align such that the central fibers are orientated anteroposteriorly with the p eriphery forming a ring like orientation. Detamore et al ., Tensile Properties of the Porcine Temporomandibular Joint Disc. ASME 2003.

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35 Figure 1 6 Viscoelastic compressive biomechanics of the porcine TMJ disc. Lumpkins et al. Regional variations in the viscoelastic compressive properties of the temporomandibular joint disc and implications toward tissue engineering. Journal of Biomedical Materia ls Research Part A, 2009

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36 CHAPTER 2 GENERAL MATERIALS AND METHODS This chapter describes the generic materials and methods used in these investigations, and are divided into 2 sections: experimental and analytical methods. Methods unique to each experimental chapter are described separately in the materials and methods s ection of the appropriate chapter. Experimental Methods Porcine TMJ Disc: Procurement and Dissection Partially dissected fresh porcine TMJs were isolated from male animals ages 6 9 months, purchased with IACUC approval (IACUC Protocol # 201207534) from An imal Technologies Inc. (Tyler, TX). Dissection was conducted by first separating the mandible from the temporal bone, then ligaments connecting the disc to the condyle were detached, exposing the inferior disc surface. The remaining connections to the gl enoid fossa were severed (Figure 2 1). Once removed, the mass and dimensions of each discs was measured and the superior surface and medial edge were marked using a water resistant marker (Sharpie Mean Streak Waterproof Marking Stick) for ease of later re gional sampling. After dissection discs were stored in 0.15M phosphate buffer saline (PBS, pH 7.4) at 4C until use. All samples were stored less than 12 hours before testing [65] Decellularization of the Porcine TMJ Disc After dissection of the disc from the TMJ cavity the discs are placed in phosphate buffer saline (PBS, pH 7.4) for no more than 12 hours before being transferred to a 1% sodium do decyl sulfate (SDS) solution. The TMJ discs are agitated in the SDS solution on an orbital shaker plate for 24 hours at 100rpm (Figure 2 2B).

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37 After decellularization with the SDS detergent the discs are rinsed for 5, 15, 30 minutes followed by 1, 2, 4, 6 and 12 hours in PBS (pH 7.4). Additional rinsing was done if bubbles form in the PBS rinse, indicating detergent remained on the scaffold. After PBS rinsing remaining DNA fragments and cellular debris was removed by samples incubation overnight (approx imately 8 hours) at 37 C in 50 U/mL in a desoxyribonuclease (DNase) solution (Sigma, St. Louis, Missouri, USA) and then successively rinsed again using PBS for an additional 12 hours. In the last PBS rinse the pH of each disc was normalized to between 7. 3 and 7.4 before additional scaffold processing. Lyophilization of Fibrocartilage Disc Scaffold The TMJ disc scaffolds, after pH balance, were progressively frozen first to 20 C for 6 hours followed by 80 C for 18 hours. After the freezing cycle, 24 hours in total, the scaffolds were lyophilized (freeze dried, Figure 2 2C) for 24 hours at 84 C in vacuum less than 8mTorr (<1.66 Pa) using a bench top freeze drier (Millrock Technology, Kingston, NY). solation and E xpansion F or mesenchymal stem cell (MSC) extraction, full term human placental tissues, including umbilical cords were obtained from Labor & Delivery at Shands Hospital at the University of Florida (Gainesville, FL) and processed within 12 hours of delivery. Cells w 3). The 3mm cubes and plated onto t 25 cell culture flasks. To facilitate adherence of the explanted tissue to the cultur e plastic substrate, the flask is kept up right for 2 4 hours then laid flat for further culture. Cells migrate out from the explant within 1 2 weeks cultured in t 25 cell culture flask

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38 (Falcon) with media change every 3 days. After approximately 70 80% co nfluence the cells are then passaged twice to provide highly proliferative p 3 human cells for further USA) supplemented with 1% penicillin streptomycin (Gibco Life Technologies, Grand Island, NY) and 10% fetal bovine serum complex (FetalPlex, Genimini Bio Products, West Sacramento, CA) and cultured in 5% CO 2 and 37C. Analytical Methods Biochemical Anal ysis Glycosaminoglycans content a nalysis Samples were digested in a solution containing 125 g/mL of papain (papaya proteinase I) for 24 h at 60 C to break down the extracellular matrix molecules holding the cells together (digesting the peptide bonds). G AG content was assessed using dimethyl methylene blue (Sigma Aldrich, St Louis MO, USA) and calibrated using chondroitin sulfate as a standard [66 68] T otal collagen content analysis: m odified hydroxyproli ne a ssay Total collagen content analysis was determined using a modified hydroxyproline assay. Samples were digested in a solution containing 125 g/mL of papain (papaya pro teinase I) for 24 h at 60 C. Total collagen content was determined from a modified Hydroxyproline content assay. Briefly, 100 L of sample supernatant, a series of standards (1 5 g/2 0.001N HCl mL), and a blank (0.001 N HCl) were placed in a 96 well cle ar bottom micro plate Hydroxyproline oxidation was initiated by adding 50 L chloramine T solution (Mallinckrodt, Fair Lawn, NJ) to each well. The wells were agitated slightly (hand agitation approximated 50 rpm for about 20 seconds) and let

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39 stand for 2 0 minutes at room temperature. 50 L perchloric acid to each well to deactivate the chloramine T and again hand agitated then let stand for 5 minutes. 50 L p dimethylaminobenzaldehyde solution (2g/10mL in cellosolve) (Fischer, Paris, KY) was added to eac h well then incubated at 65C for 20 minutes to develop the chromophore. Absorbance was read at 550 nm [69, 70] Instead of using hydroxyproline standards, Corporation, Westbury, NY) were chosen for a more direct comparison. Cellular Integration Analysis Fluorescent s tains Diamidino 2 phenylindole (DAPI). After overnight fixation in 2.5% gluteraldehyde, cells were washed in PBS three times for 5 minute s, then stained using 300 nM DAPI dihydrochloride (Molecular Probes #D1306; Eugene, OR) in PBS to visualize cell nuclei. Cells were washed in PBS three times for 5 minutes and imaged. Rhodamine phalloidin (RP). Cells were fixed overnight in 2.5% gluteral dehyde. After two 5 minute washes, cells were permeabilized using 0.1% (w/v) Triton X 100 in PBS. Cells were incubated in a blocking solution of 1% (w/v) bovine serum albumin (BSA) in PBS, then stained using 5 U/mL RP (Molecular Probes #R415; Eugene, OR) i n the presence of 1% BSA to visualize filamentous actin, or F actin. Cells were washed in PBS three times for 5 minutes and imaged. Live/dead stain. Viability Cytotoxicity Kit *for mammalian cells (Mole cular Probes #L 3224; Eugene, OR). Cells were incubated for 30 minutes with 2 m calcein AM (which fluoresces when enzymatically modified by intracellular esterase active only in live cells) and 2 m ethidium homodimer 1 (an intercalating dye that stains t he nuclei in dead or dying cells)

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40 in standard EC media and imaged through both GFP and DsRed filters as described below. Fluorescent imaging and a nalysis Fluorescently stained cells were imaged using a Zeiss AxioImager M2 upright fluorescence microscope coupled with an AxioCam HRm Rev. 3 monochromatic digital camera operated by AxioVision software version 4.8. Multidimensional acquisition was employed to im age cells at multiple wavelength ranges, which could then be exported as separate channel images for analysis. Histology Tissue processing After overnight fixation in 2.5% gluteraldehyde, TMJ disc samples were immersed in OCT medium within plastic base molds and rapidly snap frozen. A stainless steel beaker filled with isopentane was placed in a larger Styrofoam container with liquid nitrogen. The base mold was then lowered to the top of the chilled isopentane solution using a pair of forceps and held in place until the sample was completely frozen, which occurred in roughly 30 seconds. Snap frozen tissue samples were stored at 80C until cryo sectioning. Frozen TMJ disc scaffolds were cut into 8 m sections using a cryostat, and collected on glass slide s for staining. Tissue sections were stored at 80C until staining. Hematoxylin & eosin stain Sections were rinsed in DI water for 30 seconds, stained with hemotoxylin for 1 minute, dunked in tap water, and then rinsed in running tap water for up to 5 m inutes. Sections were immersed in bluing reagent for 30 seconds, and then rinsed in DI water for 30 seconds before staining with eosin for 20 seconds, dunked in tap water, and

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41 rinsed in running tap water for 1 minute. Sections were progressive dehydrated u sing 70, 80, 90, and 100% EtOH for 30 seconds each before clearing and mounting. Allen Scientific #87019; Kalamazoo, MI). Sections were rinsed in run ning DI water for 1 for 10 minutes. Sections were rinsed in running DI water for 5 minut es and stained in Biebrich Scarlet Acid Fuchsin Solution for 5 minutes. Sections were then rinsed in running DI water for 30 seconds, placed in Phosphotungstic Phosphomolybdic Acid Solution for 5 minutes, and then stained in Aniline Blue for 5 minutes. Sec tions were placed in 1% Acetic Acid, rinsed in running DI water for 30 seconds, then dehydrated in 100% EtOH twice for 1 minute before clearing and mounting. Color imaging Stained samples were imaged using a Zeiss Axio Imager.M2 microscope coupled with a Zeiss AxioCam MRc camera operated by AxioVision software version 4.8. Cellular p roliferation and metabolic a ctivity To obtain cell density within scaffolds with Quanti iT PicoGreen assay, cellularized scaffolds were prepared by initial snap freezing in l iquid nitrogen then freeze crack pulverized and suspended in 1mL of DI water. The samples were then freeze thaw cycled at 20 C and 37 C three times and sonicated in ice before evaluation to rupture the cell membranes of the samples. Samples were then ex cited at 480nm and the florescence emission intensity measured at 520nm. Once emission

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42 intensity was measured it was plotted against calibration DNA concentration as per manufactures instructions (Invitrogen, Oregon, USA). Calibration curves were produce d for known concentrations of cells to DNA that were then used to determine the DNA concentration/cell. Quanti iT PicoGreen assay was used per manufacturer instructions (Invitrogen, Oregon, USA). Metabolic activity per cell was determined by measurement of metabolic reduction of the media using a resazurin salt assay, against a calibration curve of the same cell lineage in conjunction with the PicoGreen DNA quantification. Culture media was replaced by resazurin salt solution (1:10 salt to media concen tration) with fresh media and incubated for 2 hours. Over incubation the resazurin salt solution is reduced when exposed to metabolites of cellular metabolism. After incubation the sample were excited at 570nm and the florescence emission read at 600nm Cellular metabolic activity was normalized against cell density, where the total metabolic activity of each sample was divided into the cell number. Mechanical Analysis : Cyclic Compressive A nalysis A Biomomentum Mach1 Micromechanical System (Biomomentum Inc. Laval, Quebec, Canada) was used for all mechanical testing. The testing chamber was filled with 0.15 M PBS (pH 7.4) to maintain hydration, similar to the in vivo environment, [1] and to maintain consistency with previous studies on TMJ tissue mechanics [71, 72] A stationary bottom plate and a vertically translating 2.5 cm diameter indenter of the Mach1 were used to generate the cyclic mechanical loading on the punches. Prior to testing, samples were allowed to eq uilibrate (unloaded) in PBS for 5 min at 37 1 C. Sample height was determined using a preprogrammed function of the Mach1 called find contact. In more detail this measurement is made by first setting the indenter

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43 position when in contact with the botto m plate as zero. Next we raised the indenter and inserted the sample into the test chamber and slowly (0.05mm/s) lowered the indenter until a force of 0.0075N was measured when the indenter came into contact with the sample. This value is specified as th e disc height. The 6mm central zone scaffold punches were cyclically compressed to 10% strain (Figure 2 4). The amount of vertical deformation ( L) to be applied was calculated using the strain equation, with the measured height (L 0 ) known from earlier measurement. The Mach1 measures resulting force (F Z ) experience by the indenter during the compressive loading. The resulting compressive stress on each sample is defined by where F Z is the compressive force a nd A is the cross sectional area of the sample [65] From the stress and strain data the compressive modulus of elasticity (E) was calculated using the equation Imaging Scanning electron microscopy (SEM) TMJ disc samples were placed in the well bottoms of 48 well plates for tissue processing. Samples were fixed in 2.5% gluteraldehyde for 24 hours and washed three times in PBS. A 45 second microwave cycle followed by a one minute bench top incubation were used for the following solutions: fixation in 1% osmium tetroxide solution, wash twice in PBS, wash once in distilled water, and pro gressively dehydrated in 25%, 50%, 75%. 85%, 95%, and 100% (three times) ethanol solutions. Samples were then critical point dried, sputter coated with gold/palladium, and imaged using a Hitachi S 4000 FE SEM (10.0 kV).

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44 Transmission electron microscopy ( TEM) Samples were fixed with Trumps, pH 7.2 (formalin, gluteraldehyde, and 0.2M graded e thanol (25, 50, 75, 95 and three times 100%) and infiltrate cold 100% ethanol then mounted for sectioning in the ultramicrotome ( ~ 70nm). Sections were collected and pos examined on a Hitachi H 7000 at 100kV and imaged with Olympus Veleta camera and iTEM software.

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45 Figure 2 1. Porcine Temporomandibular Dissection. Porcine TMJs are dissected from 6 9 month old pigs using a technique that removes the disc by severing the connective tissue surrounding the disc. First the muscular attachments are dissected from the skull revealing the joint between the temporal bone an d the condyle (A). The joint is then carefully resected by severing the retrodiscal tissue at the posterior of the joint and puncturing the synov ial capsule of the TMJ disc, allowing the condyle to be dissected away from the rest of the skull (B) To rem ove the disc from the condyle the connective tissue surrounding the disc is severed t hus the disc structure is dissected undamaged (C) Testing of the discs are completed within 24 hours of slaughter. Dissected discs are kept in 7.4 pH phosphate buffer saline at 4deg C until use. Figure adapted with permission from the Journal of Biomedical Materials Research Part A.

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46 Figure 2 2. Preliminary processing the native TMJ disc for laser ablation micro patterning. The native disc is decellularized using 1% sodium dodecyl sulfate agitated on an orbital shaker plate at 100rpm. The remnant detergent is progressively removed by phosphate buffer saline (PBS, pH 7.4) washes. Latent DNA fragments are removed from the extracellular matrix of the acellular scaf fold by a 2 hour incubation of the scaffolds in DNase and then a repeat of the progressive PBS rinses. The acellular disc is then frozen at 20C for 18 hours followed by 6 hours at 80C then freeze dried at 84C in vacuum less than 8mTorr (<1.66 Pa). The dry scaffold was then rehydrated using 20mL PBS at room temperature for and evaluated for change in dimension and mass. Photo courtesy of Cassandra Juran.

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47 Figure 2 3. Human umbilical cord histology cross section identifying the Wharton r egion mesenchymal stem cells are derived from for these experiments The human umbilical cord possesses four key regions: the amnion, a thin boundary layer of the cord; the umbilical vein, primary nutrient source from the mother to the baby; two umbilical arteries, which return deoxygenated Jelly, a connective tissue possessing developmental stem cells.

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48 Figure 2 4. Compressive mechanical testing regime. Compressive cyclic mec hanical evaluation is conducted to determine viscoelastic properties of the TMJ disc and the tissue engineered disc construct throughout experiments of this dissertation. A 6mm punch is placed on a stationary bottom plate while a translating vertical inde nter is repeatedly depressed to compress the tissue to a desired strain. The resulting stress waveform shows max stress occurs with the application of the first strain indentation (instantaneous) and progressively decreases until steady state conditions.

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49 CHAPTER 3 SHEAR MECHANICS OF THE TMJ DISC: RELATIONSHIP TO COMMON CLINICAL OBSERVATION Introduction The Temporomandibular Joint (TMJ) regulates movements of the mandible with respect to the temporal bone of the skull. The fibrocartilagenous TMJ disc has in the last decade been the subject of more extensive mechanical evaluations. The majority of published biomechanical analyses have focused on the TMJ discs compressive properties [60 63] and have fo und that the disc possesses significant regional variation [61, 64] While compressive properties are important during joint l oading, the TMJ has primarily a gliding joint function that induces compressive as well as significant shear forces. Previous studies have evaluated shear under static shear loading conditions and have shown regional variation of the TMJ disc properties [73] Current investigations of the TMJ under dynamic shear conditions have focused on the central [49, 50, 74] The relationship between repetitive shear and compressive loading o f intervertebral disc and disc damage have been alluded to by Callaghan and Iatridis where it was suggested that disc herniation and damage may be more linked to repeated flexion/extension/and shear motions than the applied joint compression [75, 76] In these investigations the regional shear and compressive characteristics of the porc ine TMJ disc and the interconnectivity and dependency of these characteristics on one another and variables relevant to physiologic function have been evaluated. We hypothesized that not only is the shear modulus (G) of the TMJ disc dependent on frequency shear, and compressive strain but that the compressive modulus of elasticity (E) is also dependent on the application of cyclic shear loads [65] Additionally we

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50 hypothesized that the interdependence of the compressive and shear elastic moduli will reveal material trends consistent with damage seen clinically in patients with Temporomandibular Disorders. Materials and Methods Specimen Preparation Pa rtially dissected fresh porcine TMJs were isolated from male animals ages 6 9 months, purchased with IACUC approval (IACUC Protocol # 201207534) from Animal Technologies Inc. (Tyler, TX) and dissected as described in Chapter 2. Once removed, the mass and dimensions of each discs was measured and the superior surface and medial edge were marked using a water resistant marker (Sharpie Mean Streak Waterproof Marking Stick) for ease of later regional sampling. After dissection discs were stored in 0.15M phosph ate buffer saline (PBS, pH 7.4) at 4C until use. All samples were stored less than 12 hours before testing [65] Immediately before mechanical tes ting a circular stainless steel punch with a 6mm inner diameter was used to extract 3 samples from each disc: anterior, intermediate, and posterior samples (Figure 3 1B). Anterior to posterior (A P) and medial to lateral (M L) directions were labeled usin g a water resistant marker on each disc sample as shown in Figure 3 1B(insert) in order to orient the sample correctly for testing within the hydrated testing chamber. Sample Groups The TMJ discs were divided into 3 testing groups: anterior, intermediate, and posterior, each undergoing 27 testing procedures: frequency (F) variation (.5 Hz, 1Hz, variati on (1%, 3%, and 5%) with 9 specimens to each testing procedure. The order of

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51 the testing procedure was randomized for each sample group to minimize sample error (Figure 3 7). Disc Geometry and Sample Height Before mechanical testing the dimensions (length in the anterior to posterior and medial to lateral directions) of the whole disc were recorded and were 17.96 2.12mm and 27.34 3.43mm respectively. The sample height measurement is made by first setting the indenter position when in contact with the bottom plate as zero. Next we raised the indenter and loaded the sample into the test chamber and slowly (0.05mm/s) lowered the indenter until a force of 0.0075N was measured when the indenter came into contact with the sample. This value is specified as the disc height. Physiologic Load Choice The compressive strain loading of 5% to 15% was chosen in accordance to the maximum amount of joint space reduction experienced while talking to maximum jaw clenching [77] Additionally each sample was tested at 0.5 Hz to 5 Hz which corresponds with passive mastication frequency of humans [78] to average rushed speaking respectively. The shear strains were chosen from 1 to 5% because 1% shear is frequently seen in forming facial expressions and 5% strain represents loading generated during a chewing cycle [79, 80] The shear strain in the medial to lateral direction was tested at the same strains to maintain experimental continuity. Figure 3 4 illustrates the results of the experimental mechanical testing associated with each disc region in response to shear strain loading in either the anterior to posterior or medial to lateral direction.

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52 Profile Ap plication Application of the strain profile was initiated with a 3% compressive strain to prevent slippage during subsequent shear strain application, and to simulate the reduction in joint space during dynamic physiologic function between the articulatin g surfaces as the mandible rotates with respect to the stationary fossa (Figure 3 1A and 3 1C(1)) [77] A 3% compressive strain was determined to be an adequate vertical load to inhibit slipping (Figure 3 6). The disc sample is then sheared to a specified shear strain displacement depending on samples test group. A shear application under these lightly compressed load conditions simulates the gliding and rotation of the mandibular condyle with respect to the fossa (Figure 3 1C ( 2)). Once displaced axially the compressive strain was increased to either 5%, 10%, or 15% Figure 3 1C ( 3). This compression simulates the reduction of the upper joint space as the mandible slides forward with respect to the ar ticular eminence [79, 80] The disc was exposed to 10 cycles of shear loadin g, simulating physiological jaw action (Figure 3 1C ( 4)). The shear load was then removed followed by removal of the compressive load and a recovery period of 2.5 minutes. The recovery period of 2.5 minutes (unloaded and submerged in PBS (pH 7.4, 37C1 C) bath) was found adequate for recovery of compressive and shear mechanical properties in preliminary studies (Recover Time, Figure 3 5). Testing Order Evaluation In order to ensure the validity of mechanical measurements made on a single sample of disc t ested repeatedly we established that a recovery time of 2.5 min (section above). Here we tested that the order of the mechanical tests had no significant effect on the results. We randomly designed 3 testing orders with a single in common

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53 mechanical vari each strain protocol with 2.5 min resting time between each successive mechanical test (Figure 3 7). Each test was conducted with 3 different intermediate sample punches to develop results that could be evaluated for statistically significance. The results show that the shear moduli, of the chosen common mechanical variable application, are the same through each of the three testing regimes. Biomechanical Testing Cyclic Shear Load ing A Biomomentum Mach1 Micromechanical System (Biomomentum Inc. Laval, Quebec, Canada) was used for all mechanical testing. The Mach1 is a multiaxial modular mechanical testing rig which can measure or impart compressive and shear loading. The testing c hamber was filled with 0.15 M PBS (pH 7.4) to maintain hydration, similar to the in vivo environment, [1] and to maintain consistency with previous studies on TMJ tissue mechanics [71, 72] An axially translating bottom plate and an axially stationary vertically translating 2.5cm diameter indenter were used to generate the shear and compressive loads. Prior to testing, samples were allowed to equilibrate (unloaded) in PBS for 5 min at 371C. Sample height was determined using a preprogrammed function of the Mach1 called find contact as described in Disc Geometry and Sample Height. Using the strain profile illustrated in Figure 3 1C, compressive and shear strain deformations (indenter displacement; L position, and axially translating bottom plate; x position) were applied to the tissue and assessed using the calculations and thickness and is the axial displacement in the shear direction. Both parameters relative to the initial thickness were needed to produce the desired stra in range as

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54 described in Profile Application. The Mach1 measures resulting force (F X and F Z ) experience by the indenter during the compressive and shear loading. The resulting compressive stress on each sample is defined by where F Z is the compressive force and A is the cross sectional area of the sample and the resulting shear stress is where F X is the resulting axial force and A is again the cross sectional area of the sample [65] cycli c loading tests were analyzed by calculating the hysteresis, peak stresses, and the instantaneous and steady state compressive ( ) and shear moduli ( ). The general ca lculations for the compressive and shear moduli are and respectively. Recovery Time In vivo cartilaginous tissues are repeatedly loaded and unloaded with surprising resilience to fatigue. To ensure multiple mechanical tests could be conducted using the same tissue sample we evaluated recovery time between subsequent cyclic shear mechanical test s. To evaluate recovery time anterior, intermediate, and posterior Figure 3 1C and allowed to recover for variable recovery times (15 second steps from 0 to 120 secon ds). After 75 seconds the mechanical properties are fully recovered (Figure 3 5A) and become repeatable to 15 applications of the stain profile (Figure 3 5B). We included a factor of safety of 2 into the recovery time of the experimental

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5 5 evaluations to e nsure full recovery is established in every iteration of the mechanical testing, making the total recovery time used for the subsequent experiments 2.5 minute. Slippage Evaluation To evaluate slippage during the shear testing paradigm, intermediate punches were compressed to different strain values (1%, 2.5%, and 5% compressive strain) and sheared to 20% shear strain (1.2mm for the 6mm diameter punches). The position and force in the axial and vertical directions were measured by the Mach1. The resulting load/position data is presented in Figure 3 6 and illustrates that for 0.5% to 2% compressive strain the disc punches experience slippage and punches exposed to 2.5% compressive strain or greater experience no slippage. The data collected concluded that a t a compressive strain of 3%, without any adhesive, no slippage is experienced by the TMJ disc punches. Statistical Analysis Each set of test group data was calculated based on testing of nine samples (n = 9). Standard Deviation and one way analysis of va riance (ANOVA) testing were calculated to determine statistical significance between test groups for biomechanics. Significance was established using the Tukey Results The average thicknesses reported in millimeters of the tested regions were 3.66 0.24, 2.00 0.29, and 3.88 1.45mm for the anterior, intermediate, and posterior regions respectively. Figure 3 2 illustrates that shear modulus was evaluated for the 1 st strain cycle ( ) and for the 10 th strain cycle ( ) to show the changes frequency, compressive and shear strain have on the material properties over the load application.

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56 When shear loads were displaced in the A P direction the stiffness of all regions decrease from to with the most noteworthy change occurring with frequency increase (Figure 3 2A). Change in evaluated solely as a function of F increase, from 0.5Hz to 5Hz, was on average an 8.6% incre experienced a similar increase in shear modulus (Figure 3 was increased the shear modulus decreased becoming more el astic (Figure 3 2C). Most noticeably for the posterior region tested at the high frequency (5Hz) and high compressive strain (15%) condition (data not represented in Figure 3 2C), = 1.556 0.327MPa and = 0.79 = 1.078 0.158MPa and instantaneous to the steady state condition. Similar to the shear modulus change 2(C), the compressive modulus also became more elastic with repeated shear in all regions of the disc (Figure 3 3). The cycle dependent compressive modulus of the posterior region shows that with each shear cycle application t he compressive modulus increased by nearly 5% of the value before continuing the decreasing trend. The average P direction was 1.5 0.0kPa, and in the M L direction was 0.40 0.03kPa in the posterior region, and became more regular as the compressive modulus reached a steady state condition (cycle 6 through 10). This phenomenon was also seen in the anterior region but only when shear strain

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57 was applied in the M L direction, a nd the magnitude was less than 25% of the posterior 3, table). The shear modulus characteristics are described in Figure 3 4. Only the posterior region statistically maintains its shear modulus from to when sheared in the A P direction. All regions when sheared in the M L direction maintain within 20% of their stiffness over the strain application cycle. Discussion The ability of the TMJ disc to act as a bu mper between the articulating skeletal structures of the TMJ is fundamental to jaw movement, thus it is paramount to understand the mechanical response of a healthy TMJ disc when exposed to loading similar to that experienced in vivo The current investig ation focused on the mechanical characteristics of the disc during cyclic shear testing when the magnitude of shear strain parameters, values described in Physiologic Load Choice. Previous investigations by Tanaka have shown the disc experiences shear [50, 81] Figure 3 2 depicts t he dependency of the disc regions on frequency, compressive strain, and shear strain. Shear stiffening was hypothesized to be a combination of two effects. The first due to interstitial fluid or bulk matrix outflow as pressurized fluids are squeezed from the point of strain application; in fluid resorption between the successive strain applications. These conclusions are further supported by our results and expanded to be a universal trend for the regions

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58 tested. The shear softening with increased shear strain however was only observed in the anterior and intermediate regions of the disc. It has previously been hypothesized that observed shear softening is due to th e proteoglycan (glycosaminoglycan (GAG) component) and water matrix within the disc possessing Non Newtonian characteristics. resistance to pressure. The lack of significant shear s oftening in the posterior region then is in agreement with this hypothesis as the posterior band of the disc contains a lower GAG content than the anterior or intermediate zones [44, 82] (Figure 3 4). A difference shear properties of the porcine disc at porcine core temperature (39C) and our evaluation was done at human body temperature. This temperat ure difference may account for the variances in our mechanical results as higher temperatures reduce stiffness and strength of the disc as many ECM components (collagen and proteoglycans) are temperature sensitive [58] Application of cyclic shear in the A P direction decreases the E until a steady the mechanical profile (Figure 3 3C). We hypothesized that the increase in compressive stiffness associated w ith these bumps represents a material property that acts as a breaking system (force). Compression causes outflow of interstitial fluid or a shifting of the bulk matrix from the point of strain application. As the fluid matrix shifts, the periphery swell s to maintain the tissue volume and causes increased hoop stresses and hydraulic pressures that inhibits the force dispersion and acts to recoil the fibers back toward their undistorted orientation. Compressive properties of the disc are

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59 strongly dependen t on interstitial fluid flow [71] and with fewer paths for tissue fluid to escape when the disc is under shear, E will respond in intermittent increased stiffness. A possible limitation of these investigations is we have evaluated dis c punches that do not necessarily retain the boundary conditions of the whole disc; however, the ECM fiber alignment superstructure remains intact. This trend is only observed in the anterior and posterior zones likely due to fiber arrangement, where coll agen fibers are oriented in a mediolateral arc that increases hoop stresses and hydrostatic forces [45, 83] By co mparison the intermediate region, where the collagen fibers are aligned in the anteroposterior direction, is shown to culminate in channels that shuttle interstitial fluid quickly within the region. The mechanical consequence is the intermediate zone is m ore elastic than the periphery of the disc [61] Perforation is often associated with disc derangement or osteoarthritis; however, tearing of the posterior lateral region is also seen in asymptomatic discs [84, 85] The results of the current study show the steady state shear modulus is stiffest in the discs posterior regi on, indicating this region is different structurally and/or in composition. These mechanical results are in agreement with clinical observations of regional disc damage. It is clinically accepted that TMJ disc fatigue occur most frequently in the discs la teral posterior region [85] We hypothesize that this retained stiffness with repeated from the impact point, leading to greater residual localized stress summation. It is failure, seen clinically as disc perforations.

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60 Shear strain in the M L direction is less prominent in healthy TMJ disc loading [86, 87] However shear strain is associated with non pathological teeth grinding and jaw clenching that is seen in bruxism which can potentially lead to TMDs [88 90] Sometimes simplified as hyperactivity of the lateral pterygoid, clenching and bruxi ng mechanics are more completely described as parafunctional activity of one or more of the major masticatory muscles (Masseter, medial pterygoid, temporalis, and lateral pterygoid) [90, 91] the M L direction under high compressive load is meaningful to assess any potential relationship between bruxing and TMJ disc perforation. Similar to the shear modulus characteristics of the posterior region under A P shear, all regions of the disc have an ob servable trend of retaining their shear modulus from G int to G ss when load is applied with M L loading (Figure 3 4). Conclusions In conclusion, these results confirm that mechanical characteristics of the TMJ disc are highly dependent on the ECM microenvi ronment and its regional make up. The posterior region of the disc, which is the most commonly observed zone in which the disc fatigues, has been shown to maintain its stiffness when compressed or sheared cyclically. While there is no direct association between theoretical or experimental models to the clinic, these results are in agreement with mathematical modeling results that have shown large stresses developed in the posterior region of the disc and retrodiscal tissue during prolonged clenching, and higher still in these regions when antero lateral internal derangement is included [91, 92] The hypothesis that there is a observation of TMJ disc damage is supported by the data collected in these works.

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61 Figure 3 1 Joint anatomy and simplified free body diagram, regional sampling, and testing method. Figure 1A illustrates the gross anatomy of the TMJ and the placement of the disc within the glenoid fossa. The insert illustrates the position of the TMJ disc when the mandibular condyle slides forward with respect to the articular eminence when the jaw opens. Figure 1B illustrates the sampling regions of the TMJ disc and the directional notation used to properly orient the sample for testing (grey li ne anteroposterior direction, black line mediolateral direction). A representative testing strain profile is shown in 1C (description in text body) and 1D (dashed arrows indicate active loading and solid arrows indicate sustained loading) depicts the sequential testing procedure used to generate the strain profile of 1C. Figure adapted with per mission from the Journal of Dental Research

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62 Figure 3 2 Change in Shear Modulus with frequency, compressive strain, and shear strain. Mean values of the instantaneous (1) and steady state (2) shear moduli as function of frequency with = 1% and = 10% (A), compressive displacement, F = 1Hz and = 1% (B), or shear displacement, = 10% and F = 1Hz (C). Samples are shear strained in the anterior to poste rior direction for all data presented. Increase in frequency and compressive strain cause shear stiffening and the application of greater shear strain induces the shear modulus to become more elastic in the anterior and intermediate region of the disc n= 9, e rror bars represent standard deviation. Figure adapted with per mission from the Journal of Dental Research

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63 Figure 3 3 Change in compressive modulus when cyclic shear strain is applied in the anterior to posterior or medial to lateral direction. Representative zonal compressive modulus evolution over the load application are shown in A, B, and C. Mean values of the compressive modulus of each region of the TMJ disc are shown in the table. The anterior and intermediate regions have less reducti on of their compressive modulus retaining their compressive modulus even when repeatedly sheared. The posterior region becomes more elastic with application of shear strain. All regions have significant reduction in initial to steady state modulus when s heared in the medial lateral direction (P < 0.05 n=9 ) Figure adapted with per mission from the Journal of Dental Research Anterior posterior shear strain application data are represented by the black curves and medial lateral shear strain application ar e represented by the grey curves.

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64 Figure 3 4 Mean regional values of the instantaneous and steady state shear modulus of each disc region when strained in the anterior to posterior direction and the medial to lateral direction. The anterior and central regions experience a significant decrease in shear modulus when strained in the Anterior Posterior (A P) direction. This increased elasticity is not seen in the posterior region ( n=9, *, P < 0.05; **, P <0.01). Figure adapted with per mission from the Journal of Dental Research

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65 Figure 3 5 Recovery time evaluation. A recovery time of 75 seconds or greater produces repeatable instantaneous shear strain modulus values (A). B shows that if the sample is allowed to rest for 75 seconds between successive stain applications the mechanical abili ties are retained to 15 strain profile applications (n=9, error bars represent standard deviation) Figure adapted with per mission from the Journal of Dental Research

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66 Figure 3 6 Representative Stress Strain plot depicting axial slippage at lower c ompressive strain values. Slippage was evaluated at 1, 3, and 5% compressive strain over a 20% shear strain axial displacement. At 1% compressive strain and a shear strain of 20% applied at 0.05mm/s the compressive stress experience erratic decreases due to tissue slippage in the test chamber. These decreases are not seen in the 3% or 5% compressive strain test conditions Figure adapted with per mission from the Journal of Dental Research

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67 Figure 3 7 Random testing order strain profile applicati on. The instantaneous shear 3% and F = 1Hz were evaluated from three randomly designed sequential strain profile applications. The modulus of the evaluated strain cycle appl ications from profile 1 (A), profile 2 (B), and profile 3 (C) are shown to have no statistical difference from one another. This implies that multiple mechanical test can be conducted in any order to produce repeatable meaningful results (n=9, error bars represent standard deviation) Figure adapted with per mission from the Journal of Dental Research

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68 CHAPTER 4 MECHANOBIOLOGICAL ASSESMENT OF TMJ DISC SURFACES: A NANOINDENTATION AND TEM STUDY Introduction The temporomandibular joint (TMJ), the functional joint of the jaw, is arguably the most mechanically active and mechanically complex joint in the human body. The functional loading between the highly incongruent articulating skeletal structures is regulated and facilitated by the small fibrocartilage T MJ disc. Mechanical characterization of the disc has recently become an area of investigation and reports have shown the disc to be mechanically robust, exhibiting highly elastic compressive, shear, and tensile moduli. These properties are a consequence o f the very dense and structurally organized extracellular matrix (ECM) matrix which are adapted to support the repeated sliding action during functional loading of the joint [51, 58, 61, 64, 93] Shear multi axial lo ading evaluation showed that shear loading is primarily distributed at nanoindentation wil l reveal critical design parameters for further TMJ disc tissue engineering efforts. Structural imaging investigations have posited that the TMJ disc possesses such robust mechanical ability due to the intricate architecture of the ECM fibrils, specifica lly collagen. Minarelli in 1997 [94] reported that the collagen fibril arrangement of the intermediate zone (central area of the disc) anterioposteriorly, laterolaterally and during occlusal loading. These observations were in disagreement with the works of Jagger in 1980 [95] where fibrils were observed only oriented in an anterior to posterior

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69 direction. De Bont [96] also observed that the articular surface of the disc was composed of densely packed colla gen fibrils oriented anteroposteriorly, in addition they also observed thin small coiled fibrils at the most superficial layer of the surface. More recently Detamore et al., [45] found that collagen fibers around the porcine TMJ disc have a ring like structure around the periphery and are orientated anterior to posterior in the intermediate zones, agreeing with the work of Jagger et al., [95] and other human TMJ disc tissue studies [1, 38, 56, 78, 97] The porcine model has become prevalent in publica tion due to the similarities of joint and disc structure as well as physiologic action and chewing modalities [38] These works hypothesize that the s tructural complexities seen in the imaging investigations and the robust mechanical ability of the TMJ disc may be due to regional or layered differences through the thickness of the intermediate surface zones. To test this hypothesis we utilized ultra mi croindentation in conjunction with detailed Transmission Electron Microscopy (TEM) imaging to assess these structures .These are the first investigations to use these evaluation techniques to assess the TMJ disc and as such provide unique insight into stru cture function relationships of the TMJ disc. Micro and nanoindentation techniques offer researchers unprecedented resolution of mechanical properties at the micron and nanometer scale. These techniques offer the advantages of sub micron sized contact tips that increase spatial methodology for investigating the fine details of tissues interaction surfaces. Gupta in 2005 [98] investigated the role of the zone of calcified cartilage within the human patella and how these zones influence load transfer from articular cartilage to subchondral

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70 bone. Using nanoindentation results were correlated to mineral content of th e various zones to provide a detailed material property map, information invaluable for further whole joint characterization. Zone specific micromechanical evaluation of human trabecular and compact bone by Zysset at al., [99] and growth plate cartilage by Radhakrishnan et al., [100] i llustrated that these tissues could be described as an assembly of distinct micromechanical and structural units with seemingly homogeneous material properties. Indentation properties of the superior zone of the TMJ disc and temporal fossa articular cart ilage were compared by Kim in 2003, [77] and found 60% greater recovery in the articular cartilage; Indicating the disc could not tolerate phy siologic loading without the cushioning effect of the articular cartilage. A major difference between this a 2mm diameter flattened indenter tip with a step resolution CSM 0.001nm and noise The geometric properties of nanoindentation will allow the resolution needed to evaluate the correlation of micro architectural el ements of the disc to depth dependent mechanical properties immediately adjacent to the surfaces in contact with the articulating skeletal components. These works investigate the microscale mechanical ability of the disc surface and near surface regions using a nanoindentation approach and correlate the mechanical findings to the structural architecture illustrated by scanning and transmission electron microscopy.

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71 Materials and Methods Depth Dependent Nanoi ndenter Micromechanical Testing Porcine TMJ discs were collected as described in Tissue Dissection section of Chapter 2. 6mm diameter samples were removed from the discs central zone using a biopsy punch, then immediately loaded for mechanical evaluation. The discs superior surface and the anterio r to posterior direction was marked with water resistant marker to maintain correct orientation during testing. Samples were then placed into a hydrated test chamber (PBS, pH 7.4) and aligned such that the anteroposterior direction was orientated the same relative direction. All mechanical tests were performed using a tip. Quasi static and creep tests conducted on the superior and inferior surfaces were accomplished by load analysis and unloading allowing measurable recovery. Data was tabulated to report load versus depth and resulting stress/strain to determine compressive modulus of elasticity and % hysteresi s. Progressive multi cycle indentation was used to capture the change in mechanical properties versus indentation depth of the superior surface region. Five indentation depths were used to evaluate depth dependent mechanical characteristics. The microinde ntation methodology is illustrated in Figure 4 characterization of each depth the indent applied held for 5 seconds and then unloaded and allowed to recover for 5 seconds before initializing the next loading cycle. After running 5 cycles of indentation the

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72 indenter dept cycle regime was initiated at the new depth. The force difference from start to final load Data was processed to tabulate mechanical properties of clinical and research significance. The load (F Z ) and indentation depths (x) we used to calculate stress and strain using and respectively, with A being the cross sectional area of the TMJ disc in contact with the indenter and x 0 being the total thickness of the disc methods for SEM and TEM described in Chapter 2. Statistical Analysis Data for each test conducted was calculated bas ed on testing of four samples (n=4). Standard deviation and one way analysis of variance (ANOVA) testing were calculated to determine statistical significance between data sets for biomechanics. Significance was established using the Tukey (p = 0.05, n=4). Results The average thickness of the intermediate zone was 1.43 0.12mm with a corresponding mass of 3.36 0.21g. SEM images in Figure 4 1 show a cross section of the TMJ central zone at low magnification illustrating the biconcave ultr astructure of the disc. Magnified micrographs of the surface zones show that each surface is structurally unique. The superior SEM suggests a layered structure with ECM fibrils most highly linearized and compacted at the joint interface and fibrils more undulant/helical with distance from the surface. Relative to the superior surface, the

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73 inferior surface and underlying structures exhibited a more homogenous fibril structure with regular and linearized collagen bundles displaying a relatively consistent porosity. Microindentation step creep comparison between the superior and inferior surface regions is shown in Figure 4 2 with corresponding high magnification TEM micrographs of the former and latter surfaces. The superior surface modulus is more elastic at 1.34MPa compared to the inferior at 2.49MPa. Hysteresis values displaying dissipated energy show the superior zone to be 2.12X the inferior surface. Structurally the superior surface has fibril orientation anterioposteriorly and obliquely while the i nferior surface is more isotropic, linearized and less compacted with little cellular presence. Mechanical evaluation by microindentation of the superior surface is depicted in Figure 4 3A and the resulting stress profiles and compressive modulus of elasti city shown in 4 3B and 4 3C. Data shows the disc has a distinct surface zone of 4 6 microns (pictorially loop a b), which elicits the majority of the superior surface strength but has a reduced hysteresis of 31.37 3.37%. Loops c d, e f and modulus valu es seen in Figure 4 3C (2 5) exhibit more elastic properties than the surface zone (loop a b and modulus Figure 4 3C (1)). TEM images of the superior surface (Figure 4 4) indicate three structurally distinct layers: a highly compacted and anisotropic fibr il orientation of the surface interface layer, a less compacted linearized and anterior to posterior orientated fibril subsurface layer containing greater cellular concentration and evidence of minor calcification, and a helical or bundular layer possessin g few cells and an undulating fibril network.

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74 Discussion The position of the disc between the highly incongruent skeletal structures of the TMJ with differing superior and inferior joint spaces indicate that the surfaces of the disc are likely to have diff ering mechanical structural and cellular composition. SEM examination of the bi zones have markedly different ECM architecture. The superior disc surface is arranged in layered depth depe ndent zones identifiable in the superior regional zone scanning electron micrograph in Figure 4 1. Unlike the superior disc surface the inferior surface is composed of well defined linear collagen fibril meshes with uniform porosity and no significant cel l populations adjacent to the surface (Figure 4 1 inferior regional zone SEM). This may indicate that this zone is less involved in recovery of the tissue after mechanical loading and acts more as an anchoring structure possessing compressive resistance. mechanical responsiveness. The inferior surface presents resistive response to mechan ical loading, with an elastic modulus of 2.5MPa (Figure 4 2 mechanical assessment). These findings correlate to structural observation from TEM inspection of the inferior surface zone (Figure 4 2 inferior TEM insert) showing collagen fibers have a mor e linear orientation anterior to posterior. Limited cell populations are identified in the inferior TEM micrographs; we posit that the lack of cellular presence in the immediate inferior surface is due to the smaller inferior synovial pocket and lesser deg ree of articulating motion between the disc and the condyle in comparison to the superior surface of the disc and the temporal bone. If, as has been previously

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75 postulated, mechanical movement encourages diffusive shuttling of nutrients through the disc an d the inferior disc surface has less mechanical movement as well as a smaller source of nutrients (synovial space and fluid volume) then cells along this surface would be less effective at recovery after joint loading [101] Micrographs of the peripheral inferior zone displayed remnants of dead cells (Figure 4 5) which supports our hypothesis that the infer mechanically recover from loading but instead provides the robust resistance to compressive loading (stiffness) seen in whole tissue mechanical evaluation of the disc. The superior surface of the dis c demonstrates increased elastic qualities compared to the inferior surface (Figure 4 2 mechanical assessment) and greater hysteresis indicating that this surface has greater energy dissipative ability after loading. These mechanical findings are suppor ted by high magnification TEM (Figure 4 2 superior TEM insert) showing that the superior surface has fiber orientation both anterioposteriorly but also obliquely indicating that shear loading in multiple directions can be accommodated. Motion of the dis c within the superior joint space is predominantly anteroposterior correlating to mouth opening and closing; however, the disc also translates mediolatterally with chewing and teeth grinding [78, 102] The multi axial loading of the disc during jaw movement hypothetically associates with the obliquely aligned collagen fibers observed in the superior surface micrograph. Investigating further the complex architecture of the superior disc surface, ultra microindentation was applied to define depth dependent mechanical regions. SEM was used to broadly define structural layers to estima te indentations depths. Based on these

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76 Mechanical stress as a function of indentation depth (Figure 4 3B), shows that between each of the 5 indentation depths (labeled 1 5) three mechanically distinct layers exist (labeled as loops). The layer with the highest rigidity and the most resistance to mechanical strain was shown to be immediately adjacent to the surface (loop a b). This layer, which we designated as a surface int erface layer, has little energy recuperative ability with a % hysteresis of 31.37 3.37%. This region structurally has a linearized ECM fibril network (Figure 4 4 regional segment 1). While not evaluated in these works we have previously evaluated the shear properties of the disc showing that the center zone of the disc is responsible for the majority of the shear strength in the disc [51] In concert with this previous data we hypothesize this sur face interface layer (4.5 articulation fossa of the temporal bone, less than compressive resistance. Cyclic compressive loading at indentation depths 2 and 3 (8.5 ths) produce a stress curve with similar slopes, indicating that these two indentation depths are within the same structural layer. This subsurface layer offers the least compressive resistance eliciting a decreased compressive elastic modulus of 1.98 0 .43MPa. This layer also presents higher cell concentrations than the surface interface layer where few cells were observed. This is likely the region responsible for cellular regeneration after disc surface damage resulting from impact, dysfunction, or d isease. Observations from the TEM imaging (Figure 4 4 box 3) show a near acellular region with an undulating fibril structure, similar to observations of de Bont. The depths associated with these structural observation correlate to indentation depths 4 and 5, which mechanically behave similarly with compressive modulus properties exhibiting

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77 greater stiffness than depths 2/3 but more elastic qualities than depth 1. The helical bundles presented in this layer have an elastic modulus comparative to the sur face interface layer, having the highest % hysteresis of all layers. The greater energy dissipation (64.61 2.44%) within this layer is likely due to the crimped architecture of the collagen bundles. As compressive loading is experienced the helical bun dles linearize to absorb the load. Then as the load is removed the bundles regain their original crimped conformation ready to absorb the next load. From these mechanical findings we hypothesize this is the major structural supportive layer of the superi or disc surface regions acting to support resistance to compression and functions to absorb and disseminate repetitive loading without structural impairment. Conclusions These findings derived through the use of micro and nanoindentation techniques furt her our understanding of the finer detail of the mechanical architecture of articulating tissue. These works demonstrated that the central zone of the TMJ disc is structured in isometric depth dependent layers which provide different mechanical properties that impart normal physiological function The inferior surface (interfacial) has a minimal cellular composition with little structural variation and offers comparatively less energy dissipation in comparison to the superior surface. The superior surfac e presents three distinct mechanical and structural layers each responsible for different mechanical characteristics supporting the action of articulation within the TMJ. This type of characterization is imperative for further advances in the field of fun ctionalized tissue engineering as researches are beginning to grasp the importance of the interface mechanobiology in governing whole tissue integration, adaptation, and regeneration.

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78 Figure 4 1 Joint and Disc Anatomy with Depth Dependent Regional Zone Highlighted. Schematic Joint Anatomy with Disc Placement and orientation elucidated and SEM micrographs of t he TMJ disc. The superior and inferior disc architecture has key differences which can b e seen at low magnification and are accentuated at higher magnification. The superior disc surface is segmented into what appear to be several distinct surface regions before presenting the bulk architectural ECM matrix construction while the inferior su rface appears to have one dense uniform surface region which appears more fibrous and linearized.

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79 Figure 4 2 Representative s urface dependent step creep response and TEM correlations. The inferior surface of the disc is mechanically stiffer and h as less energy dissipation through indentation indicating that this side of the disc is primarily responsible for resistance to loading. The superior surface has a more elastic modulus and greater hysteresis indicating that this surface has more recoverab ility after loading. These mechanical findings are supported by high magnification TEM showing that the superior surface has fiber orientation both anterioposteriorly but also obliquely indicating that shear loading in multiple directions can be accommoda ted. These findings correlate with knowledge of joint function and architecture: The inferior disc surface doe s not have much relative motion to the mandible while the superior disc glides within the fossa and over the eminence of the temporal bone during function.

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80 Figure 4 3 Cyclic ultra micro indentation mechanical characterization. The micro indentation methodology is illustrated in A) describing loading [1], holding [2], and unloading [3] then repeating for 5 cycles to obtain steady state mechanical properties [4] of the TMJ disc. Mechanical characterization of the surface, subsurface, and helical bundles layers are described in B) and C). Although five micro indentation depths were tested (numbers 1 5) three distinct mechanical regions have been separated in B) depicted by loops a b, c d, e f, with loop a b exhibiting the greatest resistivity to loading and loops c d and e f representing the surface regions with the greatest energy dispersion (hysteresis). The hysteresis loops correlate to the steady state compressive m odulus of elasticity data shown in C) ( n=4

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81 Figure 4 4 TEM of the superior disc surface. A) illustrates that the disc architecture adjacent to the surface has three distinct regional segments: 1, a surface interface layer; 2, a subsurface region; and 3, a helical bundles layer. B) is a magnified selection of A) more clearly denoting the three separable regions indicated by boxes 1 2 and 3.

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82 Figure 4 5 Apoptotic cell zone at inferior surface periphery. Imaged at the periphery of the inferior surface TEM slice this image shows apoptotic retraction of cells from the ECM (1a and 1b). Also lack of organelle structure is seen in necrotic cell (2) on path to apoptosis. While not representative of the inferior surface in the center zon e this degenerative microarchitecture was seen in samples taken from the extremities of the intermediate zone (peripheral zones).

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83 CHAPTER 5 LASER MICRO PATTERNING SCAFFOLD DEVELOPMENT AND BIOMEDICAL ASSESMENT Introduction Investigations concerning shear and surface structure/function characterization established target qualities for TMJ disc tissue engineering efforts. The next primary consideration for TMJ disc engineering is elimination of the potential for immune reaction and foreign body encapsulati on indicative of xenogeneic tissues implanted in human patients without immunosuppressant therapies. Previous studies described in Chapter 1 established that the major ECM biochemical composition of the porcine and human discs are statistically identical [41, 44] Thus the major immune epitope of the pig disc is the porcine disc cells themselves. Striping of cells and soluble glycosaminoglycans from the porcine disc results in a naturally derived human homolog ECM produced by decellularization. Decellula rized scaffolds have gained attention both in research and clinical application as methodologies have developed to remove immune epitopes and cellular debris. Additionally early studies into the addition of cytokine markers to improve the acellular matrix advancement of the technology. Thin scaffolds (<1mm) developed using decellularization techniques ranging from osmotic shock (hyper to hypotonic solution) decellularization, freeze t haw cycling, alcohol washing, to common detergents have been shown to be effective and allow repopulation of the scaffold with host cells after implantation. However many applications of acellular whole tissue scaffolds require thicker matrices to suppo rt the mechanical strain of function.

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84 While many studies have shown that decellularization is accomplishable on thick tissues, and even whole organs, with the aforementioned decellularization techniques, repopulation of the scaffold has proven problemati c. In vitro testing of cellular repopulation of whole tissue scaffolds after decellularization have revealed that cells experience a variety of detrimental influences including: ECM deterioration by corrosive chemical interaction, residual ionic charge o n the scaffold stifles cellular adhesion, remnant destructive agents not properly removed during steps subsequent to decellularization, and lack of mass transport of nutrients to the interior of thick scaffolds to support cell population growth. Fibrocar tilage is a complex 1.5 4mm thick tissue which exhibits complementarily structural strength and geometric flexibility. Fibrocartilage is responsible for regulating the articulation of three of the most heavily and repeatedly loaded joint in the body: the intervertebral discs in the vertebral joints, the menisci of the knee joint, and the disc of the temporomandibular joint (TMJ). The TMJ disc scaffold has been shown to be decellularized completely by several decellularization techniques, with findings tha t 1% sodium dodecyl sulfate detergent decellularization retained native properties of the disc most effectively [65] The McFetridge lab previously assessed the effectiveness of 1% sodium dodecyl sulfate (SDS), Triton X 100, and an EtOH/Acetone to strip cells and soluble ECM components to minimize immunogenicity. While each of the three approaches removed most of the cellular material significant differences were noted in the discs mechanical properties [65] Results have shown that porcine discs treated with SDS most closely matched the energy dissipation capabilities and resistance to deformation of the native tiss ue. Treatments using Triton X 100 caused the resultant

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85 tissue to become relatively softer with inferior energy dissipation capabilities, while treatment using acetone/ethanol led to a significantly stiffer and dehydrated material. These findings supported the potential of a porcine derived scaffold decellularized by SDS as a xenograft for TMJ disc reconstruction [65] Fibrocartilage is predominantly avascular; however recent studies have shown that a diffusive network exists within the TMJ discs which supplies nutrients via diffusive pumping of the joint during activity. This network, while useful in dispersi ng chemokines and small molecules, is not structurally significant enough to accommodate cellular dispersal through the thick ECM matrix during recellularization of the decellularized scaffold. Thus even if the structural integrity of the disc is retained through larger molecule permeation during initial remodeling. These works will describe a novel scaffold preparation method in which microporosity will be incorpo rated into an acellular naturally derived TMJ disc fibrocartilage scaffold to improve permeability and diffusivity without significant alteration of compositional or mechanical ability. To incorporate artificial porosity a CO 2 laser micro ablation metho d was chosen. Application of CO 2 LMP incorporation into tissue engineered scaffolds performed on biomimetic gels have shown that incorporation of laser ablated pores enhances cellular infiltration [132 ] The major differences between previous studies and these works are these works laser ablate a natural ECM material which retains biochemical and biomechanical properties of the target tissue. Also these works ablate through a tissue

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86 >2mm in thickness while previous studies have been limited to under 1mm thickness [103 105] Materials and Methods Experimental Methods Laser micro p atterning The TMJ discs were isolated, decellularized, and lyophilized as described in Chapter 2 before laser ablation. Laser ablation on the freeze dried TMJ disc scaffolds was done using a 40W CO 2 laser engraver (Full Spectrum Lasers, Las Vegas NV). Incorporation of the LMP into the TMJ disc scaffold was done by setting the laser to pulsed firing with pulse duration set at 15ms and 10% total power and the delay between pulses was set to 30ms to reduce thermal damage. These settings were determined experimentally optimizing for hole diameter dimension and pore penetration through the thickness of the scaffold (data not shown). Four 8x16 LMP test patterns with different hole sizes and corresponding hole separations were incorporated into TMJ corresponding hole separation (centerline to centerline distance) i s defined as five times the hole size. After laser working, scaffold samples were excised from the intermediate zone of the whole disc using a 6mm biopsy punch. These samples were used for all further evaluation. Hydration Dry scaffolds samples from all LMP hole diameter groups were rehydrated using PBS (pH 7.4) and their changes in mass and length was recorded at 2, 5, 10, 30 minutes and every 2 hours following for 48 hours to gauge the rehydration rate for physiologically representative inert fluid. Ad ditionally solutions of 3M sodium chloride

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87 and 0.055 mg/mL resazurin salt were used as hydration media to evaluate how different molecular densities and relative charge would affect rehydration rate. Analytical Methods Biochemical q uantification Total coll agen content and total glycosaminoglycan (GAG) content were evaluated as described in Chapter 2. Permeability coefficient a nalysis The ease of fluid flow through our porous sample was evaluated using a custom built testing apparatus for hydraulic conductiv ity measurement. It consists of a two chambered isolation setup with the TMJ disc scaffold punches acting as a permeable barrier between the two chambers. The two cylindrical chambers (internal volume, 2.5 cm 3 ) were vertically oriented such that the uppe r chamber when filled with fluid created a head pressure which could be calculated at the beginning of the experiment. (5 1) Where Q is the volumetric flow rate measured as the upper chambers internal volume divided by the time it took for the fluid to traverse the thickness of the scaffold, permeation area of the sa mple (for our study the surface area of the 6mm disc punch), and t is the thickness of the sample. COMSOL computational fluid m odeling To evaluate the wall shear and fluid flow conditions within the LMP pore COMSOL Multiphysics Modeling version 4.3b was us ed. The scaffold matrix was

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88 modeled as a poroelastic model with a porosity coefficient of 0.7 a permeability of 7.5E 15 m 2 3 The fluid permeating the scaffold and the LMP pore has properties dynami c viscosity 8.9E 4 1E 3 kg/m 3 The interactions were modeled using the equations for capillary action through porous material (5 2) (5 3) (5 4) (5 5) Where S is the sorptivity sectional area, V is the cumulative volume of absorbed liquid at time t, I is the cumulative liquid inta ke, f is the porosity, and x is the wetted length or the fraction of volume occupied by the liquid, Mechanical e valuation Biomechanical characteristics were evaluated by two different methods: whole scaffold mechanical testing using a Biomomentum Mach1 mu lti axial mechanical testing rig (Biomomentum, Quebec, Canada) and micro mechanical testing by a CSM nanoindenter (CSM Instruments, Boston, MA). Both mechanical tests were conducted in unconfined compression of the central region of the disc. Compressive mechanical evaluation of the TMJ disc scaffold punches was conducted as described in Chapter 2. Briefly testing conducted within a hydrated

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89 chamber (PBS, 7.4) and scaffold punches were cyclically compressed to 10% compressive strain between a stationary b ottom plate and a vertical indenter. To identify the micro scale zone of mechanical consequence due to laser ablation axially from the ablated hole periphery on the TMJ disc the 120m LMP scaffold was evaluated using CSM Instruments Ultra Nanoindentation tester loaded with Z ) measured. Stress/strain were calculated using where l o conveyed by the indenter tip. The resultant force (F Z with A being the cross sectional area of the indenter in contact with the sample. From these data the compressive modulus of elasticity (E) w as computed by Statistical A nalysis Each set of test group data was calculated based on testing of six samples (n = 6). Standard Deviation and one way analysis of variance (ANOVA) testing was used to determine statistical significance between test groups for biomechanics. Significance was established using the Tukey Results The present study presents a novel methodology to generate an acellular naturally derived fibrocartilage scaffold with included laser micro ablation to improve permeability while maintaining mechanical resiliency. Scaffold Optimization for LMP The average mass and thickness in the central zone of the scaffold discs are respectively: native, 3.68 0.63g and 2.00 0.26mm; SDS decellularized 4.45 1.00g

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90 and 3.85 0.61mm; and the freeze dried and rehydrated disc, 4.03 0.90g and 2.67 0.21mm. The macroscopic and histological effect of decellularization and lyophilization on the TMJ fibrocartilage disc is illustrated in Figure 5 1. The n ative tissue has cell rich pockets and geometrically regular ECM fibrils. After SDS decellularization the cell pockets are removed completely and obvious structural change to the ECM network have occurred. Post decellularization ECM fibrils, while mainta ining their orientation, are compressed. Lyophilization and subsequent rehydration (such that histologic samples could be taken) was shown to act toward restoration of the ECM network with the fibrils regaining much of their contouring. The mechanical im pact of the scaffold processing illustrates that the SDS decellularized scaffold increases in stiffness with an increase in compressive elastic modulus from native properties (Figure 5 2). The compressive modulus of the scaffold decreases to statistically native properties after freeze drying and rehydration. Laser Micro Patterning (LMP) Analysis Incorporation of LMP into the freeze dried TMJ disc scaffold was assessed using SEM imaging of each of the 4 hole geometries. Figure 5 3 shows the 120m hole siz e images both top and side view. The LMP holes measure within 120 15 microns of the desired pore size. Rehydration rate and permeability are demonstrated in Figure 5 4(A) with screen captures of a rehydration video which illustrates that the LMP holes do traverse the thickness of the scaffold that the laser working does not change the hydrophilic quality of the ECM scaffold, and the pores do not collapse during hydration. The results of permeability evaluation are presented in the table insert of Figu re 5 4. SDS decellularization significantly decreases the hydraulic permeability coefficient (indicating the permeability is greater than in native tissue). The non LMP scaffold

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91 (SDS decellularized freeze dried and rehydrated with no LMP) has the shows t he least permeability of all the scaffolds tested with a significantly slower rehydration rate than the LMP scaffolds. While the incorporation of LMP does increase permeability only the LMP 200m hole size shows a higher permeability than the SDS acellula r. The computational capillary hydration model assessment of fluid velocity and flow directionality depicted in Figure 5 4(B) shows the maximum fluid velocity during hydration of the scaffold when hydrated with PBS (pH 7.4) is 0.0286 m/s, with general flui d direction matching the empirical hydration images shown in the progression in Figure 5 4(A). The whole scaffold mechanical properties are illustrated in Figure 5 5. Statistically only the SDS decellularized and 200m hole sizes show difference from the native. The general trend is the mechanical integrity of the scaffold with the LMP is retained until the hole size reaches 160m, at which point the scaffold structure is compromised by tissue removal via ablation. Micromechanical assessment of the surf ace region between two LMP holes shows that mechanical consequence due to thermal damage by the laser tissue interaction is limited to the immediate surroundings of the laser tissue interface. Figure 5 6 demonstrates that mechanical consequence due to the rmal damage to the ECM matrix affects only a 65m region surrounding the hole. 65m was the measurement resolution for this experiment and the decrease in compressive modulus values of the surface region of the scaffold was exponential between the hole pe riphery and 65m. It follows that the mechanical effect due to thermal damage affects only 18 23m by linear regression of the average curve (n=3).

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92 Discussion Here we report a scaffold processing methodology which generates a TMJ disc scaffold possessing structural, biochemical, biomechanical, and porosity appropriate for engineered regeneration by host cells after implantation or by culture cells in vitro One of the primary advantages in using whole tissue scaffolds for TMJ disc tissue engineering is t he disc already possesses the correct ECM structure and composition for physiologic function. The problem lies in removing the cellular debris and immune epitopes by decellularization methodologies; and how those techniques then distort the ECM fibril con formation causing the disc to lose it mechanical integrity. Previous study conducted by our research group demonstrated that sodium dodecyl sulfate (SDS) detergent decellularization generates a mechanically viable scaffold showing that no statistical diff erence was found between the compressive modulus of 1% SDS treated discs and the native untreated tissue. However, histologic inspection of the ECM structure of the SDS treated disc illustrated compacted collagen bundles with a loss of the underlying undu lating microarchitecture of the native TMJ disc [92] The results of the present investigations identified the same linearizing of the ECM fibrils after SDS decellularization and subsequent rinses (Figure 5 1). But this structural defect was recovered af ter lyophilization. Lyophilization works by removing water molecules from a material via sublimation creating vacuum forces between the ECM protein fibrils and removing the inter bundular spaces created during decellularization. In effect bringing the p rotein bundles into proximity with one another before hydration. During hydration water molecules reintroduced reconstitute hydrogen bonding between the collagen bundles

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93 resulting in the recovery of the individual bundular conformation and the overall undu lating architecture of the ECM. Bundular protein configuration is primarily organized by hydrogen and ionic bond forces developed between ECM protein fibrils. The recuperation of the ECM conformation also has restorative effects on the mechanical proper ties of the disc. Representative stress/strain loops show that the freeze dried TMJ disc displays energy distribution and deformation resistance comparable to the native disc (Figure 5 2). After the development of a suitable decellularized fibrocartilag e matrix, we used laser ablation drilling to incorporate an engineered microporosity which we hypothesize will act to overcome the hindrance of sub cellular porosity. The freeze dried disc possesses qualities optimized for laser micro patterning (LMP). I t is a dry material so no refraction or reflection of the laser will occur. Table 5 1 elucidates that decellularization, lyophilization, and LMP do not significantly diminishes the collagen content of the disc, and only the SDS treatment step reduces the glycosaminoglycan (GAG) concentration. GAG present in the TMJ disc have previously been hypothesized to play a role in the biomechanics of the tissue [132] compressive deformation with increased concentration. While t his mechanical consequence is evident in Figure 5 2 SDS curve the material recovers its elasticity after freeze drying but the GAG content is lost. We hypothesize that SDS treatment releases into the inter fibril spaces the soluble GAGs while not removi ng the insoluble GAGs. It would then follow that the soluble GAGs which take up within the inter fibril spaces would act to stiffen the matrix and increase the compressive modulus. After the soluble GAGs are rinsed from the inter fibril spaces and the ti ssue lyophilized and

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94 hydrated the insoluble GAGs not lost to decellularization become the primary affecters of the compressive properties of the disc. Although the xenogeneic SDS decellularized and freeze dried TMJ disc scaffold has mechanical ability si milar to the native tissue, the processing technique only addresses the removal of cellular debris and immunogenic epitopes. To create a scaffold which can be re cellularized either by cultured cells in vitro cells in vivo we needed to incorporate into the acellular TMJ scaffold a path of infiltration for cells initially and for metabolites and small molecules during remodeling. To accomplish this we utilized a high precision 50W CO 2 laser ablation system (Full Spectrum Lasers, Las V egas, NV) optimized to drill pores through the thickness of the TMJ disc scaffold (Figure 5 3). For a penetrating (through the entire thickness) hole geometry we chose to use the upper (superior) disc surface as the focal plane of the laser (Figure 5 3 superior surface view). This created a hole with the maximum Figure 5 3 cross sectional vi ew The choice of the optimum hole size for retaining mechanical ability whilst improving porosity and creating an environment to encourage uniform cell s eeding was critical for further development and application of this scaffold. Permeability and rehydration rate was assessed for each of the hole sizes and patterns (Figure 5 4 and tabulated data Table 5 2). The native tissue had a permeability coefficie nt of 1.85. The SDS decellularized scaffold demonstrated the lowest hydraulic permeability coefficient

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95 (least resistance to traversing fluid) due to the altered ECM structure and the highly ionic residual charge left on the ECM proteins by the SDS. Incre asing the porosity by drilling decreased the permeability coefficient indicating the material was more permeable to fluids. While this conclusion was obvious the dramatic decrease in nce of the do not vary as significantly. We hypothesize that the dramatic difference in the ension we utilized COMSOL Multiphysics modeling software to model the fluid mechanics within the pores of the various LMP hole sizes. Our hypothesis is defended by the results of the COMSOL model which resulted in predictions showing a higher degree of geometries (Figure 5 4 2). The mechanical consequence of the laser ablated micro patterns is shown in Figure 5 6 with the experimental control native mechanics illustrated as solid horizontal lines with the shading representing the standard deviation. The instantaneous (first loop modulus) and steady state compr essive moduli (average modulus of loop 7 10) of LMP pattern also showed no difference to th e controls when evaluating the E INST but displays a much stiffer compressive modulus at steady state conditions. The greater

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96 agrees with the results of the permeability and hydr ation evaluation discussed previously, stipulating that the higher mechanical modulus value is due to surface tension forces created between the hole walls causing greater resistance to compressive loading. The peak hysteresis results agree with the compr essive modulus retaining approximately only one fourth that of the native tissu LMP scaffold critically disrupts the mechanical ability of the disc due to loss of the continuity of the ECM fibril microarchitecture and LMP with hole diameters above r development. These results indicate that when considering permeability and mechanical the effects of the laser ablation such that an ideal LMP diameter can be conclud ed, nanoindentation of the immediate surroundings of the ablated hole was conduc ted utilizing a CSM Ultra nanoi ndentation system. The results demonstrate that mechanical consequence due to thermal damage to the ECM matrix affects only a 65m region surrou nding the hole. 65m was the measurement resolution for this experiment and the decrease in compressive modulus values of the surface region of the scaffold was exponential between the hole periphery and 65m. It follows that the mechanical effect due to thermal damage affects only 23.12m by linear regression of the average curve. Conclusions In summary laser micro patterning by ablation is a high throughput, high efficiency, and high precision methodology for inclusion of engineered microporosity to f acilitate cellular incorporation and support the diffusive needs of a scaffold during early

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97 remodeling. This technique creates a scaffold with the appropriate biochemical and structural environment and tunable compressive mechanical resilience. These eva luations indicate that when engineering for biochemical composition, permeability, and mechanical integrity appropriate for generating a suitable TMJ disc replacement the micro ablation pattern is optimal. This technology provides an effective solution to diffusive limitations of decellularized scaffolds and of large metabolite (nutrient and wastes) mass transport during early remodeling. Further studies regarding the potential of the LMP scaffold for engineering the disc, including cell seedi ng and early regeneration of the acellular scaffold, should be explored in the future. Overall these results support the potential of a porcine derived acellular engineered laser ablated microporosity scaffold as a xenograft for TMJ disc reconstruction.

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98 Figure 5 1. TMJ disc scaffold preparation and optimization for laser micro patterning. Mechanical consequence of lyophilization. The bright field histologic image of the native tissue shows thick and undulating ECM fibrils and the native macroscopic image shows healthy disc morphology. The 1% SDS decellularized ECM fibers demonstrate compressed and linearized structure indicative of this detergent lyophilization and rehydration the ECM fibrils have recovered general morphologic configuration of the native tissue but with less stain retention due to loss of cellular material and soluble glycosaminogly can content. The schematic insert demonstrates the rationale of lyophilization before laser ablation to produce regular and penetrating holes.

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99 Figure 5 2 Mechanical consequence of decellularization and lyophilization on fibrocartilage TMJ disc. Th e application on cyclic compressive loading illustrates the mechanical consequence of 1% SDS decellularization and The d ecellularization process results in an increase in the max stress of the first indentation and an increase in the steady state stress from native tissue properties. The freeze drying and rehydrating process after decellularization yields recovered mechanical ability toward native properties.

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100 Figure 5 3. Laser Micro Patterning schematic and final printed TMJ patterns with dimensional data. The four LMP patterns on the TMJ disc are imaged in the top figure. A SEM of the superior surface for the 200 m diameter hole demonstrat es the regularity of dimension and spacing produced by laser ablation. The cross sectional view illustrates that the laser ablation penetrates the thickness of the disc, with the LMP hole fully traversing the Photo courtesy of Ca ssandra Juran.

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101 Table 5 1. Biochemical evaluation of the native TMJ disc and acellular TMJ disc scaffolds

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102 Figure 5 4. Rehydration and p ermeability experiment and COMSOL Multiphysics modeling fluid mechanics within the LMP pore. Rehydration of the 120 m LMP scaffold with resazurin salt solution (blue solution) was recorded and video stills taken approximately every 14 seconds. Rehydration through the LMP pores took approximately 105 seconds for all three vid eos and total hydration to scaffold saturation took approximately 2 minutes. Inset Table 5 2 summarizes the hydration and permeability data derived from these videos and the permeability chamber testing. To evaluate the fluid flow through the LMP holes a finite element study utilizing COMSOL software was conducted simulating hydration of the pore with a PBS (pH 7.4) computational homolog. Results of the 80 m hole simulation are shown above. Photos courtesy of Cassandra Juran.

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103 Figure 5 5. Whole scaf fold evaluation of LMP mechanical consequence. Representative and modulus values. The 120 and 160 m LMP scaffolds result in mechanical properties statistically identical to the native tissue ( n=6, error bars represent standard deviation)

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104 Figure 5 6. Nano indentation evaluation of thermal damage to ECM scaffold. 120 m LMP samples were evaluated using a CSM Ultra Nanoindentation system to assess the mechanical result of laser ablation. These results show that the thermal consequence is lim ited to approximately 23 m by linear regression (n=3, error bars represent standard deviation)

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105 CHAPTER 6 MESENCHYMAL STEM CELL CHOICE, VALIDATION, AND NOVEL SEEDING METHODOLOGY Introduction The native TMJ disc has been reported to possess approximately 6 81 197 cells/mm 2 and t w o distinct cell populations [43] Fibroblast like cells are found throughout the disc and in highest density in the peripheral zones (anterior and posterior bands). Chondrocyte like cells are found predominantly in the intermediate zone of the disc preferentially nearer the superior sur face. These two cell types and their phenotypic de differentiated state (in comparison to articular cartilage cells or fibrous tendon cells) complicate tissue engineering cell choice. Cultured native porcine TMJ disc cells grown on culture plastic tend t o take on a fibroblast appearance as they proliferate and do not secrete cartilage matrix markers when cultured in well plates. This indicates that cultured TMJ disc cells are a poor choice for seeding an engineered construct because they lose their chondrogenic phenotype during proliferation [106, 107] Several studies have shown that dermal fibroblasts can be tr ansdifferentaited into chondrocyte cells [108, 109] Many other works have shown that adipose stem cells can be transdifferentiaed into osteoblasts, chondrocytes, and fibroblasts [110] The fibrobla st and chondrocyte similar differential pathway from mesenchymal stem cells (MSCs) suggests their use as a multi potent lineage cell choice for TMJ disc tissue engineering. C ells following the mesenchymal ste m lineage are able to transdifferentiate, agai n supporting their use as a multi potent cell choice and because they are derived from a neo natal source they have been shown to be less immunogenic compared to adult derived cell lines

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106 Jelly of the human umbilical cord h ave proven multi potent [111] The McFetridge lab has used these cells to produce functionalized (contractile) smooth muscle cells for use been demonstrated to possess the ability to be differentiated into fibrochondrocyte representative cells by Wang et al. [112] The native disc cells are not exposed to any immediate microvasculature, as most tissue types are. The cells are instead predominantly supplied nutrients by a mechanistic diffusive pumping which forces oxygen and other small molecules to traverse the thickness of the poroelastic disc [113, 114] Shi et al., investigated the correlation between anisotropic diffusivity of fluorescein through the TMJ disc a nd the 2 /s with statistically less diffusivity in the superoinferior plane than along the fiber orientation for all regions but the anterior (medial, intermediate, and lateral diffusivity in the anteroposterior direction and the poste rior diffusivity in the mediola teral direction) [101] These works demonstrate that within the disc exists an architecture devoted to shuttling small molecules to supply nutrients to cells. These findings are corroborated by recent high resolution diffusion tensor imaging MRI (magnetic resona nce imaging). Benavides et al., using diffusion tensor imaging confirmed that in the anteroposterior direction the intermediate zone had grater diffusivity than the periphery, but also that the medial and lateral regions of the intermediate zone had highe r diffusivity than the central region [115] These results indicate that the region which undergoes the most repetitive and strenuous mechanical loading is the region which is least supplied with

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107 nutr ients by diffusive action alone. Providing further credence of the diffusive pumping hypothesis. The mechanism of cellular metabolism in the TMJ disc poses several problems when considering tissue engineering development of a functional disc: First, the d iversity of specifically functionalized chondrocyte like and fibroblast like cell populations require either strategic co cultures or a cu lture of multipotent stem cells ; osity allowing mass transport of small molecules but not the incorporation of new cell communities beyond the immediate surface regions; and lastly, the superoinferior plane is not the direction in Our efforts to create a scaffold which is modified to overcome the limitation of subcellular porosity and lack of appropriate diffusive paths was described in Chapter 5 These works will describe the methods used to : isolate and validate the stemness of SCs; incorporate suitable cell population within the laser micro patterned (LMP) scaffold by a novel seeding methodology which utilize hydration physics to include a uniform cell density within the LMP scaffold; and show that cells receive nutrients over t he course of short duration culture (7 days) through the laser incorporated artificial porosity and the subcellular matrix diffusive pathways retained from the native disc Methods These experiments were conducted in three independent studies and the resul ts correlated in the findings presented in this chapter: first, the human mesenchymal stem cells (MSCs) validation as an appropriate cell choice for TMJ disc engineering; second, a computational study of hydration seeding modality optimizing for cell reten tion and

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108 distribution; and third, an empirical assessment of the hydration seeding method compared to both the computational model findings and experimental control traditional pipet seeding experimental results. Human Mesenchymal Stem Cell (h MSC) Isolati on and Differentiation births at Shands Labor and Delivery Ward (Gainesville, FL) and expanded as described in Chapter 2. Briefly, the umbilical cord was resected from the placenta and dissected. 25 flasks for 3 4 weeks with standard culture media (DM EM, 10% FBS and 5mL pen/strep). After cells reached 60 75% confluence (Figure 4 1a) they were enzymatical ly (Acutase) detached from the culture plastic and pelleted at 1,000 RPM for 5 minutes then re suspended and plated in a T 75 an allowed to proliferate for 3 days. After 3 days the cells were hemistry MSC staining section below. Mesenchymal stem cells have been shown to differentiate into a variety of mesenchymal lineage restricted cell types [116] We utilized an established derived MSCs into TMJ chondrocyte like cells [117] The differentiation media was composed of low glucose Eagle medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 1% insul in transferrin selenium (Invitrogen), 0.15% lipid rich bovine serum albumin (Albumax, Invitrogen), 0.1 nM dexamethasone (Sigma), 10 mM ascorbic acid 2 phosphate (Sigma), 1% penicillin streptomycin (Fisher), 2% fetal bovine serum (FBS, HyClone; Logan, UT), 10 ng/mL of recombinant human epidermal growth factor, and 10 ng/mL of human platelet derived growth factor BB (R&D Systems, Inc.). The

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109 differentiation media was applied to day 3 p 1 hWJMSCs after the cells reach 60 75% confluence. Cells used for validat ion of differentiability were cultured approximately 6 7 weeks after initial explant before immunohistochemical evaluation The medium was changed every 3 days until differentiation could be established by observation of morphology (approximately 9 days a fter incubation with differentiation media ) and immunohistochemistry of secreted matrix surrounding the cell colonies as described below in the immunohistochemistry of chondrogenesis section Immunohistochemistry of MSCs After cryosectioning samples were h ydrated with phosphate buffer saline (PBS, pH 7.4) for five minutes three times before incubated with 8% bovine serum albumin for 2 hours to inhibit non specific binding. Primary antibodies for : the CD44 antigen; a receptor for hyaluronic acid, osteopotin, and a variety of MMPs, CD90 antigen; a glycophosphaidylinositol anchor and cell surface protein associated with multipotent stem cells, and CD105 antigen; also known as Endoglin a proliferation associated glycoprotein part of the tumor g antibodies were incubated at assay dependent dilution (1:10,000 for CD44, 1:40,000 for CD90, and 1:2,000 for CD105) on the samples overnight in a humid chamber then rinsed with PBS (5 minutes 3X) befo re florescent conjugated secondary antibody were incubated for two hours and rinsed again (5 minutes 3X) then imaged. Hydration Seeding Computation and Live/DEAD Viability Evaluation All computational studies were performed using COMSOL Multiphys i cs Mode ling Software Version 4.3 b. Multiphysics modeling was used to both evaluate the scaffold material and evaluate the fluid forces during resorption. The TMJ LMP disc scaffold was treated as a porous vi scoelastic material in Chapter 5 thus in this chapter we

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110 represent the TMJ disc construct using the bi phasic theory. The scaffold was represented by poroelastic constitutive equations developed in the COMSOL software and the interstitial fluid properties relevant to the hydration media (values of Material Pr opertie s used for model shown in Table 6 1). The poroelasticity modeling equations used to generate the finite element matrix were: ( 6 1 ) ( 6 2 ) ( 6 3 ) ( 6 4 ) ( 6 5 ) Qm for thermal conductivity ; the temperature is 20 ; and g representing gravity. The dependent variables pf for pressure and the position variables u, v, w, and the operator Equation 6 1 des representi ng the material total discharge per unit area This equation mod els the proportional relationship between the instantaneous discharge through the porous medium, the dynamic viscosity of the fluid, and the pressure drop over a given distance. This equation is important as it contains the independent variables which can 6 2 generates the strain quantities for each element due to flux incurred in hydration. Equation 6 3 is the fluid mass balance relationship which described the fluid as incompressible within the porous m atrix. Equation 6 4 defines the work increment

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111 associated with the strain increment which can be used to find the shear moment stress at the contact between fluid and solid associated with local velocity gradient in the fluid (Equation 6 5). Equation 6 5 can be optimized to reduce shear moment at the matrix fluid interface, important when engineering to eliminate cell death associated with membrane rupture caused by excessing shear [118, 119] T he initial conditions are a starting head pressure of H p of .04m, no displacement field; u=v=w=0, no velocity field the initial pressure is pf=100000Pa, and no fluid flow ( 6 6 ) The boundary loads are described as ( 6 7 ) ( 6 8 ) Where P bc is 0.05 N/m 2 When particle tracking was enabled the diffusion equations followed Brownian motion combined with drag forces governed by ( 6 9 ) ( 6 10 ) ( 6 11 ) ( 6 12 ) With m p representing the particle mass, is the particle velocity response, are the fluid velocities derived from the poroelastic model, e time step

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112 taken by the solver, r P b is the zero and unit standard variation created by the program for each particle at each time step. Independent variable values and initial and boundary conditions for the particle tracing can be found in Table 6 2. Hydration Seeding Empirical Evaluation 6mm samples of the central zone of the acellular lyophilized LMP TMJ disc were used to evaluate cell attachment, retention, and metabolism during the first 24 hours and after 7 days of culture. The samples were divided into two test groups: the hydration seeding group and the pipet (control) seeding group. Both samples were pla ced in 48 well plates and seeded with 900 cells/mm 2 seeding density. The hydration seeding group dry LMP scaffold samples were placed in wells of a 48 well of media was added to submerge the sample. The pipet seeding group samples were 2 After 24 hours the PBS was removed and the scaffolds seeded with 1mL of 900 cells/mm 2 density media. The cultures were 2 for 24 hours before evaluation. Feedback Optimization An initial experiment revealed a large degree surface cell death inspected by Live/DEAD imaging. Evaluation of the computation model revealed hydration of the scaffold by a fluid with properties representing standard culture media (media sup plemented with 10% fetal bovine serum (FBS) and 1% penicillin) resulted moment shear field values correlating to published val ues with induce cell membrane rupture

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113 (values 10 2 to 10 5 depending on cell lineage). Thus the hydration media properties were augmented by varying the fluid viscosity in the computation model until moment shear field values were bellow membrane rupture (see Table 6 1). Experimentally the media was supplemented with dextran (MW: 70,000; Sigma Aldrich #39310; St Louis MO) to increase the viscosity until value matched the computation model and the hydration seeding vs. pipet seeding experiment was repeated with the d extran supplemented media. 7 day c ulture Samples of both the hydration seeding and the pipet seeding methodologies were cultured for 7 days to validate stability of the cell population. At 24 hours after seeding the seeding media was removed an d fresh culture media (no cells) added. Three days later the culture media was changed again and at six days later the samples CO 2 in the wells of a 48 well plate envir onmentally isolated by micropore tape to inhibit infection. Evaluations PicoGreen Quanti IT DNA quantification (described in Chapter 2) was used to determine cell attachment (2hrs), retention (24hrs), and culture (7 days) of the test groups. In conjunct ion with DNA quantification cellular metabolism was also assessed using resazurin salt reduction (described in Chapter 2) at 2 and 24 hours and 7 days to establish cellular viability. Imaging of the cells distribution through the scaffold was established by DAPI/rhodomine phalloidin florescence staining and Live/DEAD imaging (also described in Chapter 2).

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114 Results MSC Isolation and E valuation approximately 13.1 7.1 x 10 3 cells per twelve 2 mm x 2mm morsel which after passaging and three days further culture expand to an approximately 5.1 1.4 x 10 6 cells. Immunohistochemistry shown in Figure 5 1 show mesenchymal positive markers (CD 44, CD 90, and CD 105). Application of the chondrogenic differentiation media and culture 9 14 days proved adequate for MSC differentiation into chondrocyte ECM secreting cells quantified by total collagen and total GAG secretion (0.340g/L and 0.011g/L respecti vely) Computational Hydration Seeding and Live/DEAD Viability Evaluation experienced by the particles) generated as the fluid moved through the poroelastic TMJ disc representative material. C omputation model of hydration seeding by standard culture m edia The moment field illustrated in Figure 6 3 A&B show that surrounding the periphery of the h ole the largest strains are experienced with peak value approximately 70x10 8 4 N/m) when tissue is hydrated with media homolog fluid properties. This shear moment value falls within the values of published cell membrane rupture shear moment corroborated by Live/DEAD imaging of the scaffolds surface which shows significant cell death surrounding the hole periphery. Results of Feedback Optimization to reduce the shear moment to 0.9x10 5 N/m provide a require d fluid Addition of 6% dextran ( MW 70,000, Sigma Aldrich #31390,

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115 St Louis, MO) to standard culture media appropriately increases the media viscosity to the desired value [120, 121] Live/DEAD imaging confirmed that increasing the culture d cell death at the LMP hole periphery (image not shown). All further computational and experimental studies use the 6% dextran supplemented media solution in evaluation of cell seeding. Computation m odel o f hydration seeding by dextran modified culture m edia The particle tracing model computed the streamline velocity field, and positional data with time and mapped the particles movement across the imaged planes (Figure 6 magn itude 10 8 color represented in red in Figure 6 4) to the slowest (order of magnitude 10 10 color represented in blue in Figure 6 4) for all cut planes. The cross section view through the holes (Figure 6 4 C) show parabolic velocity fields concurrent wi th wall shear fields seen in capillary tubes. An H&E histologic image of the hydration seeding method compared to controls is shown in Figure 6 5. Hydration Seeding and 7 Day Culture Experimental A ssessment All seeding and 7 day culture experiments were conducted with the dextran modified culture media. DNA quantification and cellular metabolism data of the cells incorporation into the LMP scaffold for hydration and pipet seeding within the superior, mid, and inferior regions is presented in Figure 6 6 DNA quantification demonstrates statistical differences between the hydration and pipet seeding group for the mid and superior regions at both 2 and 24 hours of culture. The inferior zone presents no statistical difference between the seeding techniques f or either culture time. The resazurin salt reduction, indicative of cellular metabolism, of the pipet seeding technique showed the highest metabolism at 2 hours and decreases from 2 to 12 hours culture in

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116 linear fashion ( Figure 6 6 A ). Between 12 and 24 hours of initial culture the pipet seeded cells shift toward a less metabolically active state ( 4.32 4.31 % reduction/cell tabulated average metabolism per cell at 12 and 24 hours respectively compared to 6.89% reduction /cell at 2 hours ) while the hydratio n seeded cells retain highly metabolic state ( 6.12 % reduction/cell at 24 hours compared to 7.87 2.12 % reduction/cell at 2 hours ) The DNA quantification and cellular meta bolism data of the hydration seeded scaffolds show greater retention (14.11 2.2 1 x10 3 cells at 2 hours and 12. 8 0 3.31 x10 3 cells at 24 hours) compared to pipet seeded scaffolds (10.31 3.61 x10 3 cells at 2 hours and 8.97 2.04 x10 3 cells at 24 hours). R egional DNA distr ibution show cells integrate throughout the scaffold more effectively with the hydration seeding method than the control pipet seeding method ( Figure 6 6 ) After 7 days in culture the final cell density with the hydration technique has proliferated to approximately four times the initial density measured at 24 ho urs (44.41 7.11 x 10 3 cells/scaffold and 12.80 3.31 x 10 3 cells/scaffold respectively). This high degree of proliferation is not observed in the pipet seeded group with a final cell density of 20.43 4.43 x10 3 cells/scaffold. The % reduction of resa zurin salt seen in the h ydration seeded scaffold at 7 days is statistically higher t han the pipet seeded scaffold ( 4.14 0.21 x 10 3 %reduction/cell and 3 .24 0.42 x 10 3 %reduction/cell, p* < .01). Discussion Tissue Engineering has been represented as a simplified three step process: scaffold development; cell choice; and culture to induce function. While this model is highly simplified it implies the importance of appropriate cell choice in the development of a tissue engineered construct. The native T emporomandibular Joint disc contains two distinct cellular populations, fibroblast like and chondrocyte like, which are

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117 distributed at differing compositions throughout the major regions of the disc. Cells isolated from the porcine TMJ disc and grown on c ulture plastic have been shown to lose their variation and instead take on a de differentiated non functional state. Also these cells were isolated from healthy TMJs and after the animals sacrifice. Attaining cells from a patient with a damaged TMJ would involve much more complication than experienced through this ex vivo study. Thus the use of autologous cells for TMJ disc engineering purposes is unlikely. Consideration of an appropriate cell lineage for use in TMJ disc tissue engineering stems from the cells origin in healthy TMJ development. Developmentally the TMJ disc matures during the late fetal stage from osteochondral progenitor cells of the cranial neural crest derived from intraembryonic ectoderm cells. Also developed along this pathway are p re chondrocytes, condylar condensati on cells, and glenoid fossa polymorphic progenitor cells. This highly multi potent pathway in TMJ disc developmental lineage suggests the use of a multi potent stem cell, for connective and cartilage tissue cells the ce ll choice is mesenchymal stem cells (MSCs). MSCs can differentiate into cartilage, bone, adipose, and connective tissue cells. If untreated on culture plastic MSCs have been shown to take on a fibroblast morphology and genetic phenotype. However, recent research has indicated that fibroblast differentiation is a complicated cascade reaction much like the differentiation of MSCs into other cell types. highly proliferative MSCs. Also because they are a fetal tissue they lack many of the immune epitopes which cause rejection in transplant patients. Thus these cells are an ideal choice for TMJ disc engineering purposes Figure 6 1 illustrates the isolation and

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118 validation analyses used for these works. Immunohistochemistr y of the hWJ MSCs grown on culture plastic show positive stain for CD44, CD45, CD90, and anti integrin beta 1 (CD29). After expansion the hWJ MSCs still express the stem markers proving that they are a viable stem cell source through passage 3. After chondrogenic differentiation and expansion on culture plastic the cells no longer express any CD44, CD45, and only minimal CD90 and CD29. The chondrogenically differentiated cells have also begun to secrete matrix representative of chondrocytes including collagen type I and II and chondroitin sulfate glycosaminoglycan. Incorporation of the MSCs into the dense ECM of the decellularized TMJ disc matrix through sub cellular porosity without mechanical or nutrient gradient is unlikely. The use of the laser micro cells was shown in Chapter 4 ; however evaluation of the cell attachment and viability has not yet been quantified. Utilizing the lyophilized state of the scaffold we hypothesized that cells could be drawn into the scaffold with more uniformity if the scaffold was hydrated with cell rich media (Fig ure 6 2). The concept is that the cells would be drawn into the scaffold along with t he fluid which is absorbed much like a sponge. To test the validity of this hypothesis we conducted a computation study with COMSOL Multiphysics Modeling Console incorporating capillary action, vacuum forces, and diffusive mechanics into a hydration model and simulated the TMJ disc as a poroelastic material with properties described in Table 6 1. We then incorporated into the fluid model particles with cell dimensional and geometric qualities into the fluid description described in Table 6 2. The model r (pressure, velocity, moment) experienced within the poroelastic material and tracing of

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119 the particles movement over 5 minutes of hydration. Results show that particles incorporate themselves through the thickness of the scaf fold with the hydration model (Figure 6 4 florescence and Figure 6 6 DNA quantification). Also the model predicts that the cells will experience at the cusp of the LMP holes a m oment shearing of approximately 70x10 8 4 N/m). Reported value s of cell surface tension causing membrane rupture range from 0.06N/m to 10 5 N/m. Thus at the hole periphery at the superior and inferior surfaces cell rupture will occur. This computational finding was dramatically corroborated by Live/DEAD florescence imaging which shows cell death at the hole periphery only ( Figure 6 3 ). DNA quantification and cellular metabolism evaluation of the scaffold substantiate the models findings that cells will incorporate throughout the scaffolds thickness (from superior to inferior along the LMP holes) and also that the majority of those cells are alive and metabolically viable when compared to a culture on plastic seeded with the same density. Our experimental evaluation also demonstrate a statistical difference between the hydration and pipet seeding techniques in the attachment and metabolic quality of the cell population incorporated. The pipet seeded technique has significantly less cell attachment but comparable metabolic activity form 2 6 hours post seeding ( Figur e 6 6 A). After 6 hours the metabolic activity decreases in the pipet seeded cells. A decrease is not seen in the hydration seeded cells over the 24 hours of initial culture, but does occur at 3 days of culture. We hypothesize that the retained level of metabolic activity over the initial 24 hours of culture is because the hydration seeding technique is more traumatic for cells than the pipet seeding method.

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120 But the efficiency of cell retention Figure 6 7 ). The regional assessment of the scaffolds show that the hydration seeding method has cells incorporated in the mid region after 2 hours of culture while the pipet seeding method shows statistically no cell presence in this region at 2 hours. After 24 hour s the regional distribution of the hydration seeding and pipet seeding is proportional but the quantification data shows that the pipet seeding method scaffold have retained less of the initial seeding density than the hydration technique ( Figure 6 6 ). C onclusions These works evaluate cell choice and test a novel seeding method witch was conceived to utilize the mechanics of hydration experienced by the lyophilized LMP appr opriate for TMJ disc tissue engineering as they have the ability to differentiate into chondrocyte representative cells and fibroblast representative cells. How to incorporate these MSCs was evaluated using a computation model to predict the fluid forces which cells would be exposed and estimate a pattern with which cells would diffuse into the poroelastic material scaffold. These results show that MSCs seeded with the hydration mechanism would traverse the scaffold and attach with a relative degree of un iformity. Unfortunately the model also predicted that the MSC would experience detrimental shear forces at the LMP hole periphery on the superior and inferior surfaces. These computational findings were corroborated by an empirical evaluation which also c ompared hydration seeding to the control pipet seeding technique. Results of the experimental evaluation demonstrated the advantage of the hydration seeding technique over initial culture (24 hours) and extended culture (7 days). These

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121 evaluations demons trate that utilizing the novel hydration seeding methodology to seed human MSCs is viable for TMJ disc tissue engineering.

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122 Figure 6 1 differentiation. TEM images demonstrate the morphological differences between the chondrocyte like and fibroblast like cells of the native TMJ disc. Human Mesenchymal Stem Cells (MSC) of the human umbilical cord and cultured in a T 25 culture flask (Fisher Sci entific). The bright field image below the culture flask picture images the MSC after 3 weeks culture. Evaluation for stemness, or the quality of stem cell properties, was conducted using three MSC protein markers: CD 44, a receptor for hyaluronic acid, opteopontein, and MMPs; CD 90, a protein associated with multipotency and a glycophosphatidylinositol anchor cell surface protein; and CD 105, better known as endoglin a proliferation associated protein. Photos courtesy of Cassandra Juran.

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123 Table 6 1. Definition of COMSOL Multiphysics computational modeling independent variables for the TMJ disc poroelastic matrix material properties.

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124 Table 6 2. Definition of COMSOL Multiphysics computational modeling independent variables for the TMJ disc fluid material and particles properties. variable name variable symbol variable value variable unit Dynamic viscosity 8.9E 4 Feedback Optimized Computational Dynamic Viscosity 3.0E 3 K 0.1667 --------Compressibility Q m 1 --------Particle mass m p 56 P g Particle radius r p 5 m constant k b 1 ---------

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125 Figure 6 2 Schematic of hydration seeding modality and COMSOL Multiphysics Hydration seeding is hypothesized to draw cells into the laser ablated holes and subsequently into the poroelastic material following the hydration media. A finite element computational experiment conducted using COMSOL Multiphysics software elucidated the mechanism of hydration could be used to facilitate seeding. Photo courtesy of Cassandra Juran.

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126 Figure 6 3 COMSOL Multiphysics Modeling of the Moment Field associated with hydration seeding. A) Illustrates the moment field and B) illustrates the associated moment vectors. C) is a Live/DEAD florescence image taken at 5 minutes post seeding showing cell death i s experienced in the zone surrounding the LMP holes which represent the high moment and high shearing vectors.

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127 Figure 6 4 Cut away views of COMSOL Multiphysics Modeling tracing cell scale particles during initial (5min) rehydration seeding and flui d forces cell florescence microscopy of the same cut away views.

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128 Figure 6 5. H&E histology of the hydration seeded and control pipet seeded constructs at 2 hours of culture. The traditional pipet seeding (control) method results in a dense encapsulation of the pore surface but migrate little further. The hydration seeding technique has uniformly seeded the scaffold between two LMP holes creating a constant cellular population.

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129 Figure 6 6 DNA quantification and cellular metabolism of the scaffold and regional variation of DNA quantification over 24 hours of culture. A) shows the cellular metabolism and DNA quantification of adherent cells during the initial 24 hours of culture. B) illustrates the regional variation of cell integration between the hydration and pipet seeding methods at 2 and 24 hours of culture.

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130 Figure 6 7 Variation in DNA quantification and cellular metabolism of the hydration seeded and pipet seeded scaffolds cultured over 7 days. The hydration seeding technique demonstrates greater cell proliferation at days 3 and 7 and higher cellular metabolism at all evaluated time points compared to the pipet seeding method controls.

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131 CHAPTE R 7 CELLULAR ADHESION AND CYTOCOMPATABILITY PROOF Introduction The goal of these investigations was to evaluate the viability of the laser micro patterned (LMP) TMJ disc sca ffold, developed in Chapter 5, seeded by the hydration method, described in the Chapter 6, compared to a non LMP disc scaffold control. The LMP construct (scaffold + cells) has previously been evaluated for permeability and mechanics. This study conclude evaluated) for the desired characteristics. While in that same study a computational investigation into the optimal pore size for cell attachment had not been considered. Murphy et al., evaluated cell adhesion and proliferation as a function of pore size (85 325m) and found that a mean pore size of 120 m enhanced for initial cell attachment and proliferation (48 hours) [122 124] Thus the spot size using this approach falls within the optimum window for cellular integration. Other laser ablation studies performed on biomimetic gels have shown that incorporation of laser ablated pores enhances cel lular infiltration [ 132 ] The major differences between previous studies and these investigations are these works ablate a natural ECM material which retains biochemical and biomechanical properties of the target tissue. Also these works ablate through a tissue >2mm in thickness while previous studies have been limited to under 1mm thickness [103 105] An ideal bioactive TMJ disc implant, that aims to restore joint function, needs to be mechanically robust, enough to wi thstand joint loading during initial remodeling and

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132 have a porosity that encourages cellular integration by improving mass transport conditions throughout the thickness of the scaffold [125] Methods and Materials Scaffold Culture Porcine TMJ discs were decellularized, rinsed, lyophilized, laser ablated, and seede d via the hydration technique utilizing methods previously described (Chapters 2, 5, and 6). After 24 hours the seeded constructs were transferred into 24 well TC plates (Fisher Scientific) and cultured to 7, 14, and 21 days under traditional culture condit ions (5% CO 2 Biomechanical A nalysis A Biomomentum Mach1 Micromechanical System (Biomomentum Inc. Laval, Quebec, Canada) was used for all mechanical testing as described in Chapter 2 for compressive mechanical testing The testing chamber was filled with 0.15 M PBS (pH 7.4) to maintain hydration, similar to the in vivo environment [1] and to maintain consistency with previous studies on TMJ t issue mechanics [71, 72] The force and displacement raw data values were recorded and used to calculate stress, strain, and compressive modulus of elasticity as described previously. Hydraulic P ermeability The ease of fluid flow through our porous sample was evaluated using a custom built testing apparatus for hydraulic conductivity measurement. It consists of a two chambered isolation setup with the TMJ disc scaffold punches acting as a permeable b arrier between the two cha mbers, as described in Chapter 5

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133 Cellular I ncorporation Cellular Adhesion was evaluated over the first 24 hours of culture at time points 10 mi nutes, 2, 6, 12, and 24 hours using the Quanti iT PicoGreen assay (Invitrogen, Oregon, USA) as described in Chapter 2 Calibration curves were produced for known concentrations of cells to DNA that were then used to determine the DNA concentration/cell. At 2 hours and 24 hours the distribution of cell number was evaluated through the thickness of the sample by dividing the punch into 3 approximately equal segments (superior, middle, and inferior) and quantifying DNA content in each of these regions to app roximate the uniformity of cell seeding through the thickness of the scaffold. DAPI and calcei n am LIVE/DEAD Florescence Assay were imaged at 24 hours to illustrate seeding density of the disc scaffold. Metabolic activity per cell was determined by measu rement of metabolic reduction of the media using a resazurin salt assay, against a calibration curve of the same cell lineage in conjunction with the PicoGreen DNA quantification again as described in Chapter 2 General Methods Briefly, culture media wa s replaced by resazurin salt solution (1:10 salt to media concentration) with fresh media and incubated for 2 hours. Over incubation the resazurin salt solution is reduced when exposed to metabolites of cellular metabolism. After incubation the sample we re excited at 570nm and the florescence emission read at 600nm. Cellular metabolism was measured at 1, 7, 14, and 21 days in culture. Cellular metabolic activity was normalized against cell density, where the total metabolic activity of each sample was divided into the cell number.

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134 Statistical A nalysis Each set of test group data was calculated based on testing of nine samples (n = 9). Standard Deviation and one way analysis of variance (ANOVA) testing was used to determine statistical significance betw een test groups for biomechanics. Significance was established using the Tukey Results The present study reports that utilizing, in conjunction, sodium dodecyl sulfate (SDS) decellularization and optimized scaffold working for laser micropatterning (LMP) a TMJ disc scaffold can be generated which possesses biochemical and biomechanical properties similar to the native tissue, as well as an artificial porosity providing diffusion of nutrients and wastes to and from cells dur ing initial remodeling. Mechanical Evaluation Representative stress strain hysteresis in Figure 7 1 shows that an 8x16 grid of rehydrated scaffold relative to native tissue or the control non LMP scaffold (decellularized, lyophilized and rehydrated in PBS, pH 7.4). Cellular Adhesion and Regional Seeding Efficacy Cellular adhesion to the LMP discs and control samples without LMP (non LMP) 24 hours after seeding are shown in Fi gures 7 2 7 3 and 7 4 Figure 7 2 illustrates the differences in total cell adhesion between LMP and non LMP constructs throughout the initial 24 hours of culture. 10 minutes after seeding 63.68% of the total seeding concentration had adhered to the LMP scaffold while the non LMP cell adhesion was lower with 48.90% adhesion. From the initial seeding to 24 hours of culture both

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135 scaffolds experience significant loss in total cell adhesion with the LMP dropping to 51.21% of the total seeding concentration and the non LMP retaining only 21.05%. The uniformity of cellular adhesion through the thickness of the scaffold is shown in Figure 7 3 sections representing the superior, mid and inferior zones of each construct are isolated and analyzed separately. DNA quantification results indicate that increased densities of cells adhere to surface zones (inferior and superior zones) of the LMP compared to the non LMP constructs and only LMP samples show the presence of cells in the mid zone 24 hours after seeding. In Figure 7 4 (A and B), calcein AM florescence images illustrate that cells adhere at high densities to the surface of the naturally derived scaffolds. Adherent cells lay flat and extended adjacent to the surface ECM fibrils of the non LMP scaffold; while cells adherent to the LMP scaffold have a less organized (anisotropic) orientation. DAPI and rhodomine phaloidin florescent staining of the scaffolds cross sections ( Figure 7 4 (C and D)) show that cells of the LMP scaffold have adhered to the walls of the hole traversing the thickness of the scaffold while cells are only localized on the scaffold periphery with the non LMP samples. Cellular Integration and Early Remodeling Cell Proliferation data in Figure 7 5(A) shows that the LMP samples had greater sust ained cell proliferation and metabolic activity (per cell) compared to non LMP constructs. Cell density in the non LMP constructs increased between day 7 and day 14 followed by a steady state population with no statistical difference in density, nor metab olic activity, through the remaining time course. The mechanical consequence of cellular integration through the thickness of the scaffold is illustrated in Figure 7 5(B) Data shows the non LMP scaffold to increase its elastic properties, exhibiting line ar geometric decrease in modulus with time. A non significant increase is seen in the non

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136 LMP modulus between day 1 and day 7 which we attribute to increased cell density in the scaffold periphery. The LMP scaffold is significantly strengthened during i nitial cell incorporation (between day 1 and day 7) before eliciting typical scaffold mechanical weakening seen during static culture. Histologic sectioning seen in Figure 7 6 show cellular migration from the pore periphery (initial adhesion site) into the scaffold and between LMP holes after 21 days in culture. Cell nuclei are apparent as far as 1000 m from the edge of the sample and up to 235 m from the edge of pattern holes ( Figure 7 6 (A and B)). With non LMP samples no cell nuclei were present mor e than 500 m from the sample edges ( Figure 7 6 (D and E)). Only surface zones have high cell density in the non LMP samples ( Figure 7 6 (D and E)) and no cell populations are observed within the constructs interior. Both H&E and the florescent images show the ECM fiber alignment of the LMP scaffold has changed significantly from the initial adhesion architecture ( Figure 7 6 (A and B). Fibers are oriented anisotropically around the hole perimeter rather than oriented linearly as seen in the non LMP scaffold. Discussion It has been previously stated that requirements to engineer a successful TMJ disc replacement are: (1) a biodegradable or biocompatible scaffold with mechanical capability to withstand joint loading without fatigue or fragmentation; (2) a repr esentative cell source incorporating chondrocyte like populations and fibroblast like populations; (3) suitable surface chemistry for cell attachment, proliferation, and differentiation; and (4) cellular integration and microenvironment conducive to mass t ransport through the scaffold, as the native disc is primarily avascular and cells within the native disc receive

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137 glucose and oxygen from mechanical pumping of synovial fluid within the joint space. [46, 126, 127] These works describe the application of a novel scaffold development addressing the hypothesis that incorporation of an art ificial path of infiltration for cells, nutrients, wastes, oxygen, and other small molecules will improve a naturally derived acellular cal and biomechanical properties. A constraint of many naturally based scaffolds is a consequence of the dense fibrous ECM networks with subcellular pore sizes that inhibit cell migration as well as limiting mass transport conditions. These limitations are exacerbated as populations increase further impeding cell migration, proliferation, ECM secretion, and remodeling. To circumvent these limitations artificial pores were integrated into the acellular TMJ disc scaffold using a CO 2 laser. The mechanical con sequence of these processing steps (decellularization, lyophilization, LMP and rehydration) was evaluated using unconfined cyclic compressive loading with compressive modulus of elasticity values presented in Table 7 1 and hysteresis in Figure 7 1 The me chanical ability of the LMP scaffold is within 5% of the control SDS decellularized FD and rehydrated scaffold and possesses energy dissipation trends similar to the native tissue, illustrating that our LMP scaffold has mechanical qualities comparable to t he native disc. Creation of a uniformly cell dense scaffold is imperative for fibrocartilage tissue engineering as the native tissue is largely avascular and nutrients are only obtained through diffusive mechanism; also pockets or zones of high cell densit y may result in hypoxia and inappropriate tissue regeneration during early remodeling events. Cell

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138 adhesion and retention was found to be improved with LMP constructs compared to non patterned constructs after 24 hours of culture, however initial adhesion was less than optimal with only 45% and 61% adhesion for non LMP and LMP scaffold respectively ( Figure 7 2 ). The more important finding from a construct developmental perspective is that cells were present throughout the thickness within LMP samples ( Fi gure 7 3 (B)). Cell concentration within the mid layer of the scaffold proportionally increased relative to superior and inferior layers which displayed a decrease in cellular presence between 2 and 24 hours while cell concentration of the LMP construct sh owed no statistical difference. This resulted in a near uniform cell distribution through the thickness of the 2.06 0.26mm LMP scaffold at 24 hours ( Figure 7 3 (C)). While the non LMP scaffold show a cell dense surface layer, no significant cellular infi ltration was noted with DNA analysis ( Figure 7 3 (B and C)) nor by florescence imaging of the constructs cross section ( Figure 7 4 (D)). Construct development relies on the sequence of cellular adhesion, infiltration, colonization and remodeling by metabolic ally active cells to synthesize an appropriate ECM. Metabolic activity of the cells within the constructs was evaluated at 1, 7, 14, and 21 days of culture. Both LMP and non LMP scaffolds demonstrated an increase in metabolic activity of cells between da y 1 and day 7, likely a transition period in which the cells adhere and acclimate to the scaffolds initial ECM environment ( Figure 7 5(A) dashed lines). Cells adhered to the LMP scaffold exhibit a greater than 4 fold increase in metabolic activity despit e no significant increase in cell density over the initial 7 days of culture indicating that proliferation is secondary to stabilization within the natural matrix. The cell density quantified in the LMP constructs was significantly higher than

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139 the non LMP likely due to increased surface area and the availability of nutrients due to improved mass transport through the thickness of the scaffold. The mechanical consequence of the constructs dense cellular repopulation is elucidated in the comparison of the LMP modulus against the non LMP ( Figure 7 5(B) ). The non LMP constructs experience the typical mechanical degeneration over time as seen in unfixed natural matrix materials. The LMP scaffold experiences a mechanical strengthening before the degenerative trend is observed. The significant increase in elastic modulus seen only in the LMP scaffold and not the non LMP constructs is a consequence of the laser working and more uniform cellular repopulation. As cells infiltrate a natural biopolymer they secret e digestive enzymes and new matrix to cocoon themselves in an environment conducive to proliferation [128] Cells seeded on the ex vivo derived scaffolds used in these investigations would undergo very similar processes during initial culture (1 7 days), mildly interrupting the fibril alignment of the disc causing mechanical weakening of the ECM superstructure during early integration. Then, to a more significant consequence, filling the void space created by decellularization and laser ablation with their newly secreted matrix ( Figure 7 6 ). This occurs also in the non LMP scaffold; howe ver, only at the cell dense surface layer where direct interaction with culture media is possible. Both constructs, the LMP and the non LMP, exhibit the cell dense surface layer at 21 days of culture; however, observation of cellular presence through the t hickness of the constructs after 21 days illustrated the continuity of cell distribution throughout the LMP construct while cells on the non LMP construct were limited to a periphery zone of only 200 50m (Figure 7 6 ). Finding increased cell populations within the interior of

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140 the LMP scaffold implies that a pore diameter of 120m is adequate to allow cell infiltration and adhesion without detachment due to fluid forces. Also that the pores are eration and limit nutrient transfer to and from cells within the constructs interior during initial remodeling. Future studies will incorporate mechanical stimulation to encourage mechanotransduction of MSCs and guide matrix remodeling of the scaffolds s uperstructure. Moreover further investigation of cellular enzymatic degeneration and ECM secretion will be imperative to develop conclusions about cell activity during early and extended remodeling events. Conclusions An ideal fibrocartilage scaffold fo r TMJ fibrocartilage disc replacement will exhibit a balance of biocompatibility, mechanical ability, and porosity [129] The metho dology described in these works develop a novel scaffold which retains the mechanical resilience and biochemical microenvironment of the native TMJ disc and is augmented for cell adhesion and mass transport to support uniform cellular integration. These i nvestigations prove that laser micro patterning into the natural ECM structure of the acellular TMJ improves permeability of the dense ECM matrix scaffold. This improved permeability then supports uniform cellular integration and encourages cellular remod eling by providing a path of infiltration for metabolite diffusive action while not lessening the mechanical ability compare to the non LMP. Early remodeling events are not inhibited by either the decellularization process or laser ablation but are accomp lished with greater uniformity through the thickness of the scaffold. The techniques developed in these investigations can be applied to a multitude of naturally derived grafts as the laser micro patterning can be incorporated into thick whole tissue

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141 scaf folds (bone, ligament, cardiovascular, etc.) for direct implantation or further in vitro study.

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142 Figure 7 1. Mechanical Effects of the scaffold processing technique. The mechanical ability of the TMJ disc at each stage of the scaffold processing tec hnique was evaluated by cyclic compressive testing. 10% compressive strain was applied by a flat indenter and the resulting force was measured. From the force and surface area measured the stress was calculated and is presented in this hysteresis. Highe r stress and hysteresis are seen in the SDS decellularized tissue due to ECM protein conformational disruption by the highly ionic SDS molecule.

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143 Table 7 1. Hydraulic Conductivity and Theoretical Permeability of the native and worked TMJ disc scaffold. Compressive Fluid Solid Properties of the TMJ scaffolds illustrate decreased hydraulic permeability coefficient for all processed scaffolds in comparison to native and an increased compressive modulus. Extremes for both properties are observed after SDS decellularization and recovered toward native after freeze drying and rehydration. Hydraulic Permeability Coefficient (m 4 /Ns) Compressive Modulus of Elasticity (MPa)

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144 Figure 7 2 Cell Adhesion over initial 24 hours. Scaffolds were seeded with a density of 900 cells/mm2 (approximately 25500 cells total per scaffold)and incubated at 5% CO2 and 37C for 10 minutes to 24 hours and then evaluated for cellular adhesion using the Quanti iT PicoGreen assay. Nearly 60% of cells were adherent to the LMP scaffold after 10 minutes and retained nearly 70% of those cells after the 24 hours of incubation. The non LMP scaffold however only retains about 50% of the initial cells adhered (10 minute cell density). n=9, *, P < 0.05, **, P < 0.01, error bars represent standard deviation.

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145 Fig ure 7 3 DNA quantification and cell seeding density as a function of thickness. Cell adhesion was further evaluated by conducting DNA quantification with the Quanti iT PicoGreen assay regionally from the superior surface through the scaffolds thickness at 2 hours aft er seeding (B) and 24 hours after seeding (C). The analysis reveals that both the LMP and the non LMP scaffolds retain a large cell population from seeding, however the LMP scaffold has cell infiltration into the middle region of the tissue, identified in illustration (A), while the non LMP scaffold has no cell infiltration past the surface regions (n=9, *, P < 0.05, **, P < 0.01, error bars represent standard deviation)

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146 Figure 7 4 Cellular Adhesion to the surface layer and through the thickness of the TMJ disc scaffold. Live/dead florescence staining of the top surface of the LMP (A) and non LMP (B) scaffolds imaged 24 hours after seeding. DAPI staining identifies the nuclei of cells in cross sectional histologic slides taken at the centerline (3 mm from the edge) of both scaffolds (C and D). The cross sectional images clearly illustrate that the LMP scaffold have greater cellular adhesion because of the additional surface area the cells are exposed to, while the non LMP scaffold has cell adhesion only at the periphery of the cross sectioned sample.

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147 Figure 7 5 Cellular Proliferation an d Metabolism and mechanical characterization over culture period A) Both the LMP and the non LMP scaffolds show increase in cell number and cellular metaboli sm during the culture period with the LMP scaffold exhibiting greater increases in both, likely due to higher initial cell adhesion because of the laser holes. B) Compressive mechanical testing reveals that the compressive modulus of both the LMP and the non LMP scaffolds at day 1 are comparable to the native disc properties. By day 7 of culture the compressive properties of the LMP scaffold has significantly increased indicating a stiffening of the matrix, however at day 14 and day 21 the mechanical prop erties begin to decrease linearly similarly to the non LMP scaffold (n=9, *, P < 0.05, **, P < 0.01, error bars represent standard deviation).

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148 Figure 7 6 Histological evaluation of culture at day 21. A and B show DAPI/rhodamine phaloidin staining, illustrating that cellular presence within the constructs. B and E show H&E imaging at the same magnification as the florescence images and C and F are higher magnification sections of B and E. A and C show cellular infiltration has spread between the LM P holes and has created a relatively uniform cell population; while C and E show that the non LMP construct has only a dense layer of cells at the construct periphery and F shows no recognizable cells in the interior of the non LMP construct. Sampling was taken from 500m from the superior surface of the samples for comparative consistency.

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149 CHAPTER 8 PROJECT CONCLUSIONS, CLINICAL IMPACT, AND FUTURE DIRECTION Project Conclusions The work presented in this dissertation focuses the development and functio nalization of a xenogeneic derived human cell populated Temporomandibular Joint (TMJ) disc construct Specifically these studies first define key native tissue characteristics necessary to tissue engineering efforts of the morphologically complex TMJ disc then evaluate the effect incorporation of an artificial path of infiltration for cells and nutrients into an acellular porcine derived TMJ disc has on remodeling potential. The major conclusions of the project are as follows: Shear and disc surface struct ure/function properties elucidate the mechanical complexity required of an engineered implant; The decellularized porcine TMJ modified by laser ablation incorporated microporosity retains the biochemical composition and mechanical ability of the native dis c; The laser micro porosity (LMP) can be used to modulate permeability mechanical ability, and cellular integration; MSCs are an appropriate multi potent cell source for TMJ disc tissue engineering; The incorporation of these cells can be computationally modeled to express relative seeding confluency within the LMP scaffold; Viability of MSCs seeded onto the LMP disc scaff old has been established up to 3 weeks in static culture demonstrating improved remodeling potential to compared to the non LMP control ; The seeded disc construct is a viable in vitro model for development of a TMJ disc replacement. Project Clinical Impact The Temporomandibular Joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment

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150 options and debilitating nature of severe Temporomandibular Disorders has been the primary driv ing force for the introduction and development of TMJ disc tissue engineering as an approach to alleviate this priority clinical issue. This projects clinical significance is twofold: 1, to create a viable TMJ disc replacement for those afflicted with sev ere TMDs; 2, to further our understanding of TMJ disc physiology and function in healthy and dysfunctional states. These works generated an in vitro model culture system which regenerates a decellularized porcine TMJ to a physiologically representative s tate including appropriate cell populations and ECM architecture. Project Summary Chapter 3 assessed regional variation in the discs shear loading characteristics under physiologically relevant loads and to associate those mechanical findings to common c linical observations of disc fatigue and damage. Porcine TMJ discs were compressed between an axially translating bottom platen and a 2.5cm diameter indenter within a hydrated testing chamber. Discs were cyclically sheared at 0.5, 1, or 5Hz to 1, 3, or 5% shear strain. Within the anterior and intermediate regions of the disc when sheared in the anteroposterior direction both shear and compressive modulus experienced a significant decrease from instantaneous to steady state; while the pressive modulus decreased approximately 5% no significant loss of shear modulus was noted. All regions retained their shear modulus within 0.5% of instantaneous values when shear was applied in the mediolateral direction. The results nal shear mechanics suggest an observable and predictable link with the common clinical observation that the posterior region of the disc is most often the zone in which fatigue occurs which may leads to disc damage and perforation.

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151 Chapter 4 use ultra nan oindentation to assess tissue zones in conjunction with detailed Transmission Electron Microscopy (TEM) to define structural attributes that influence disc function. These are the first works that demonstrate that the central zone of the TMJ disc is struct ured in isometric depth dependent layers, each of which provide disc architecture adjacent to the superior surface was shown to have three distinct regional segments: 1, a surface interfa ce layer; 2, a subsurface region; and 3, a layer of helical ECM bundles. Each of these layers being responsible for different mechanical characteristics that support joint articulation. In inferior surface displayed an interfaces layer (to 20 um) that sho wed limited cell populations with little depth dependent structural variation. By comparison to the superior surface, the inferior surface has a stiffer elastic modulus and displayed reduced energy dissipation properties. These data indicate that the infe rior surface is responsible more for structural resistance to compression than load distribution during joint motion. These works focus on the fine mechanobiology of the surface layers of the TMJ disc, properties imperative for future tissue engineering ef forts focused on restoring function to the joint. Chapter 5 demonstrated that laser ablation through the thickness of the TMJ disc was an appropriate method to include artificial porosity into the acellular disc. These works describe a novel scaffold prep aration me thod which microporosity has been incorporated into an acellular naturally derived fibrocartilage scaffold to improve permeability and diffusivity without significant alteration of compositional or mechanical ability. These works describe the d evelopment of the acellular temporomandibular joint (TMJ) disc fibrocartilage scaffold and the mechanical consequence of alteration to the

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152 sc affold, after decellularization and after incorporation of the artificial porosity The LMP methodology and result ing mechanical and permeability effects are described for both the whole tissue scaffold and the micro scale alteration to the ECM properties at the periphery of the LMP hole. The study also evaluated how the mechanics could be tuned by controlling the di ameter of the laser micro patterned (LMP) holes. The evaluations indicated that when engineering for biochemical composition, permeability, and mechanical integrity appropriate for generating a suitable TMJ disc replacement the micro ablation pattern is optimal of the patterns investigated The technology developed provides an effective solution to diffusive limitations of decellularized scaffolds and of large metabolite (nutrient and wastes) mass transport during early remodeling. Overall th ese results support the potential of a porcine derived acellular engineered laser ablated microporosity scaffold as a xenograft for TMJ disc reconstruction. Chapter 6 are an appropriate cell source for TMJ disc tissue engineering. Chapter 6 also described, evaluated, and computationally optimized a novel seeding methodology to populate the sample uniformly by hydration mechanisms. Computational results demonstrated that shear moment forces at the LMP holes periphery cause cell membrane rupture and ultimate cell death. To eliminate this problem we used the computational model to solve for a known shear moment (which does not result in cell membrane rupture) varying the had to be increased for the system to present a solution. Experimental evaluation

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153 results agreed with out computational model for each study conducted. The final finding of these works was the hydration seeding method created a viable uniform cell population throughout the LMP construct. Chapter 7 aimed to evaluate the efficacy of laser micro patterning (LMP) ex vivo derived TMJ disc scaffolds to enhance cellular integration, a major limitation to the development of whole tissue implant technology. LMP was incorporated into the scaffold structure using a 40W CO 2 laser ablation system to drill a n 8x16 pattern with a bore diame human neonatal derived mesenchymal stem cells (MSC) differentiated into chondrocytes at a density of 900 cells/mm 2 then assessed on days 1, 7, 14 and 21 of culture. Results derived from histology, PicoGreen DNA quantification, and cellar metabolism assays indicate the LMP scaffolds improve cellular remodeling compared to the unworked scaffold over the 21 day culture period. Mechanical analysis further supports the use of the LMP sho wing the compressive properties of the LMP constructs closely represent native disc mechanics. The addition of an artificial path of infiltration by laser micro patterning culminated in improved chondrocyte adhesion, dispersion and migration after extende d culture aiding in recapitulating the native TMJ disc characteristics. Project Future Directions Aim 1: It has been demonstrated that c ompressive mechanical stimulation encourages chondrocyte matrix secretion from the cultured MSCs Figure 8 1 describes several experiments utilizing mechanical stimulation to functionalize and regulate remodeling of the cultured TMJ disc construct. Preliminary investigations of continuous compression mechanical stimulation for 5 weeks culture reveal matrix

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154 secretion is up regulated in comparison to the static culture. But like in the static culture the remodeling is unorganized at the LMP hole periphery; Inclusion of a tensile modality in the compressive mechanical stimulation retains the chondrocyte matrix secretion f rom the compression stimulation study but also directs remodeling to an ECM architecture representative of the native disc At 5 weeks culture under tensile/compressive mechanical stimulation secretion from the cultured MSCs has closed the LMP holes and t he remodeled matrix possesses a radial fibril alignment characteristic of the functional TMJ disc. Future direction for study utilizing the mechanical stimulation systems include simulation of disease conditions including hyper mechanical environment homol og of Bruxism A commonly presented temporomandibular disorder. Aim 2 : Evaluate the effects of controlled biochemical and biomechanical culture environments on human MSC incorporated within a laser drilled acellular TMJ disc scaffold. Cellular migration and remodeling activity are critical elements of the wound healing process. The goal of this aim is to define environmental conditions that promote a functional fibrochondrocyte phenotype, with a goal to drive the constructs mechanical and bioche mical properties toward native characteristics. We will assess how control of nutrient and gas gradients as well as mechanical loading modulates cellular and whole scaffold function. Aim 3 : To incorporate a region specific co culture of fibrochondrocytes and vascular support cells to assess in vitro conditions that enhance natural disc morphology and potential (physiological) integration. While the central zone of the healthy TMJ disc is predominantly avascular, the medial and posterior regions are

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15 5 connec ted to highly vascularized connective tissues that are the primary source of diffused nutrients. We hypothesize that by incorporating vascular cells into the disc after implantation by initiating prevascularization. We will also test the hypothesis that the presence of chondrocytes and/or compressive loading inhibits vascular cell invasion using discrete and localized co culture systems. Aim 4 : Test the hypothesis that details of the chemical and fluid mechanical environment are critical to ensure fibrochondrocyte cells adopt a phenotype that reduces disease potential by minimizing mineral deposition The mechanism of TMJ calcification by chondrocytic cells has been li nked to excessive (or prolonged) mechanical loading, theoretically linking TMJ calcification and bruxing (jaw clenching) a joint loading experienced by many TMD patients. We will quantify and replicate the excessive loading conditions associated with bru xism to develop a disease model as described in Aim 1, which will further our understanding of cartilage remodeling and regeneration and importantly, can be extrapolated toward the development of conditional and pharmaceutical treatments. The environ mental considerations from Aim 2 can be used to evaluate other scaffolding biomaterials. Aim 3 posits vascularization and inclusion of support cells to expedite graft integration into the surrounding joint anatomy future works will assess innervation in c oncert with vascularization TMDs are often only diagnosed because of presenting pain due to the sensory nerves that infiltrate the joint and the disc periphery. Evaluating nerve ingrowth and dysfunction in our bioreactors will aid in the development of t reatment modalities beyond the scope of whole tissue grafting. The disease model

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156 can be expanded to include internal derangement, bone cancers, or organic degeneration of the articular surfaces or any number of other conditions thought to cause TMD. Lastl y, all data generated will be used to develop evaluation parameters for pre clinical stud ies We have approached the notion of pre clinical work and have found, through our own work with animal model studies and through publication of animal TMJ disc studi es, that without the proper assessment questions little useful data would be generated beyond success/failure of the graft. These works will provide testing criteria and a true scale of successes during the implantation period

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157 Figure 8 1. Experimen tal design and preliminary results comparing cyclic compression and combined cyclic tension/ compression mechanical stimulation to promote wound healing mechanisms of the MSCs and functionalize the LMP TMJ disc construct. The schematic drawings illustrate t he mechanism of loading for each culture condition while the assemblies below pictorially describe the culture plate systems. Resulting scaffold florescence imaging at 5 weeks in culture are shown demonstrating that the static culture has little matrix re modeling but retains the cell population. The cyclic compression mechanical stimulation results in mass remodeling of the matrix; however the remodeling is unorganized and the LMP holes have not closed. The cyclic radial tension and compression mechanica lly stimulated samples showed significant remodeling and organized fibril alignment radiating from the LMP hole which at 5 weeks stimulation has nearly filled with newly laid matr i x.

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169 BIOGRAPHICAL SKETCH Cassandra Marie Juran began her academic studies in aerospace e ngineering, focusing on control systems engineering, at the University of Florida. She graduated Cum Laude in May 2008 with dual b in mechanical and aerospace e ngineering and a minor in b iomechanics from the University of Florida (Gainesville, FL ). After undergraduate laboratory work in the field she chose to change disciplines to biomedical e ngineering, utilizing her undergraduate training and specializing in biomechanics. She receiv ed a Master of Engineering in biomedical e ngineering in May 20 10 and late that year began doctoral research under the guidance of Dr. Peter S. McFetridge at the J. Crayton Pruitt Family Department of Biomedical Engineering at the University of Florida. Her first task was to aid the newly hired Dr. McFetridge and his doctoral student s in establishing the Tissue Engineering and Regenerative Medicine Laboratory in the Biomedical Sciences Building. Cassandra graduated with a Doctor of Philosophy in biomedi cal engineering in May 2014 Her dissertation is focused on the g eneration of a naturally derived Temporomandibular Joint disc graft, mechanical pre conditioning strategies for improved cellular mechancotransduction and early remodeling of the graft, and in vitro modeling of disease conditions which degenerate the nativ e TMJ disc.



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http://jdr.sagepub.com/Journal of Dental Research http://jdr.sagepub.com/content/92/2/193 The online version of this article can be found at: DOI: 10.1177/0022034512468749 2013 92: 193 originally published online 19 November 2012 J DENT RES C.M. Juran, M.F. Dolwick and P.S. McFetridgeShear Mechanics of the TMJ Disc : Relationship to Common Clinical Observations Published by: http://www.sagepublications.com On behalf of: International and American Associations for Dental Research can be found at: Journal of Dental Research Additional services and information for http://jdr.sagepub.com/cgi/alerts Email Alerts: http://jdr.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? Nov 19, 2012 OnlineFirst Version of Record Jan 15, 2013 Version of Record >> at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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193 DOI: 10.1177/0022034512468749 Received May 8, 2012; Last revision October 19, 2012; Accepted October 23, 2012 A supplemental appendix to this article is published elec tronically only at http://jdr.sagepub.com/supplemental. International & American Associations for Dental ResearchC.M. Juran, M.F. Dolwick, and P.S. McFetridge*J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, JG-56, Biomedical Sciences Building, Gainesville, FL 32611-6131, USA; *cor responding author, pmcfetridge@bme.ufl.edu J Dent Res 92(2):193-198, 2013ABSTRACTThe temporomandibular joint (TMJ) is a complex hinge and gliding joint that induces significant shear loads onto the fibrocartilage TMJ disc during jaw motion. The purpose of this study was to assess regional variation in the discs shear loading characteristics under physiologically relevant loads and to associate those mechanical findings with common clinical observations of disc fatigue and damage. Porcine TMJ discs were compressed between an axially translating bottom platen and a 2.5-cm-diameter indenter within a hydrated testing chamber. Discs were cyclically sheared at 0.5, 1, or 5 Hz to 1, 3, or 5% shear strain. Within the anterior and intermediate regions of the disc when sheared in the anteroposterior direction, both shear and compressive moduli experienced a significant decrease from instantaneous to steady state, while the posterior regions compressive modulus decreased approximately 5%, and no significant loss of shear modulus was noted. All regions retained their shear modulus within 0.5% of instantaneous values when shear was applied in the mediolateral direction. The results of the discs regional shear mechanics suggest an observable and predictable link with the common clinical observation that the posterior region of the disc is most often the zone in which fatigue occurs, which may lead to disc damage and perforation.KEY WORDS: tissue engineering, temporomandibular disc, joint disease, jaw biomechanics, extracellular matrix (ECM), biomechanics.INTRODUCTIONThe temporomandibular joint (TMJ) regulates movements of the mandible with respect to the temporal bone of the skull. The fibrocartilagenous TMJ disc has, in the past decade, been the subject of more extensive mechanical evaluations. The majority of published biomechanical analyses have focused on the TMJ discs compressive properties (Tanaka et al., 2003a,e; Allen and Athanasiou, 2005; Lumpkins and McFetridge, 2009) and have found that the disc possesses significant regional variations (Lumpkins and McFetridge, 2009; Kuo et al., 2010). While compressive properties are important during joint loading, the TMJ has primarily a gliding joint function that induces com pressive as well as significant shear forces. Previous studies have evaluated shear under static shear loading conditions and have shown regional variations of the TMJ disc properties (Lai et al., 1998). Current investigations of the TMJ under dynamic shear conditions have focused on the central region of the discs intermediate zone (Tanaka et al., 2003b, 2004; Koolstra et al., 2007). The relationship between repetitive shear and compressive loading of the intervertebral disc and disc damage has been alluded to by Callaghan and Iatridis and colleagues, who suggested that disc herniation and damage may be more linked to repeated flexion extension and shear motions than to applied joint compression (Callaghan and McGill, 2001; Iatridis and ap Gwynn, 2004). In these investigations, the regional shear and compressive characteristics of the porcine TMJ disc and the interconnectivity and dependency of these characteristics on one another and variables relevant to physiologic function have been evaluated. We hypothesized that not only is the shear modulus (G) of the TMJ disc dependent on frequency, shear, and compressive strain, but also that the compressive modulus of elasticity (E) is dependent on the appli cation of cyclic shear loads (Lumpkins et al., 2008). Additionally, we hypoth esized that the interdependence of the compressive and shear elastic moduli will reveal material trends consistent with damage seen clinically in patients with temporomandibular disorders.MATERIALS & METHODSSpecimen PreparationPartially dissected fresh porcine TMJs were isolated from male animals ages 6 to 9 mos, purchased with IACUC approval (IACUC Protocol # 201207534) from Animal Technologies Inc. (Tyler, TX, USA). Dissection was conducted as previously described (Lumpkins et al., 2008; Appendix 1). After dissection, discs were stored in 0.15 M phosphate-buffered saline (PBS, pH 7.4) at 4C until use. All samples were stored for fewer than 12 hrs before being tested (Lumpkins et al., 2008). Immediately before mechanical testing, a circular stainless steel punch with a 6-mm inner diameter was used to extract Shear Mechanics of the TMJ Disc: Relationship to Common Clinical Observations at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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194 Juran et al. J Dent Res 92(2) 20133 samples from each disc: anterior, intermediate, and posterior samples (Fig. 1B). Anterior-to-posterior (A-P) and medial-tolateral (M-L) directions were labeled by means of a waterresistant marker on each disc sample (Fig. 1B, insert), to orient the sample correctly for testing within the hydrated testing chamber.Sample GroupsThe TMJ discs were divided into 3 testing groupsanterior, intermediate, and posterioreach undergoing 27 testing proce dures [frequency (F) variation (0.5 Hz, 1 Hz, and 5 Hz), com pressive strain () variation (5%, 10%, and 15%), and shear strain () variation (1%, 3%, and 5%)], with 9 specimens for each testing procedure. The order of the testing procedure was randomized for each sample group to minimize sample error (see Appendix 7, Appendix Fig. 3).Biomechanical Testing Cyclic Shear LoadingA Biomomentum Mach1 Micromechanical System (Biomomentum Inc., Laval, Quebec, Canada) was used for all mechanical testing. The Mach1 is a multi-axial modular mechanical testing rig which can measure or impart compressive and shear loading. The testing chamber was filled with 0.15 M PBS (pH 7.4) to maintain hydra tion, similar to the in vivo environment (Piette, 1993) and to maintain consistency with previous studies on TMJ tissue mechanics (Tanaka et al., 2003c; Allen and Athanasiou, 2006). An axially translating bottom plate and an axially stationary vertically translating 2.5-cm-diameter indenter were used to generate the shear and compressive loads. Prior to being tested, samples were allowed to equilibrate (unloaded) in PBS for 5 min at 37 1C. Sample height was determined by a pre-programmed function of the Mach1 called find contact (see Appendix 2). With the strain profile illustrated in Fig. 1C, compressive and shear strain defor mations (indenter displacement, L position; and axially translat ing bottom plate, x position) were applied to the tissue and assessed according to the calculations = L / L0 and = x / L0, where L is the decrease in sample thickness and x is the axial displacement in the shear direction. Both parameters relative to the initial thickness L0 were needed to produce the desired strain range (see Appendix 6). The Mach1 measures resulting force (FX and FZ) experience by the indenter during the compressive and shear load ing. The resulting compressive stress on each sample is defined by = FZ/A, where FZ is the compressive force and A is the crosssectional area of the sample, and the resulting shear stress is = Fx/A, where FX is the resulting axial force and A is again the cross-sectional area of the sample (Lumpkins et al., 2008). Samples were sheared under sinusoidal strain defined by = sin(t), where and are the respective strain amplitude and frequency, and t is time. We analyzed results of the cyclic loading tests by calculating the hysteresis, peak stresses, and the instantaneous and steady-state compressive (Eint,Ess) and shear moduli (Gint,Gss). The general calculations for the compressive and shear moduli are E = / and G = / respectively.Statistical AnalysisEach set of test group data was calculated based on the testing of 9 samples (n = 9). We calculated standard deviations and used one-way analysis of variance (ANOVA) testing to determine statistical significance between and among test groups for bio mechanics. Significance was established by the Tukey-Kramer test (p = 0.05, n = 9). Figure 1. Joint anatomy and simplified free-body diagram, regional sampling, and testing method. (A) The gross anatomy of the TMJ and the placement of the disc within the glenoid fossa. The insert illustrates the position of the TMJ disc when the mandibular condyle slides forward with respect to the articular eminence when the jaw opens. (B) The sampling regions of the TMJ disc and the directional notation used to orient the sample properly for testing (gray line, anteroposterior direction; black line, mediolateral direction). A representative testing strain profile is shown in (C) (description in text body), and (D) depicts the sequential testing procedure used to generate the strain profile of (C) (dashed arrows indicate active loading, and solid arrows indicate sustained loading). at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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J Dent Res 92(2) 2013 Shear Mechanics of the TMJ Disc Relative to Clinical Observations 195 RESULTSThe average thicknesses reported for the tested regions were 3.66 0.24, 2.00 0.29, and 3.88 1.45 mm for the anterior, intermediate, and posterior regions, respectively. Shear modulus was evaluated for the 1st strain cycle ( Gint) and for the 10th strain cycle ( Gss) to show the changes exerted by frequency and compressive and shear strain on the material properties over the load application (Fig. 2). When shear loads were displaced in the A-P direction, the stiffness of all regions decreased from Gint to Gss, with the most noteworthy change occurring with fre quency increase (Fig. 2A). Change in Gint, evaluated solely as a function of frequency increase from 0.5 Hz to 5 Hz, was, on average, an 8.6% increase for all and conditions evaluated. When compressive strain was increased and the other test condi tions were maintained, the disc experienced a similar increase in shear modulus (Fig. 2B); however, when the shear strain was increased, the shear modulus decreased, becoming more elastic (Fig. 2C). Most noticeably, for the posterior region tested at the high frequency (5 Hz) and high compressive strain (15%) (data not represented in Fig. 2C), Gint = 1.556 0.327 MPa and Gss = 0.797 0.315 MPa at = 1% and Gint = 1.078 0.158MPa and Gss = 0.686 0.072MPa at = 5%, an average decrease of 22.5% from the instantaneous to the steady-state condition. Similar to the shear modulus change with (Fig. 2C), the compressive modulus also became more elastic with repeated shear in all regions of the disc (Fig. 3). The cycle-dependent compressive modulus of the posterior region shows that, with each shear cycle application, the compressive modulus increased by nearly 5% of the Eint value before continuing the decreasing trend. The average magnitude of the E (peak height) when sheared in the A-P direction was 1.5 0.0 kPa, and in the M-L direction was 0.40 0.03 kPa in the posterior region, and became more regular as the compressive modulus reached a steady-state condition (cycles 6 through 10). This phenomenon was also seen in the anterior region, but only when shear strain was applied in the M-L direction, and the magnitude was less than 25% of the posterior regions E (table in Fig. 3). The shear modulus characteristics are described in Fig. 4. Only the posterior region statistically maintained its shear modulus from Gint to Gss when sheared in the A-P direction. All regions when sheared in the M-L direction maintained within 20% of their Gint stiffness over the strain application cycle.DISCUSSIONThe ability of the TMJ disc to act as a buffer between the articu lating skeletal structures of the TMJ is fundamental to jaw movement; thus, it is paramount to understand the mechanical response of a healthy TMJ disc when exposed to loading similar to that experienced in vivo. The current investigation focused on the mechanical characteristics of the disc during cyclic shear Figure 2. Changes in shear modulus with frequency compressive strain, and shear strain. Mean values of the instantaneous (1) and steady-state (2) shear moduli as a function of frequency, with = 1% and = 10% (A), compressive displacement, F = 1 Hz, and = 1% (B), or shear displacement, = 10% and F = 1 Hz (C). Samples were shear-strained in the anterior to posterior direction for all data presented. Increases in frequency and compressive strain caused shear stiffening, and the application of greater shear strain induced the shear modulus to become more elastic in the anterior and intermediate regions of the disc. Error bars represent standard deviations. at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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196 Juran et al. J Dent Res 92(2) 2013testing, when the magnitude of shear strain (), compressive strain (), and frequency (F) are varied under broadly physiolog ical parameters (see Appendix 5). Previous investigations by Tanaka and co-workers have shown that the disc experiences shear stiffening, or increase in elastic modulus, as or F is increased, and, conversely, shear softening with increasing (Tanaka et al., 2003b, 2004). Fig. 2 depicts the dependency of the disc regions on frequency, com pressive strain, and shear strain. Shear stiffening was hypothe sized to be a combination of 2 effects: the first due to interstitial fluid or bulk matrix outflow as pressurized fluids are squeezed from the point of strain application; and the second due to the frequency of application being increased, which results in a lag in fluid resorption between the successive strain applica tions. These conclusions were further supported by our results and can be expanded to be a universal trend for the regions tested. The shear softening with increased shear strain, however, was observed only in the anterior and intermediate regions of the disc. It has previously been hypothesized that observed shear softening is due to the proteoglycan [glycosaminoglycan (GAG)] component and water matrix within the disc possessing non-Newtonian characteristics. GAGs are highly hydrophilic sugar chains that act to maintain the discs resistance to pres sure. The lack of significant shear softening in the posterior region, then, is in agreement with this hypothesis, since the posterior band of the disc contains a lower GAG content than the anterior or intermediate zones (Nakano and Scott, 1996; Almarza et al., 2006) (Fig. 4). A difference between our experi mental design and that of Tanaka et al. is that the latters work evaluated the shear properties of the porcine disc at porcine core temperature (39C), and our evaluation was done at human body temperature. This temperature difference may account for the Figure 4. Mean regional values of the instantaneous and steady-state shear moduli of each disc region when strained in the anterior-toposterior direction and the medial-to-lateral direction. The anterior and central regions experienced a significant decrease in shear modulus when strained in the anterior-posterior (A-P) direction. This increased elasticity was not seen in the posterior region (* p < 0.05; ** p < 0.01). Figure 3. Changes in compressive modulus when cyclic shear strain was applied in the anterior-to-posterior or medial-to-lateral direction. Representative zonal compressive modulus evolutions over the load application are shown in (A), (B), and (C). Mean values of the compressive modulus of each region of the TMJ disc are shown in the table. The anterior and intermediate regions had less reduction of their compressive modulus, retaining their compressive modulus even when repeatedly sheared. The posterior region became more elastic with application of shear strain. All regions had significant reduction in initial to steady-state modulus when sheared in the medial-lateral direction ( p < 0.05). Anteriorposterior shear strain application data are represented by the black curves, and medial-lateral shear strain application data are represented by the gray curves. at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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J Dent Res 92(2) 2013 Shear Mechanics of the TMJ Disc Relative to Clinical Observations 197 variances in our mechanical results, since higher temperatures reduce stiffness and strength of the disc, since many ECM com ponents (collagen and proteoglycans) are temperature-sensitive (Detamore and Athanasiou, 2003). Application of cyclic shear in the A-P direction decreased the compressive modulus until a steady state was reached; however, in the posterior region, significant bumps were observed in the mechanical profile (Fig. 3C). We hypothesized that the increase in compressive stiffness associated with these bumps represents a material property that acts as a breaking system (force). Compression causes outflow of interstitial fluid or a shifting of the bulk matrix from the point of strain application. As the fluid matrix shifts, the periphery swells to maintain the tissue volume and causes increased hoop stresses and hydraulic pressures that inhibit the force dispersion and act to recoil the fibers back toward their undistorted orientation. Compressive properties of the disc are strongly dependent on interstitial fluid flow (Allen and Athanasiou, 2006), and with fewer paths for tissue fluid to escape when the disc is under shear, E will respond in intermit tent increased stiffness. A possible limitation of these investiga tions is that we evaluated disc punches that did not necessarily retain the boundary conditions of the whole disc; however, the ECM fiber alignment superstructure remained intact. This trend was observed only in the anterior and posterior zones, likely due to fiber arrangement, where collagen fibers were oriented in a mediolateral arc that increased hoop stresses and hydrostatic forces (Detamore et al., 2005; Almarza and Athanasiou, 2006). By comparison, the intermediate region, where the collagen fibers were aligned in the antero-posterior direction, was shown to culminate in channels that shuttled interstitial fluid quickly within the region. The mechanical consequence is that the inter mediate zone was more elastic than the periphery of the disc (Lumpkins and McFetridge, 2009). Perforation is often associated with disc derangement or osteoarthritis; however, tearing of the posterior-lateral region is also seen in asymptomatic discs (Kuribayashi et al., 2008; Liu et al., 2010). The results of the current study showed that the steady-state shear modulus was stiffest in the discs posterior region, indicating that this region is different structurally and/or in composition. These mechanical results are in agreement with clinical observations of regional disc damage. It is clinically accepted that TMJ disc fatigue occurs most frequently in the discs lateral-posterior region (Kuribayashi et al., 2008). We hypothesized that this retained stiffness with repeated shear strain applications limits the discs ability to distribute (disseminate) loads away from the impact point, leading to greater residual localized stress summation. It was further hypothesized that these localized stresses result in material fatigue and even tual failure, seen clinically as disc perforations. Shear strain in the M-L direction is less prominent in healthy TMJ disc loading (Fushima et al., 1995; Athanasiou et al ., 2009). However, shear strain is associated with the nonpathological tooth-grinding and jaw-clenching that are seen in bruxism, which can potentially lead to TMDs (Kuboki et al., 1996; Nagahara et al., 1999; Gallo et al., 2000). Sometimes simplified as hyperactivity of the lateral pterygoid, clenching and bruxing mechanics are more completely described as para functional activity of 1 or more of the major masticatory muscles (masseter, medial pterygoid, temporalis, and lateral pterygoid) (Nagahara et al., 1999; Hirose et al., 2006). The mechanical testing of the discs elastic moduli when sheared in the M-L direction under high compressive load is meaningful for the assessment of any potential relationship between bruxing and TMJ disc perforation. Similar to the shear modulus characteris tics of the posterior region under A-P shear, all regions of the disc have an observable trend of retaining their shear modulus from Gint to Gss when load is applied with M-L loading (Fig. 4). In conclusion, these results confirm that the mechanical char acteristics of the TMJ disc are highly dependent on the ECM microenvironment and its regional composition. The posterior region of the disc, which is the most commonly observed zone in which the disc shows fatigue, has been shown to maintain its stiffness when compressed or sheared cyclically. While there is no direct association between theoretical or experimental mod els and the clinical experience, these results are in agreement with mathematical modeling results showing that large stresses developed in the posterior region of the disc and retrodiscal tissue during prolonged clenching, and higher still in these regions when antero-lateral internal derangement is included (Hirose et al., 2006; Nishio et al., 2009). The hypothesis that there is a relationship between the discs regional mechanical properties and common clinical observations of TMJ disc damage is sup ported by the data collected in these works. These results sup port further investigation of fluid movement within the disc scaffold and greater development of a physiologically represen tative testing regime.ACKNOWLEDGMENTSWe gratefully acknowledge the National Institute of Dental and Craniofacial Research at the US National Institutes of Health (NIH; 1R21DE022449) and the J. Crayton Pruitt Family Department of Biomedical Engineering at the University of Florida for funding. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. en-GBREFERENCESAllen K, Athanasiou K (2005). A surface-regional and freeze-thaw charac terization of the porcine temporomandibular joint disc. Ann Biomed Eng 33:951-962. Allen K, Athanasiou K (2006). Viscoelastic characterization of the porcine temporomandibular joint disc under unconfined compression. J Biomech 39:312-322. Almarza A, Athanasiou K (2006). Effects of hydrostatic pressure on TMJ disc cells. Tissue Eng 12:1285-1294. Almarza A, Bean A, Baggett L, Athanasiou K (2006). Biochemical analysis of the porcine temporomandibular joint disc. Br J Oral Maxillofac Surg 44:124-128. Athanasiou KA, Almarza AA, Detamore MS, Kalpakci KN (2009). Tissue engineering of temporomandibular joint cartilage. San Rafael, CA: Morgan & Claypool Publishers. Callaghan J, McGill S (2001). Intervertebral disc herniation: studies on a porcine model exposed to highly repetitive flexion/extension motion with compressive force. Clin Biomech (Bristol, Avon) 16:28-37. Detamore M, Athanasiou K (2003). Tensile properties of the porcine tem poromandibular joint disc. J Biomech Eng 125:558-565. Detamore M, Orfanos J, Almarza A, French M, Wong M, Athanasiou K (2005). Quantitative analysis and comparative regional investigation of at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research

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198 Juran et al. J Dent Res 92(2) 2013the extracellular matrix of the porcine temporomandibular joint disc. Matrix Biol 24:45-57. Fushima K, Gallo L, Krebs M, Palla S (1995). Medial and lateral TMJ space variations during mastication. J Dent Res 74(Spec Iss):588, abstract #1504. Gallo L, Nickel J, Iwasaki L, Palla S (2000). Stress-field translation in the healthy human temporomandibular joint. J Dent Res 79:1740-1746. Hirose M, Tanaka E, Tanaka M, Fujita R, Kuroda Y, Yamano E, et al. (2006). Three-dimensional finite-element model of the human temporoman dibular joint disc during prolonged clenching. Eur J Oral Sci 114:441448. Iatridis J, ap Gwynn I (2004). Mechanisms for mechanical damage in the intervertebral disc annulus fibrosus. J Biomech 37:1165-1175. Koolstra JH, Tanaka E, Van Eijden TM (2007). Viscoelastic material model for the temporomandibular joint disc derived from dynamic shear tests or strain-relaxation tests. J Biomech 40:2330-2334. Kuboki T, Azuma Y, Orsini M, Takenami Y, Yamashita A (1996). Effects of sustained unilateral molar clenching on the temporomandibular joint space. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 82:616624. Kuo J, Zhang L, Bacro T, Yao H (2010). The region-dependent biphasic viscoelastic properties of human temporomandibular joint discs under confined compression. J Biomech 43:1316-1321. Kuribayashi A, Okochi K, Kobayashi K, Kurabayashi T (2008). MRI find ings of temporomandibular joints with disk perforation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 106:419-425. Lai W, Bowley J, Burch J (1998). Evaluation of shear stress of the human temporomandibular joint disc. J Orofac Pain 12:153-159. Liu XM, Zhang SY, Yang C, Chen MJ, Y Cai X, Haddad MS, et al. (2010). Correlation between disc displacements and locations of disc perfora tion in the temporomandibular joint. Dentomaxillofac Radiol 39:149156. Lumpkins S, McFetridge P (2009). Regional variations in the viscoelas tic compressive properties of the temporomandibular joint disc and implications toward tissue engineering. J Biomed Mater Res A 90:784-791. Lumpkins S, Pierre N, McFetridge P (2008). A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc. Acta Biomater 4:808-816. Nagahara K, Murata S, Nakamura S, Tsuchiya T (1999). Displacement and stress distribution in the temporomandibular joint during clenching. Angle Orthod 69:372-379. Nakano T, Scott P (1996). Changes in the chemical composition of the bovine temporomandibular joint disc with age. Arch Oral Biol 41:845853. Nishio C, Tanimoto K, Hirose M, Horiuchi S, Kuroda S, Tanne K, et al. (2009). Stress analysis in the mandibular condyle during prolonged clenching: a theoretical approach with the finite element method. Proc Inst Mech Eng H 223:739-748. Piette E (1993). Anatomy of the human temporomandibular joint. An updated comprehensive review. Acta Stomatol Belg 90:103-127. Tanaka E, del Pozo R, Tanaka M, Aoyama J, Hanaoka K, Nakajima A, et al. (2003a). Strain-rate effect on the biomechanical response of bovine temporomandibular joint disk under compression. J Biomed Mater Res A 67:761-765. Tanaka E, Hanaoka K, van Eijden T, Tanaka M, Watanabe M, Nishi M, et al. (2003b). Dynamic shear properties of the temporomandibular joint disc. J Dent Res 82:228-231. Tanaka E, Kawai N, van Eijden T, Watanabe M, Hanaoka K, Nishi M, et al. (2003c). Impulsive compression influences the viscous behavior of porcine temporomandibular joint disc. Eur J Oral Sci 111:353-358. Tanaka E, Kikuzaki M, Hanaoka K, Tanaka M, Sasaki A, Kawai N, et al. (2003d). Dynamic compressive properties of porcine temporomandibular joint disc. EurJ Oral Sci 111:434-439. Tanaka E, Kawai N, Hanaoka K, van Eijden T, Sasaki A, Aoyama J, et al. (2004). Shear properties of the temporomandibular joint disc in relation to compressive and shear strain. J Dent Res 83:476-479. at UNIV OF FLORIDA on February 13, 2013 For personal use only. No other uses without permission. jdr.sagepub.com Downloaded from International & American Associations for Dental Research