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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2013-04-30.

Permanent Link: http://ufdc.ufl.edu/UFE0042816/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2013-04-30.
Physical Description: Book
Language: english
Creator: DOUGHTIE,CAMDEN BENTON
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by CAMDEN BENTON DOUGHTIE.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Morris-Wiman, Joyce.
Electronic Access: INACCESSIBLE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042816:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042816/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2013-04-30.
Physical Description: Book
Language: english
Creator: DOUGHTIE,CAMDEN BENTON
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by CAMDEN BENTON DOUGHTIE.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Morris-Wiman, Joyce.
Electronic Access: INACCESSIBLE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042816:00001


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1 DO INFLAMMATORY CYTOKINES DIFFERENTIALLY AFFECT MASSETER AND TIBIALIS ANTERIOR MUSCLE REGENERATION? By CAMDEN BENTON DOUGHTIE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Camden Benton Doughtie

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3 To my parents for all of their support through out my education and to my A unt Gail for her intelle ctual curiosity and inspiration

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4 ACKNOWLEDGMENTS I thank my committee Dr s Joyce Morris Wiman, Charles Widmer and Calogero Dolce for their incredibl e guidance and patience and Andrew Brown for his endless support. I also thank the Southern Association of Orthodontists for their financial support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Muscle Regeneration: Satellite Cell ................................ ................................ ........ 14 Differential Regeneration of Muscle ................................ ................................ ........ 15 Acute Inflammation: A Response to Muscle Injury ................................ .................. 17 Summary ................................ ................................ ................................ ................ 20 Significance ................................ ................................ ................................ ............ 21 Hypotheses ................................ ................................ ................................ ............. 22 Specific Aims ................................ ................................ ................................ .......... 22 2 MATERIALS AND METHODS ................................ ................................ ................ 23 Overview of Experimental Design ................................ ................................ ........... 23 Myofiber Isolation ................................ ................................ ................................ .... 23 Myofiber Culture ................................ ................................ ................................ ..... 24 Immunofluorescen t Staining/ Evaluation ................................ ................................ 25 Counting and Classification of Cells ................................ ................................ ........ 25 Power Sample Size Estimation ................................ ................................ ............... 26 Statistical Analyses ................................ ................................ ................................ 26 3 RESULTS ................................ ................................ ................................ ............... 32 Do Differe nces in Cell Proportions Exist b etween MAS and TA Control Cultures? ................................ ................................ ................................ ............. 32 Do Differences in Cell Proportions Exist b etween MAS and TA Proliferation Control Cultures and Cytokine Exposed C ultures? ................................ ............. 33 Do Dif fere nces in Cell Proportions Exist b etween MAS and TA Differentiation Control Cultures and Cytokine Exposed Cultures? ................................ ............. 34 Do Differe nces Exist in the Effects of Cytokine Exposure on Cell Prolif eration and Myofiber Formation b etween MAS and TA Cultures? ................................ ... 35 4 DISCU SSION ................................ ................................ ................................ ......... 46

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6 Differences in the i n v itro Proliferative and Differentiation Potential between Presumptive Myogenic Stem Cells Isolated from MAS and TA Myofibers ........... 46 Differences in the Response to Cytokine Exposure in vitr o between Presumptive Myogenic Stem Cells Isol ated from MAS and TA Myofibers ........... 48 TNF ................................ ................................ ................................ ............... 48 IL ................................ ................................ ................................ ................. 50 IL 4 ................................ ................................ ................................ ................... 51 Differential Effects of Cyto kine on Presumptive Myogenic Stem Cells Derived from MAS and TA ................................ ................................ ................................ 53 Conclusion ................................ ................................ ................................ .............. 54 LIST OF REFERENCES ................................ ................................ ............................... 55 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 59

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7 LIST OF TABLES Table page 3 1 ANOV A: MAS and TA controls, early cultures ................................ .................... 36 3 2 ANOVA: MAS and TA controls, late cultures ................................ ...................... 37 3 3 ANOVA: E arly MAS Controls vs. treated with cytokines ................................ ..... 38 3 4 ANOVA: E arly TA contro ls vs. treated with cytokines ................................ ......... 3 8 3 5 ANOVA: L ate controls vs. treated with cytokines ................................ ................ 40 3 6 ANOVA: E arly MAS vs. TA treated with cytokines and normalized to means of controls ................................ ................................ ................................ ........... 42 3 7 ANOVA: L ate MAS vs. TA treated with cytokines and normalized to means of controls ................................ ................................ ................................ ............... 44

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8 LIST OF FIGURES Figure page 2 1 Visual representation of the stud y design and variables examined ................... 28 2 2 Visual representation of layout of each 24 well plate ................................ .......... 29 2 3 Cell phenotype categories for early cultures. ................................ ...................... 30 2 4 Cell phenotype categories for late cultures ................................ ....................... 31 3 1 Photomicrographs of MAS and TA proliferation (early) cultures and gra ph of cell phenotypes ................................ ................................ ................................ .. 36 3 2 Photomicrographs of MAS and TA d ifferentiation (late) cultures and graph of cell phenotypes ................................ ................................ ................................ .. 37 3 3 Graphs illustrating differences in controls vs. treated prolifer ation (early) cultures exposed to cytokines ................................ ................................ ............ 39 3 4 Graphs illustrating differences in controls vs. treated differentiation (late) cultures exposed to cytokines ................................ ................................ ............. 41 3 5 Graphs illustrating differences in MAS and TA proliferation (early) cultures exposed to cytoki nes ................................ ................................ .......................... 43 3 6 Graphs illustrating differences in MAS and TA differentiation (late) cultures exposed to cytokines ................................ ................................ .......................... 45

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9 LIST OF ABBREVIATION S AEC 3 amino 9 ethyl carbazole BrdU Bromodeoxyuridine JUN c Jun N terminal k inase DMEM Dulbecco's modified eagle's m edium EDL Extensor digitorum longus muscle EMyHc Embryonic myosin heavy chain HRP Horserad ish peroxidase IGF Insulin like growth factor IL Interleukin one beta IL 4 Interleukin four IL 6 Interleukin six MAS Masseter muscle MRF M yogenic regulatory factor MRF4 M yogenic regulatory factor fou r M yoD M yogenic determination factor one Myf5 M yogenic factor five N F Nuclear factor k appa B p38MAPK p38 mitogen activated protein kinase Pax7 Paired box protein seven PBS P hosphate buffered solution PCR Polymerase chain reaction SOL Soleus muscle TA Tibialis anterior muscle TBS Tris phosphate buffered solution

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10 TMD Temporomandibular disorders TNF Tumor necrosis factor alpha TNFR1 Tumor n ecrosis factor receptor one TNFR2 T umor necrosis factor receptor two

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DO INFLAMMATORY CYTOKINES DIFFERENTIALLY AFFECT MASSETER AND TIBIALIS ANTERIOR MUSCLE REGENE R A TION? By Camden Benton Doughtie May 2011 Chair: Joyce Morris Wiman Major: Dental Sciences Masseter ( MAS ) has a prolonged he aling response after injury compared to other muscles such as tibialis anterior (TA) However, it is not known if this diminished repair capacity is due to a decreased ability of myogenic stems cells in MAS to activate, proliferate and form myofibers or the result of a differential response to specific inflammatory me diators The purpose of this study was to : 1 ) compare the myogenic potential of MAS and TA in an in vitro model ; and 2) determine if there is a differential effect of three inflammatory cytokines TNF and IL 4 on MAS and TA myogenic potential in vitro Single muscle fibers from MAS and TA of CD 1 female mice were harvested and place d on m atrigel coated coverslips in 24 well plates. To evaluate early events, fibers were cultured for six days in a proliferation medium L ate events were evaluated by culturing the myoblasts/myotubes an additional six days in differentiation media Cytoki nes were tested individually for an effect on cell type proportions during the proliferation and differentiation phases Four cul ture replicates were evaluated for each condition A ctivation and p roliferation were assessed by immunostaining for MyoD, a myoblast marker ; m yotube and myofiber formation were evaluated by immuno staining

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12 for myogenin. Proportions of cells with distinct phenotypes were calculated for each condition. I n the in vitro model, myogenic stem cells derived from MAS and TA were capable of activation, proliferation and formation of myofibers. However, cultures derived from MAS and TA differed in myogenic cell phenotype proportions indicating differences in maturation In both MAS and TA derived cultures c ytokine a dministration affected specific myogenic cell phenotype proportions suggesting a differential effect on specific phases of myogenesis H owever when cell proportions were normalized to controls, the only significant difference between MAS and TA derived cultures exposed to cytokine was an increase in the proportion of supporting cells These results indicat e that cytokine exposure has similar effects on myogenic cells derived from both muscles T hus, t he diminished capacity of MAS to repair does not appear to be the result of an increased adverse effect of cytokines on MAS presump tive myogenic cells

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13 CHAPTER 1 INTRODUCTION T emporomandibular disorder s (TMD) is a term used to describe a group of musculoskeletal pain conditions involving the temporomandibular joint region. TMD pain involves the muscles of mastication (myofascial pain), the joint itself (arthralgia) or both (LeResche 1997) Factors associated with myofascial pain in TMD patients have been shown, through logistical regression analyses to include: clenching, head/neck trauma, anxiety and being of the female sex. Within the spectrum of TMD, the masseter (MAS) muscle is involved in nearly 60% of cases (Dworkin et al. 1990) It has been suggested that this muscle may display poor healing, which could result in ineffective treatment in some patients with masticatory muscle induced TMD. P avlath et al (1998) used a mouse model to compare the MAS muscle to a limb skeletal muscle, the tibialis anterior (TA), and found that the masseter muscle showed greatly delayed and decreased healing after a standardized freeze injury. The differential h ealing of the MAS muscle is of great interest to those managing TMD and clarifications may result in enhanced therapeutic approaches to patient care and treatment. While little is known about the cause for the differential healing, we suggest two hypothes es. The first is that there may be inherent differences in the ability of MAS muscle to regenerate when compared to other skeletal muscles. A second hypothesis is that there may be some component(s) of the environment, such as differences in the inflammato ry milieu, which may differ between the MAS muscle and other skeletal muscles. This may involve the delicate balance between cellular and chemical mediators, concentrations and temporal peaks. Preliminary studies by Morris Wiman and Widmer have shown that mast cells are increased in MAS muscle after injury and

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14 could play a role in decreased he aling (Morris Wiman and Widmer 2006). More recently in the same laboratory, MAS and TA were freeze injured and cytokine ex pression was determined (Harris 2010). The M AS muscle had an overall blunted cytokine expression compared to TA. Treatment with cromolyn, a mast cell inhibitor, partially enhanced cytokine expression within the MAS These data support the hypothesis that the differential regeneration may be due to v ariations in inflammatory milieu and the environment. The goal of the present study was to further examine the regenerative potential of MAS and TA in an in vitro model and determine what effect specific cytokines (TNF 4) have on regenerati ve potential. Muscle Regeneration: Satellite Cell Within skeletal muscle, somatic or adult stem cell populations reside that are capable of regeneration and maintenance of the muscle (for review see S hi and Garry 2006) These mononuclear cells are known as satellite cells. Satellite cells reside between the basal lamina and plasma membrane of the adjacent myofiber (Muir et al. 1965) In their normal state, these cells are quiescent an d in response to injury they become activated, proliferate and express myogenic markers. Once satellite cells express myogenic markers they are termed myoblasts. Myoblasts ultimately fuse, either to existing myofibers or to each other, to form new myofiber s (Bischoff 1994) Myogenesis of satellite cells is regulat ed by a family of muscle specific transcription factors, known as muscle regulatory factors (MRF) (Yablonka Reuveni et al. 2008) These transcription factors include: myogenic determination factor 1 (MyoD), myogenic factor 5 (Myf5), myogenin and myog enic regulatory factor 4 (MRF4) (Ludolph and Konieczny 1995) The MRFs are expressed differe ntially over time and at different stages in the activation of satellite cells (Yablonka Reuveni and Rivera 1994)

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15 Two of the MRFs, MyoD and myogenin, are upregulated during different stages of satellite cell activation. MyoD is upregulated primarily in proliferating satellite cells (Yablonka Reuveni and Rivera 1994) and occasionally in differentiating cells as well, depending on the extracellular matrix environment (Yablonka Reuveni et al. 2008) Whereas MyoD is upregulated primarily in satellite cell proliferation, myogenin is associated with satellite cell differentiation and is associated with withdrawal from the cell c ycle, fusion of myoblasts to multinucleated myotubes and a decline in MyoD (Yablonka Reuveni an d Rivera 1994; Yablonka Reuveni et al. 1999; Yablonka Reuveni and Paterson 2001) Differential Regeneration of Muscle Altho ugh all skeletal muscles contain satellite cells, and thus the potential for regeneration, it has been suggested that the MAS muscle displays poor healing compared to other skeletal muscles. Pavlath et al (1998) examined differences in regenerative capaci ties between the TA muscle (a limb muscle) and the MAS muscle (a craniofacial muscle) and reported marked differences both in timing of regeneration and quantity of fibrous connective tissue (scar tissue) formed. In the first experiment conducted by Pavlat h et al (1998), MAS and TA muscles were injured using either freeze or crush injury in a mouse model. In the TA, proliferation of myoblasts was evident within 2 days, extensive myotube formation within 4 days, normal myofiber size by day 7 and nearly comp lete architecture restoration by day 12. However, the MAS muscle displayed a markedly different course of regeneration: at day 12 large areas of injured muscle remained with little evidence of regenerated muscle fibers. At days 19 21, extensive interstitia l connective tissue remained between fibers and by day 45 regeneration of the MAS muscle was observed to be less effective than the TA muscle

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16 30 days prior. Hence, in the mouse model, the quality of regenerated tissue post crush or freeze injury was signif icantly better in the TA at all time points (through 45 days post injury). The quantity of regenerated muscle was also examined and it was concluded that MAS muscle displayed poor regeneration throughout all anatomical areas of the muscle with the most d ramatic deficits being in the core of the muscle (Pavlath et al. 1998) In an attempt to understand the reason why MAS muscle regenerated po orly, Pavlath et al. (1998) proceeded to compare regeneration of MAS muscle to two other muscles of the head and neck, the anterior belly of the digastric (a masticatory craniofacial muscle) and the sternocleidomastoid (a non masticatory craniofacial muscl e). It was found that both of these muscles regenerated similar to the TA muscle, concluding that neither masticatory muscle nor embryologic origin alone could explain differences in masseter muscle regeneration. To further examine MAS muscle injury, Pavla th et al. (1998) next examined regeneration of masseter muscle in response to endogenous injury (muscular dystrophy). In this model, a mouse with muscular dystrophy experienced a period of 3 weeks of muscle necrosis at 8 weeks of age, which targeted muscle fibers and spared nerves and blood vessels, and was followed by active regeneration of muscle. Again, MAS muscle showed significantly delayed regeneration in comparison to TA muscle. An additional analysis was performed by Pavlath et al. (1998) to examine the relative amount of myoblasts activated in damaged TA versus MAS muscle. Following similar injury, myoblast cultures were established and co stained with Bromodeoxyuridine ( BrdU) a nd MyoD (a MRF) to provide an estimate of the number of activated satellite cells. In both culture and tissue sections, TA displayed approximately

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17 2.5 times more activated satellite cells than MAS muscle (Pavlath et al. 1998) Criticisms of this experiment are that they were not able to normalize for net wet weight or fiber number. In these experiments the number of activated satellite cells was compared within its native environment either MAS or TA muscle. Therefore, one can hypothesize that the reason for decreased activation of myoblasts may be within the myoblasts themselves or may be a factor of the environment. In the last of the expe riments reported in Pavlath et al. (1998), the ability of myoblasts to fuse and express a differentiation specific protein, embryonic myosin heavy chain (EMyHC), during regeneration was assessed. The time course of expression of EMyHc was found to be simil ar for both muscles, while the amount was decreased throughout in the MAS muscle. This suggests that MAS myoblasts may be able to fuse and differentiate similar to those of TA but may be decreased in number (Pavlath et al. 1998) The reduction in number and alter ation in behavior may be inherent to MAS or may be a result of the en vironment This may lead one to hypothesize that satellite cells from the MAS are capable of differentiation yet are decreased in number. However, because the experiments were conducted with cells in their native environment, one cannot rule out a possible environmental influence. Acute Inflammation: A Response to Mus cle Injury The current understanding of muscle repair after acute trauma is that, first, there is rapid disruption of structural components in the muscle, then there is an inflammatory stage that includes removal of cellular debris and lastly there is regeneration or repair of muscle fibers (Tidball 1995) Throughout the inflammatory process there are numerous cytokines, chemokines and growth fac tors being released. Tumor necrosis factor

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18 sepsis to causing necrosis of tumors (Beutler and Cerami 1988) In a study by Warren et al. (2002), the specific role of TNF was examined in traumatic muscle injury. In particular, the levels of expression post injury were analyzed and the implications of blocking receptors for this cytokine examined In the first part of the study, Warren et al. (2002) looked at wild type control mice to examine trends in TNF freeze injury of the tibialis anterior muscle. Using real time PCR and immun o cytochemical analysis, Warren et al. (2002) showed that TNF increase post injury and peaked at 24 hours. At early time points, staining was localized primarily to inflammatory cells and at later time points there was moderate sta ining of regenerating myofibers. To further test the role of TNF knockout mice that were genetically deficient in the two main TNF and TNFR2, were subjected to a freeze injury of the TA muscle (Warren et al. 200 2). The influx of inflammatory cells was not significantly different from control mice, suggesting redundancy of TNF receptors but were administered antibodies to TNF (2 002) was that TNF regeneration. However, there are likely redundant systems at work, since muscle repair was only slightly affected by deficiencies in TNF In another study, the role of TNF cle inflammation was examined by injecting the cytokine into muscle (Peterson et al. 2006). After administering the cytokine to mice for 7 days, Peterson et al. (2006) immunocytochemically stained muscle for detection of macrophages and neutrophils. Their results revealed that administration of TNF

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19 skeletal muscle (between two fold and seven fold), but did not result in overt signs of increased muscle atrophy, injury or inflammation. While these results provide insight into the role of TNF (2006) did not follow up long term to see if signs of neutrophil induced muscle injury were present at subsequent time points. Additional studies conflict with that neutrophils do contribute to muscle injury during inflammation (Brickson et al. 2003; Pizza et al. 2005) and reperfusion of ischemic tissue (Rubin et al. 1996; Palazzo et al. 1998) In a study by Hodgetts et al (2006), depletion of host neutrophils and inhibition of TNF is in mdx mice, a model for Duchenne Muscular Dystrophy. Lastly, recent studies by Al Shanti et al. (2008) demonstrated that there is positive crosstalk between TNF 6 which may act to enhance the IGF system and promote myoblast differentiation. In sulin growth factor I (IGF 1) is a cytokine that is essential for the differentiation of satellite cells and plays an important role the anabolism of muscle fibers ( Broussard et al 2004). IL is another ubiquitous pro inflammatory cytokine. As with TNF much debate regarding the role of IL in muscle inflammation. Both TNF IL are elevated before the onset of muscular dystrophy in the murine model of this disease (Kumar and Boreik 2003). In addition, chronic administration of IL to rats has been found to reduce protein synthesis, while the administration of IL receptor antagonist prevented muscle mass deficiencies in an animal model (Cooney et. al 1999). The exact mec hanism by which IL may inhibit muscle regeneration remains unknown, but recent studies suggest that it may act by inhibiting IGF 1 and expre ssion of myogenin (Broussard et al 2004). Additional research has further demonstrated that the anti

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20 inflammator y cytokine IL 10 is capable of suppressing IL thereby ceasing it s inhibition of IGF 1 (Strle et al 2008). The cytokine IL 4 also appears to have an important role in muscle regeneration. The results of a study by Horsley et al (2003) suggest that IL 4 may act as a myoblast recruitment factor in the initial stages of myoblast fusion to form myofibers and in this way may promote the formation of new muscle fibers in muscle regeneration post injury. Harris (2010) used a mouse model to examine the diff erential expression of cytokines post injury. She found that IL 4 was increased in TA compared to masseter at baseline, decreased during early repair, and recovered in late repair. An explanation for the varying levels of expression could be that the initi al decrease was an attempt by TA to prevent premature fusion of myoblasts and allow proliferation (Harris, 2010). Given the complexity of their actions and the interactions of these inflammatory mediators, it is difficult to assign a role to any one cytoki ne. It is likely that each cytokine plays varying roles temporally to either prolong muscle damage or promote healing and that this role depends on the presence of other cyt okines and environmental cues. Summary MAS muscle damage and regeneration are important area s of research and gaining a better understanding of this topic has wide ranging implications in the treatment of masticatory muscle pain. Inflammation plays an integral role in muscle repair, but just as in flammatory mediators help promote healing they can also induce further injury and a delicate balance is needed to ensure the normal healing process. Satellite cells are multipotent stem cells that reside in muscle. Following muscle injury these cells becom e activated and proliferate and differentiate into new myofibers. Inflammatory factors affect not only key inflammatory cells, such as neutrophils and

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21 macrophages, but also regenerative cells, satellite cells. Studies by Pavlath et al. (1998 ), using a mous e model, have shown that MAS muscle displays delayed and reduced healing compared to TA muscle. This reduced healing may be due to inherent properties of the MAS muscle or may result from differences in the inflammatory milieu compared to other skeletal mu scles. In Harris ( 2010 ) the inflammatory milieu after a freeze injury was compared between MAS and TA in a mouse model. An overall blunted cytokine expression in MAS muscle was observed. The aim of the present study was to examine, in an in vitro mouse mod el, the effect of specific cytokines on the regeneration of TA and MAS muscle. In particular, the effect of three cytokines, TNF 4, was examined at proliferation and differentiation stages of the regenerative process. Significance TMD is a p revalent condition that can be debilitating and chronic. Current therapy for myofascial pain is palliative and does not address the underlying pathophysiology. A better understanding of the role of inflammatory cytokines on regeneration of MAS muscle may l ead to drug therapies that will enhance regeneration. Understanding the effect of individual cytokines is the first step in developing an accurate model of MAS muscle regeneration.

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22 Hypotheses Our hypotheses were that MAS and TA muscle fibers differ in their inherent ability to repair with the regenerative capacity of masseter musc le being significantly reduced; MAS and TA differ significantly in their response to cytokines (TNF IL 4) present in the inflammatory mi lieu during repair, with masseter regeneration being significantly more adversely affected by t he presence of these cytokines. Specific Aims Our specific aims were to e valuate inherent differences (satellite activation, myoblast proliferation, myotube a n d myofiber formation) between MAS and TA muscle regeneration in an in vitro model ; e xamine the effects of TNF 4 on satellite activation, myoblast proliferation, myotube and myofiber formation in an in vitro model of MAS and TA regeneration

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23 CHAPTER 2 MATERIALS AND METHOD S Overview of Experimental Design Our general experimental design was as follows: 1. Masseter and tibialis anterior muscles were harvested, digested in collagenase and single myofibers isolated under a dissecting microscope and via gradient separation. 2. A 10 l slurry of myofibers was placed on the center of a m atrigel coated glass coverslip in a 24 well plate. 3. For each culture replicate, a total of 4 plates were used There were 4 plates per musc le type in early (prol iferation) cultures and 4 per late (differentiation) cultures. Therefo re, there were 16 plates total (Fig 2 1). 4. Cytokines (TNF 4) were added to wells either on the first day of culture (day 1) to examine early even ts or on day 6 to examine late events. 5. Cultures were fixed either on day 6 for early events or day 12 for late events and immunostained for My oD and myogenin. 6. In a standardized microscop ic field the number of cells corresponding to a specific cell phenotype for each experimental group or control was determined by a blinded investigator. 7. Statistical analyses were performed to assess significant differences between groups. M yofiber Isolation Female CD 1 mice (n=4) were sacrificed at 8 weeks of age and the MAS and TA muscles were harvested and cleaned of connective tissue under a dissecting microscope. Muscles were then transferred to a 35 mm dish with 0.2% collagenase solution and incubated for 2 hours at 37 C. At the end of digestion, muscle was transferred to a 35 mm dish with 10% horse serum (HS) in DMEM. Under a dissecting scope, single fibers were liberated from muscles by pulled glass pipettes and by trituration in wide mouth Pasteur pipettes. Dissected fibers were transferred to 15 mL coni cal centrifuge tubes containing 10% HS (Shefer et al 2004). In this 1g gradient

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24 muscle fibers were separated from lighter fibroblasts and other supporting cells. Gradient separation was repeated 3 times and separated myofibers removed to a Petri dish wit h 10% HS. Coverslips were coated with a solution of matrigel in DMEM (1:6 least 30 minutes. A 10 l slurry of isolated myofibers was placed on the center of a coversli p in each well of the 24 well plates. Myofiber Culture Plates containing myofibers were placed in a 37 C incubator for 10 20 minutes to allow myofiber attachment to Matrigel. 500l of proliferation medium (20% FBS, 10% horse serum, 1% chick embryo extrac t) was then added to each well (Shefer et al. 2004). To assess early events in muscle repair (proliferation) cytokines were added to the wells as follows : IL (0.1, 0.4, 0.8 ng/ml, Strle et al 200 8 ; Broussard et al 2004); TNF (0.02, 0.05, 1.0 ng/m l Broussard et al 2003); and IL 4 (0.5, 1 and 2 ng/ml, Horsley et al 2003) (Fig 2 2). Cultures were maintained for 6 days in proliferation medium, with a change of medium at 72 hrs. After 6 days of culture, plates for analysis of cytokine influence on early events were rinsed in PBS and fixed in cold 4% paraformaldehyde in phosphate buffer. Plates were stored at 4 C until immunostained for MyoD and myogenin as described below. For the assessment of cytokine effects on late events in muscle repair (my otube/myofiber formation), after 6 days of culture, proliferation medium was replaced with differentiation medium (2% FCS, 10% horse serum, 0.5% chick embryo extract in DMEM) and maintained for another 6 days (12 days total)( Shefer et al 2004). Cytokine or vehicle was added at this time to assess effects on myoblast fusion and differentiation. Fresh medium with cytokine or vehicle was added every 48 hours. After

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25 6 days (12 days total) cultures were fixed in paraformaldehyde and immunostained for MyoD an d myogenin as described below. Immunofluorescent Staining/ Evaluation Coverslips were incubated in 2% normal goat serum (NGS) for 20 minutes to block non specific staining and then overnight in the refrigerator with anti MyoD primary antibody. Coverslips were rinsed in PBS and then incubated in a secondary antibody conjugated to alkaline phosphatase for 3 hours at room temperature. After rinsing in TBS, NBT/ BCIP alkaline phosphatase substrate was added to each well. The reaction was stopped by replacemen t of the substrate with TBS. After extensive rinsing in TBS, coverslips were incubated an anti myogenin primary antibody overnight in the refrigerator. After rinsing in PBS, coverslips were incubated in an appropriate secondary antibody conjugated to biot in for 3 hours at room temperature, rinsed, then incubated for 1 hour in streptavidin conjugated to HRP. Coverslips were then rinsed and finally incubated in the HRP substrate AEC. The reaction was stopped by the substitution of PBS for the substrate. We lls were rinsed with PBS, and the coverslips were carefully removed and mounted onto glass slides for viewing and image acquisition. Counting and Classification of Cells Coverslips were viewed under brightfield using a Nikon FXA microscope and a 10x field acquired using a Zeiss Mr5 digital camera and Axiovision software. The field was chosen at the interface of the regions of greatest cell density radiating from the attached myofiber and the region of cell migration. Digital images were analyzed using JMi croVision (Nicolas Roduit, ver. 1.2.7) image analysis software. This software program allowed the categorization of cells into one of the following phenotypic groups:

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26 For the early culture we cate gorized cells into the following 4 groups (Figure 2 3 ): Darkly stained cells, MyoD positive cells presumptive myoblast Medium stained cells, myogenin positive cells late stage myoblasts Lightly stained cells, supporting cells Fibers For the late culture we categorized cells into the following 6 groups (Figu re 2 4 ): small, round cells not associated with myotube or myofiber ( Class 6) thin myofibers ( Class 1) medium thickness myofibers ( Class 2) thick myofibers ( Class 3) stained myofiber s with central nuclei ( Class 4) round myofiber like ( Class 5) Intra rater r eliability of cell categorization was tested separately for early and late cultures. For both the Pearson Product Moment correlation coefficient was 0.97. Power Sample Size Estimation Sample size for the proposed cytokine experiments was based on data from Broussard et al (2004). Means and standard deviations of myosin heavy chain protein and myogenin protein levels from C 2 C 12 cells after exposure to 3 different doses of IL were used in the calculations. Using an alpha level of 0.05 and a power of 0.9, the minimum number of animals required for detection of differences among 3 different doses of a cytokine was 4 Statistical Analyses Descriptive st atistics were reported as means and standard errors. Inferential statistics were calculated using a repeated measures ANOVA with statistical significance set at 0.05. For all aims, four factors were examined: muscle, cytokine,

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27 dose and cell classificatio n. Post hoc comparisons were made using the LSD test when significant main effects/interactions were identified in the ANOVA.

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28 Figure 2 1. Visual representation of the study design and variables examined. Each of these 4 combinations was represented in a plate, and each combination was replicated 4 times (4 combinations x 4 replicates= 16 plates total).

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29 Figure 2 2 Visual representation of layout of each 24 well plate. Each plate contained either masseter or tibialis anterior muscle fibers. The top r ow was assigned to the controls and no cytokine was administered to these wells. The second, third and fourth rows were administered cytokines TNF IL 4. Each cytokine was delivered in 3 concentrations, shown here by the intensity of the pigmen tation of each box. Right and left sides of each well were duplicates.

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30 Darkly stained cells Immature myoblasts MyoD positive Medium stained cells Advanced myoblasts Myogenin positive Lightly stained cells Fibroblasts and other supporting cells Fibers All fiber types Figure 2 3. C ell phenotype catego ries for early cultures

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31 Class 6 Immature myoblasts Small dark cells not associated with myotube or myofiber MyoD positive Class 1 Thin, multinucleate myofibers Class 2 Medium thickness myofibers with lateral nuclei Class 3 Thick myofibers with lateral nuclei Class 4 Myofibers with central nuclei Blue/brown stained Class 5 Round Myofiber like cells with central nucleus Figure 2 4 C ell phenotype categori es for late cultures

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32 CHAPTER 3 RESULTS Do Differences in Cell Proportions Exist Between MAS and TA Control Cultures? Examination of MAS and TA early (proliferation) control cultures revealed distinct differences in cell phenotypes (Figure 3 1 A C ). Generally, MAS control cultures had a higher cell density and a greater quantity of cells that were medium stained or MyoD/my ogenin positive. Results of the analysis of variance, shown in Table 3 1, identified a significant interaction (muscle x classification). Further post hoc testing identified TA as having a statistically larger proportion of MyoD positive darkly stained ce lls, presumably young myoblasts (Figure 3 1 C ). MAS was found to have a significantly greater proportion of medium stained MyoD/myogenin positive cells, presumably myoblasts/myotubes (Figure 3 1 C ). Examination of late (differentiation) MAS and TA control cu ltures also revealed distinct differences in cell phenotype (Figure 3 2 A C ). As was observed in proliferation control cultures, generally, late MAS control cultures had a higher cell density than TA control cultures. Additionally, MAS differentiation cul tures appeared to have more highly developed myofibers that were well aligned with one another. Late TA control cells also possessed myofibers but in lower density and less aligned than in the late MAS controls. A significant interaction ( muscle x classifi cation ) was identified by the analysis of variance, shown in Table 3 2. Post hoc testing identified MAS as having a statistically significant greater proportion of Class 2 cells, medium thickness myofibers with lateral nuclei. TA had a significantly great er proportion of Class 6 cells, MyoD positive cells or myoblasts (Figure 3 2 C ).

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33 Do Differences in Cell Proportions Exist Between MAS and TA Proliferation Control Cultures and Cytokine Exposed Cultures? In MAS and TA proliferation cultures, in both controls and cytokine exposed, the highest proportion of cells were darkly stained, MyoD positive myoblasts, followed by medium stained MyoD/myogenin positive myoblasts and finally lightly stained supporting cells and myofibers. Statistical testing for differences among factors in MAS cytokine exposed and control proliferation cultures identified a si gnificant interaction (cytokine x classification x concentration) (Table 3 3). Significant differences in cell proportions between control and cytokine exposed MAS pro liferation cultures at different cytokine concentrations were found after post hoc testing. Administration of TNF darkly stained, MyoD positive myoblasts (Figure 3 3 A ). No d ifferences were observed at medium and high concentrations. The low concentration of IL significant differences; a significant increase in the proportion of medium stained myogenin/MyoD positive cells and a significant decrease in the proportion of darkly stained MyoD positive cells were identified. In contrast, the low concentration of IL 4 had no effect on cell proportions, but exposure to medium and high concentrations resulted in significant increases in the proportion of light sta ined supporting cells and significant decreases in the proportion of darkly stained MyoD positive cells. Statistical testing for factor differences in the TA cytokine exposed and control proliferation cultures identified a significant interaction (cytokine x classification) (Table 3 4). However, significant differences based on cytokine concentration were not detected. Post hoc testing identified significant differences between control and cytokine exposed TA proliferation cultures (Figure 3 3 B,D,F ). TNF administration did

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34 not significantly affect cell proportions in TA proliferation cultures. Exposure to IL resulted in a significant increase in the proportion of darkly stained, MyoD positive cells and a decrease in the proportion of medium stained, M yoD/myogenin positive cells. IL 4 exposure significantly affected only the proportion of darkly stained, MyoD positive cells. Do Differences in Cell Proportions Exist Between MAS and TA Differentiation Control Cultures and Cytokine Exposed Cultures? A si gnificant interaction of factors was identified in the ANOVA, shown in Table 3 5, (cytokine x classification x concentration x muscle). Further post hoc testing identified significant differences in cell proportions between control and cytokine exposed MA S differentiation cultures (Figure 3 4 A,C,E ). Exposure to TNF highest concentration resulted in a significant increase in the proportion of Class 6 cells, MyoD positive myoblasts. TNF in th e proportion of Class 4 myofibers. Exposure to high concentrations of IL decreased the proportion of Class 4 myofibers, but this decrease was concomitant with an increase in medium sized myofibers (Class 2). IL 4 exposure resulted in a decrease in the proportion of Class 4 cells, but only at the lowest concentration. At high IL 4 concentrations, the proportion of Class 5 cells was increased. Significant differences in cell proportions between control and cytokine exposed TA differentiation cultu res were also identified (Figure 3 4 B,D,F ). Exposure to TNF the highest concentration resulted in a significantly increased proportion of Class 5 cells and a concomitant decrease in the proportion of Class 4 myofibers. In contrast, exposure to TNF at the medium concentration resulted in a significant increase in Class 6 cells, MyoD positive myoblasts. IL

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35 also resulted in a significant increase in the proportion of Class 6 cells. Significant effects with IL 4 exposure were only identified at the medium concentration. Exposure resulted in an increased proportion of Class 2 and Class 3 myofibers and a decrease in Class 6 MyoD positive myoblast s Do Differences Exist in the Effects of Cytokine Exposure on Cel l Proliferation and Myofiber Formation Between MAS and TA Cultures? To compare the effects of cytokine exposure on MAS and TA cultures, the means of cell proportions for MAS and TA for each cytokine were normalized to cell proportion means of control cultu res to eliminate any differential growth effects. A significant interaction of factors, shown in Table 3 6, was found in proliferation cultures (cytokine x classification x concentration x muscle). Exposure to medium concentrations of IL and medium and high concentrations of IL 4 resulted in significant differences between MAS and TA in the proportion of light cells (Figure 3 5 B,C ). No significant differences in the proportion of different cell/ fiber types were detected between MAS and TA late, differen tiation cultures with any cytokine exposure (Table 3 7; Figure 3 6 A C ).

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36 Table 3 1 ANOVA : MAS and TA controls, early cultures Effect df F p Muscle 1 2.5 0.17 Classification 3 104.7 0.00* Muscle x Classification 3 6.3 0.00* p < 0.05 A Figure 3 1. Photomicrographs of MAS and TA proliferation (early) cultures and graph of cell phenotypes (mean + SE ). A,B) The upper panels are representative fields analyzed for MAS and TA. Thick arrows (yellow) label darkly blue stained MyoD positive cells. Thin arrows (yellow) label medium blue brown stained MyoD/myogenin positive cells. A myofiber is la beled wi th a red arrow in MAS. C) In the graph in the lower left panel differences in cell proportions between MAS and TA control proliferation (early) cultures are shown. Statistically significant differences in cell proportions between muscles are denoted by a r ed box. B. TA A. MAS C

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37 Table 3 2 ANOVA : MAS and TA controls, late cultures Effect df F p Muscle 1 9.00 1.00 Classification 5 5.75 0.00* Muscle x Classification 5 1.33 0.00* p < 0.05 A Figure 3 2. Photomicrographs of MAS and TA differentiation (late) cultures and graph of cell phenotypes (mean + SE ) A,B) The upper panels are representative fields analyzed for MAS and TA Thick arrows (yellow) label darkly blue stained MyoD positive cells. Thin arrows (yellow) label medium myofibers. C) In the graph in the lower left panel differences in cell proportions between MAS and TA control proliferation (early) cultures are shown. Statistically significant differences in cell proportions between muscles a re denoted by a red box. B. TA A. MAS C

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38 Table 3 3 ANOVA: E arly MAS Controls vs. treated with c ytokines Effect df F p Cytokine 3 Concentration 2 Classification 3 2.25 0.00* Cytokine x Concentration 6 1.57 0.00* Cytokine x Classification 9 3.92 0.00* Concentration x Classification 6 1.67 0.98 Cytokine x Concentration x Classification 18 2.38 0.01* p < 0.05 Table 3 4 ANOVA: E arly TA c ontrols vs t reated with cytokines Effect df F p Cytokine 3 1.0 0.44 Concentration 2 8.4 0.02* Classification of cells 3 88.9 0.00* Cytokine x Concentration 6 8.1 1.00 Cytokine x Classification 9 2.9 0.02* Concentration x Classification 6 0.7 0.62 Cytokine x Concentration x Classification 18 1.1 0.37 p < 0.05

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39 A C E Figure 3 3 Graphs illustrating differences in controls vs. treated proliferation (early) cultures exposed to cytokines (mean + SE) A, B) MAS and TA exposed to TNF C, D) MAS and TA exposed to IL E, F) MAS and TA exposed to IL 4. Statistically significant differences in cell proportions are denoted by a red box. C A B D E F

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40 Table 3 5 ANOVA: L ate c ontrols vs. t reated with cytokines Effect df F p Muscle 1 0 0.80 Cytokine 3 3 0.09 Muscle x Cytokine 3 0 0.97 Concentration 2 3 0.09 Concentration x Muscle 2 0 0.83 Classification 5 10 0.00* Classification x Muscle 5 27 0.00* Cytokine x Concentration 6 3 0.02* Cytokine x Concentration x Muscle 6 0 0.98 Cytokine x Classification 15 2 0.02* Cytokine x Classification x Muscle 15 2 0.09 Concentration x Classification 10 1 0.44 Concentration x Classifications Muscle 10 0 0.95 Cytokine x Concentration x Classification 30 1 0.20 Cytokine x Concentration x Classification x Muscle 30 2 0.01* p < 0.05

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41 Figure 3 4 Graphs illustrating differences in controls vs. treated differentiation (late) cultures exposed to cytokines (mean + SE) A, B) MAS and TA exposed to TNF C, D) MAS and TA exposed to IL .E, F) MAS and TA exposed to IL 4. Statistically significant differences in cell proportions are denoted by a red box. A A A B C D E F

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42 Table 3 6. ANOVA : E arly MAS vs. TA treated with cytokines and normalized to means of controls Effect df F p Muscle 1 0.32 0.61 Cytokine 2 2.52 0.16 Muscle x Cytokine 2 1.40 0.32 Concentration 2 1.09 0.39 Concentration x Muscle 2 0.55 0.60 Classification 3 1.63 0.25 Classification x Muscle 3 0.07 0.97 Cytokine x Concentration 4 1.06 0.42 Cytokine x Concentration x Muscle 4 2.23 0.13 Cytokine x Classification 6 1.70 0.18 Cytokine x Classification x Muscle 6 1.06 0.42 Concentration x Classification 6 1.46 0.25 Concentration x Classification x Muscle 6 0.63 0.71 Cytokine x Concentration x Classification 12 0.53 0.88 Cytokine x Concentration x Classification x Muscle 12 2.45 0.02* p < 0.05

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43 Figure 3 5 Graphs illustrating differences in MAS and TA proliferation (early) cultures exposed to cytokines (mean + SE) A) E xposed to TNF B) Exposed to IL C) Exposed to I L 4. For analyses, means for each muscle and cytokine exposure were normalized to control means. Statistically significant differences in fold changes between muscles at the same concentrations are denoted by a re d box. A C B

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44 Table 3 7 ANOVA: L ate MAS vs TA treated with cytokines and normalized to means of controls Effect df F p Muscle 1 0.58 0.50 Cytokine 2 0.63 0.56 Cytokine x Muscle 2 1.98 0.22 Concentration 2 1.39 0.32 Concentration x Muscle 2 1.33 0.33 Classification 5 0.57 0.72 Classification x Muscle 5 0.93 0.49 Cytokine x Concentration 4 0.53 0.71 Cytokine x Concentration x Muscle 4 1.67 0.37 Cytokine x Classification 10 1.47 0.20 Cytokine x Classification x Muscle 10 1.18 0.34 Concentration x Classification 10 0.79 0.63 Concentration x Classification x Muscle 10 0.72 0.70 Cytokine x Concentration x Classification 20 1.12 0.36 Cytokine x Concentration x Classification x Muscle 20 0.95 0.53 p < 0.05

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45 A C Figure 3 6 Graphs illustrating differences in MAS and TA differentiation (late) cultures exposed to cytokines (mean + SE) A) Exposed to TNF B) Exposed to IL C) Exposed to IL 4. For analyses, means for each muscle and cytokine exposure were normalized to control means. No statistically significant differences in cell proportions were identified A B C

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46 CHAPTER 4 DISCUSSION Previous studies have demonstrated th at injured MAS and TA differ in their regenerative response with the MAS muscle requiring three times longer to repair (Harris, 2010; Pavlath et al 1998). However, in these studies it was not determined if the flaw in repair capacity lies in the ability of satellite cells to activate, proliferate and form myofibers, or was the result of a hostile environment. Previous work in the laboratory has pro vided evidence that the cytokine/chemokine milieu differs between MAS and TA both at baseline and after injury (Harris, 2010). The present study sought to examine differences in an in vitro model and using this model to determine if responses to specific c ytokine s differed between MAS and TA. Differences in the In Vitro Proliferative and Differentiation Potential between Presumptive Myogenic Stem Cells Isolated from MAS and TA Myofibers Initially we established baseline growth characteristics for cells eman ating from myofibers isolated from MAS and TA both during early events in myogenesis (satellite cell activation, migrating, and proliferation) and during late differentiation events (myotube and myofiber formation). We observed significant differences bet ween MAS and TA in the proportion of cells representing different stages of myoblast maturation and muscle fiber formation. Early proliferation MAS cultures had a significantly greater proportion of myogenin positive cells than TA cultures. Additionally, late differentiation MAS cultures had significantly greater proportion of medium sized myofibers than TA cultures. Late MAS cultures consistently had myofibers that were more advanced, organized and more closely resembled muscle fibers. In contrast, in bo th early proliferation and late cultures, TA had a significantly greater proportion of darkly stained blue cells, presumably proliferating MyoD positive myoblasts. These results suggest

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47 that at least in vitro satellite cells in isolated myofibers from b oth MAS and TA are capable of activation and proliferation and the formation of myofibers. However, the data also suggest that, in vitro, cells derived from myofibers isolated from MAS and TA may differ in their time course towards myofibers formation. F ew previous studies have examined in vitro the relative myogenic potentials of cells isolated from MAS and limb muscle. Pavlath et al (1998) examined the proliferative potential of myoblasts isolated from freeze damaged MAS and TA and observed a decreased potential in myoblasts isolated from MAS. However, it could not be determined if the decreased potential was inherent to the MAS myoblast, the result of fewer satellite cells in MAS (Ono et al 2010) or the result of prior exposure to the inflammatory mil ieu. Ono et al (2010) compared the ability of clonal ly derived satellite cells from MAS extensor digitorum longis (EDL) and soleus (SOL) to proliferate and differentiate and reported that cells isolated from MAS proliferated more but differentiated later than cells isolated from either limb muscle. These results would appear to contradict ours. However, more likely they are the result of differences in the in vitro models. T he in vitro model used by Ono et al examined the potential of cells enzymatically isolated from myofibers and maintained quiescent for extensive periods prior to placement in a culture medium that promotes proliferation. Our in vitro model was developed to better approximate conditions during muscle repair after injury In our model, p resumptive myogenin cells (satellite cells) were isolated in situ on myofibers and allowed to activate in situ before migration, proliferate and finally proliferation. Thus the behavior of satellite cells in our model was not influenced by enzyme mediated changes in adhesion characteristics or relationships with their associated basal lamina

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48 ECM. The differences observed between our results and Ono et al. most likely reflect differences in the behaviors of isolated presumptive myogenic stems cells in an ar tificial environment and cells allowed to undergo responses to myofibers disruption in situ. Differences in the Response to Cytokine Exposure In Vitro between Presumptive Myogenic Stem Cells Isolated from MAS and TA Myofibers The effects of the addition of each cytokine to proliferation and differentiation cultures are discussed under separate headings. However, there is a wealth of data from both in vivo and in vitro studies that each of these cytokine s can elicit the expression of the other cytokines as w ell as increase their own expression in myogenic cells. Significant effects of exposure to each cytokine on myogenesis were detected in our in vitro model, but assays to specifically assess the types and quantities of cytokines present in our cultures are needed to clarify potential confounding variables and help solidify our interpretation of the roles of each cytokine. TNF Previous studies have demonstrated the complex role of TNF and muscle regeneration (Palacios et al 2010, Broussa rd et al 2003, Ground s et al 2008, Li et al 2003, Langen et al. 2004 ). TNF through its inactivation of Pax7, a gene expressed by quiescent satellite cells, through the activation of p38 MAPK (Palacios et al 2010). Ina ctivation of Pax7 allows the satellite cell to exit the quiescent state and become activated, and activates MRFs, in particular MyoD and myogenin, promoting proliferation and early stages of differentiation (Palacios et al 2010). Therefore, through its ac tivation of p38 MAPK, TNF (Palacios et al 2010). TNF B which positively influences

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49 proliferation but inhibits differentiation (Ground s et al 2008). TNF also inhibits IGF signaling through activation of c Jun N terminal kinase (JUN) (Broussard et al 2003; Ground s et al 2008) In summary, research has shown that TNF proliferation (Li et al 2003) and has varying effects on differentiation of satellite cells in vitro (Langen et al 200 3, 2004 ). Based on these studies we would expect to see an increased proportion of dark, MyoD positive cells in TNF nt increases in the proportion of dark MyoD positive cells in all TNF late) as well as in exposed late TA. The concentrations at which this effect was seen varied between proliferation and differentiation cultures, suggest ing that sensitivity to the cytokine between early presumptive myoblasts and later derivations of these stem cells differed. Cell proportions in TA early proliferation cultures were not affected by TNF Considering the delicate effect of concent ration and minimal data detailing appropriate concentrations, it is likely that we did not deliver the optimal concentration of TNF increase in proportion of MyoD positive myoblasts in both early and late MAS and late TA cultures after exposure to TNF proliferation. In addition to increasing the proportion of presumptive myoblasts, treatment with TNF differentiated MyoD/myogenin positive multinucleated myofibers ( Class 4) in late MAS (at all concentrations) and TA (at high concentration). Therefore, our results suggest that in our in vitro model TNF hich would

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50 be in accordance with TNF (Broussard et al 2003). In late TA cultures, when TNF significant increase in the proportion of small round, myogenin positive myofibers with central nuclei ( c lass 5 cells). This cell phenotype has not been previously described either in vivo or in vitro. The cells appear to be budding off established myofibers. It is unclear if these cells represent a myofiber phenotype unique to our in vitro model or are a prelude to apoptosis (Sishi et al 2011; Andrianjafiniony et al 2010 ) Several studies of the effects of TNF apoptosis. However, it ha s been demonstrated that the concentrations of TNF delivered in this study do not promote apoptosis in vitro (Broussard et al 2003) IL As with TNF of satellite cells. In addition, IL expression, hindering differentiation events possibly by inhibition of IGF 1 (Strle et al 2010; Broussard et al. 2004). However, high doses of IL in IL 6 production by myofibers, which promotes myogenesis (Strle et al 2008). Taken together these findings suggest that IL dependent effect on differentiation, with enhancement of differentiation and fusion when delivered at high doses. In our early cultures, IL presumptive myogenic stem cells derived from MAS and TA fibers. In early proliferation MAS and TA cultures we observed an increase in the proportion of medium stained, MyoD /myogenin posi tive myoblasts and a decrease in dark, MyoD positive

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51 presumptive proliferating myoblasts. In TA, this effect was seen at all concentrations, suggesting that our concentrations were on the high end and pushed myofibers towards production of IL 6 and hence differentiation. In MAS enhanced differentiation was only seen when IL low concentration delivered might actually be high for the muscle and promoted IL 6 production. However, higher concentrations may have induced IL mediated myoblast apoptosis and resulted in the minimal effect observed on differentiation when it was delivered at medium and high concentrations. An increase in the proportion of dark, MyoD positive myoblasts was observed in late differentiation cultures of TA myofibers when IL high concentration suggesting a positive effect on myoblast proliferation at these levels. In MAS, delivery at its highest concentration resulted in an increas e in the proportion of medium myofibers ( Class 3 cells). These results reiterate the varying role of IL depending on muscle type and concentration. We predict that the exposure of differentiation TA cultures to higher concentrations of IL er enhance differentiation and result in increased proportions of larger myofibers. Likewise, we predict that delivery of lower concentrations of IL would promote proliferation and result in a greater proportion of M yoD positive cells. IL 4 IL 4 acts as a myoblast recruitment factor that promotes fusion of myoblasts to form myofibers (Horsley et al. 2003). Prior in vivo studies have demonstrated that, after a freeze injury, levels of IL 4 decrease in early stages of regeneration in TA but not MAS, suggesting that levels may need to remain low in order to prevent premature fusion of myofibers (Harris 2010). Based on these studies, we would anticipate

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52 increased fusion of myofibers with the addition of IL 4. Our results support this prediction. In early cultures of MAS and TA we saw a decrease in the proportion of dark, MyoD positive, presumptive myoblasts. In TA this effect was seen at all concentrations administered, whereas in MAS it occurred only at medium and highest concentrations. Whereas in early cultures we saw a decrease in MyoD positive cells, in late cultures IL 4 appeared to push cultures towards differentiation and regeneration. The effect of IL 4 on cell phenotype in MAS differentiation cultures varied sign ificantly with concentration. When the cytokine was delivered at its lowest concentration, an increase in the proportion of MyoD /myogenin positive myofibers ( Class 4 cells) was seen, whereas delivery at highest concentrations caused an increase in the prop ortion of small, round myofiber like cells ( Class 5 cells). As there are no previous reports of IL 4 involvement in apoptosis, the increased presence of this cell phenotype would suggest that these myofiber like cells are a unique phenotype and not a prelu de to apoptosis. In late differentiation TA cultures, exposure to the medium concentration of IL 4 resulted in increased proportion of medium and large myofibers ( Class 2 and 3 cells) and a decreased proportion of MyoD positive myoblasts. Therefore, it ap pears that IL 4 enhanced differentiation in late MAS cultures (when delivered at its lowest concentration) and in late TA cultures (at medium concentration). These findings are in accordance with previous studies suggesting the positive influence of IL 4 o n differentiation and fusion of myogenic stem cells (Horsley et al. 2003). When delivered at medium and highest concentrations, there was a significant increase in MAS proliferation cultures in the proportion of lightly stained cells, which included suppo rting cells such as fibroblasts. The finding that IL 4 increases the

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53 proportion of supporting cells suggests that, in addition to the effects of these cytokines on myoblasts, another key element in their role in myogenesis may be their effect on supporting cells. Inflammatory mediators may promote proliferation of fibroblasts and supporting cells and this could push a muscle towards a state of scarring rather than regeneration (Li et al. 2004) However, the presence of supporting cells may also promote myog enic cells proliferation and differentiation ( Joe et al. 2010 ). Clearly additional studies are needed to elucidate this effect, but the unique effect of IL 4 on lightly stained cells was an unexpected observation in the present study and suggests a novel m echanism for differential muscle repair. Overall our findings suggest that IL 4 promotes differentiation and fusion of myotubes and myofibers and this is in accordance with recent studies that have demonstrated the importance of IL 4 in myogenesis (Charvet et al. 2006). In addition, we saw an effect on light ly stained supporting cells, which may be indicative of another key factor in muscle repair. Differential Effects of Cytokine on Presumptive Myogenic Stem Cells Derived from MAS and TA As cell proportions differed significantly between MAS and TA control cultures, in order to compare the effects of cytokines between MAS and TA, data was normalized to control means for each group. This eliminated any differences that might be the result of differ ences in normal growth between MAS and TA. In proliferation cultures significant differences were observed between MAS and TA in the effects of 4 on the proportion of light cells. Previous studies have demonstrated that formation of myofibroblastic cells is an adverse effect of chronic inflammation (Li et al 2004) and phenotypic changes from myoblast to non myogenic

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54 cell types have been reported in s everal in vitro models in response to a variety of environmental cues including cytokines and growth factors (Joe et al 2010; Payne et al 2010; Shefer et al 2004). No other significant effects were observed on cell phenotype proportion between MAS and TA cultures indicating that exposure to cytokines had similar effects on myogenic cells derived from both MAS and TA. These results would suggest that the diminished capacity of masseter to repair is not the result of an increased adverse effect of examin ed cytokines on presumptive myogenic cells in masseter. Conclusion The results of this study indicate that, in our in vitro model, presumptive myogenic stem cells derived from the fibers of MAS and TA are capable of ac tivation, proliferation and formation of myofibers. However, cultures derived from MAS and TA differed in cell phenotype proportions suggesting differences in maturation. Cytokine administration affected both proliferation and differentiation of presumptiv e myogenic stem cells. In general, TNF and IL 1 enhanced proliferation. I L 1 and IL 4 positively influenced proliferation and differentiation and IL 4 promoted fusion. These effects varied significantly with concentration of cytokines. E xposure to thes e cytokines has similar effects on myogenic cells derived from TA or MAS Overall, these results suggest that the diminished capacity of MAS to repair is not the result of an increased adverse effect of these cytokines on presump tive myogenic cells in MAS

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55 LIST OF REFERENCES Al Shanti, N., Saini, A., Faulkner, S.H. and Steward, C.E. (2008). Beneficial s ynergistic interactions of TNF 6 in C2 skeletal myoblasts Potential cross talk with IGF system. Growth Factors. 26 (2), 61 73. Andrianjafiniony, T., Dupr Aucouturier, S., Letexier, D., Couchoux, H. and Desplanches, D. (2010). Oxidative stress, apoptosis, and proteo lysis in skeletal muscle repair after unloading. Am J Physiol Cell Physiol. 299 307 C315 Beutler, B. and Cerami A. (1988). The history, properties, and biological effects of cachectin. Biochemistry. 27 (20), 7575 82. Bischoff, R. (1994). The satellite cell and muscle regeneration. New York, McGraw Hill. Broussard, S.R., McCusker R.H., Novakofski, J.E., S t rle, K., Shen, W.H., John son, R.W. Dantzer, R. and Kelley, K.W. (2004). IL nsulin like growth factor I induced differentiation and downstream activation signals of the insulin like growth factor 1 receptor in myoblasts. J Immunol. 172 7713 7720. Broussard, S.R., McCu sker, R.H., Novakofski, J.E., Strle, K., Shen, W.H., Johnson, R.W., Freund, G.G., Dantzer, R and Kelley, K.W (2003). C ytokine hormone interactions: tumor necrosis factor alpha impairs biologic activity and downstream activation signals of the insulin like growth factor I receptor in myoblasts Endocrinology 144 2988 2996. Brickson, S., Ji, L.L., Schell, K., Olabisi, R., St Pierre Schneider, B. and Best, T.M. (2003). M1/70 attenuates blood borne neutrophil oxidants, activation, and myofiber damage following stretch injury. J Appl Physiol. 95 (3), 969 76. Charvet, C., Houbron, C., Parlakian, A., Giordani, J., Lahoute, C., Bertrand,A., Sotiropoulos, A., Renou L., Schm itt, A., Melki, J., Li, Z., Daegelen, D. and Tuil D. (2006). New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin like growth factor 1 pathways. Mol Cell Biol. 26 (17), 6664 6674. Cooney, R.N., Maish, G.O. 3rd, Gilpin, T., Shumate, M.L., Lang, C.H. and Vary, T.C. ( 1999). Mechanism of IL 1 induced inhibition of protein synthesis in skeletal muscle. Shock 11 (4), 235 41. Dworkin, S.F., Huggins, K.H., LeResche, L., Vo Korff, M., Howard, J., Truelove, E. and Sommers, E. (1990). Epidemiology of signs and symptoms in temporomandibular disorders clinical signs in cases and controls J Am Dent Assoc. 120 (3), 273 81.

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56 Grounds, M., Radley, H.G., Gebski, B.J. Bogoyevitch, M.E. and Shavlakadze, T. (2008). Implications of cross talk between tumor necrosis factor and insulin like growth factor 1 signaling in skeletal muscle. Clinical and Experimental Pharmacology and Physiology 35 846 851. Harris, A (2010). Does an altered inflammatory response ha ve a role in delayed masseter muscle repair? (Unpublished masters thesis). University of Florida, Gainesville, FL. Hodgetts, S., Radley, H., Davies, M. and Grounds, M.D. (2006). Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNF alpha function with Etanercept in mdx mice. Neuromuscul Disord. 16 (9 10), 591 602. Horsley, V., Jansen, K.M., Mills, S.T. and Pavlath, G.K. (2003) IL 4 acts as a myoblast recruitment factor during mammalian muscle growth. Cell 113 483 494. Joe, A W Yi L Natarajan A Le Grand F So L Wang J Rudnicki M A and Rossi F M. (2010). Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol 12 (2):153 63. Kumar, A. and Boriek, A.M. (2003). Mechanical stress activates the nuclear factor pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy. FASEB J. 17 386 96. Langen, R.C., Schols, A.M., Kelders M.C., Wouters, E.F. and Janssen Heininger, Y.M. (2003). Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor FASEB J 15 1169 1180. Langen RCJ, Van der Velden JLJ, Schols AMW, Kelders MCHM, Wouters EFM, Janss en Heininer YMJ. (2004). Tumor necrosis factor differentiation through myoD protein destabilization. FASEB J 18 227 237 LeResche, L. (1997). Epidemiology of temporomandibular disorders: implications for the investigation of etiologi c factors. Crit Rev Oral Biol Med. 8 (3), 291 305. Li, Y.P. (2003) TNF Am J Physiol Cell Physiol 285 370 376. Li, Y., Foster, W., Deasy, B.M., Chan, Y., Prisk, V., Tang, Y., Cummins, J. and Huard, J. (2004) Transforming growth factor beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 164 (3), 1007 19. Ludolph, D. C. and Konieczny, S.F. (1995). Transcription fact or families: muscling in on the myogenic program FASEB J. 9 (15), 1595 604.

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59 BIOGRAPHICAL SKETCH Camden Doughtie was born in Savannah, Georgia. She grew up in Hilton Head Island, SC with her parents, Collins and Allison, and her younger brother, Logan. At the age of 12 she moved to Tampa, FL where she attended midd le school and high school. Upon graduation she enrolled in the University of Florida. While at the university, Camden majored in n euroscience and completed research on spinal cord regeneration at the McKnight Brain Institute under the mentorship of Dr. Den a Howland. In 2004, she graduated and moved to Boston, MA. There, Camden attended Harvard School of Dental Medicine and completed her dental degree. She completed research on implant supported restorations with Dr. German Gallucci. During dental school, she developed a strong interest in the field of orthodontics and applied for residency. In 2008 she enrolled again at the University of Florida, this time for a 3 year orthodontic residency. While in Gainesville, she worked with Drs Joyce Morris Wiman an d Charles Widmer on masseter muscle regeneration for her thesis. During her spare time Camden enjoys cooking, traveling and watching foreign films. After graduation in May 2011 she plans to move to a new city and begin work as a private practice orthodont ist while her fianc, Andrew Brown, attends graduate school for architecture.