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Increased Vertical Dimension Effects on Masseter Muscle Phenotype During Maturation

Permanent Link: http://ufdc.ufl.edu/UFE0024472/00001

Material Information

Title: Increased Vertical Dimension Effects on Masseter Muscle Phenotype During Maturation
Physical Description: 1 online resource (35 p.)
Language: english
Creator: Nguyen, Vo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: dimension, heavy, masseter, masticatory, mhc, mice, mouse, muscle, myhc, myosin, stretch, vdo, vertical
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

Abstract: Select orthodontic cases are facilitated by increasing the vertical dimension of occlusion (VDO). In animal studies that increased VDO by 20% or greater, masseter myosin heavy chain (MyHC) message shifts in relative expression from fast to slow. However, it is unclear what changes in MyHC protein expression may occur with a clinically relevant vertical dimension increase. Six experimental CD-1 male mice (age: 6 weeks) underwent a 10% bite opening to replicate the clinical condition using composite on the maxillary molars and were compared to six age-matched control male mice. The mice were sacrificed at day 7 and 14 after bite opening. A representative masseter transverse cryosection from each animal was examined in selected sampling regions (anterior, posterior, posterior-deep, and posterior-intermediate) to assay fiber phenotype proportions. In controls, the proportion of muscle fibers containing MyHC IIb increased in the posterior-intermediate and posterior-deep regions between 7 and 14 days (ANOVA, p < 0.05). With bite opening, the increase in proportion of MyHC IIb fibers in the bite opening group (exp-14) did not occur when compared to the control group (ctl-14) (p < 0.05). In addition, after 14 days of bite opening, the proportion of fibers positive for MyHC IIa was greater in the anterior region with bite opening compared to control masseters. Fiber diameter remained unchanged in both groups (experimental and control) and over time (p > 0.10). These data are consistent with a selective plasticity of the expression of MyHC IIb in the deep regions of the male masseter muscle.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Vo Nguyen.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Widmer, Charles G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024472:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024472/00001

Material Information

Title: Increased Vertical Dimension Effects on Masseter Muscle Phenotype During Maturation
Physical Description: 1 online resource (35 p.)
Language: english
Creator: Nguyen, Vo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: dimension, heavy, masseter, masticatory, mhc, mice, mouse, muscle, myhc, myosin, stretch, vdo, vertical
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

Abstract: Select orthodontic cases are facilitated by increasing the vertical dimension of occlusion (VDO). In animal studies that increased VDO by 20% or greater, masseter myosin heavy chain (MyHC) message shifts in relative expression from fast to slow. However, it is unclear what changes in MyHC protein expression may occur with a clinically relevant vertical dimension increase. Six experimental CD-1 male mice (age: 6 weeks) underwent a 10% bite opening to replicate the clinical condition using composite on the maxillary molars and were compared to six age-matched control male mice. The mice were sacrificed at day 7 and 14 after bite opening. A representative masseter transverse cryosection from each animal was examined in selected sampling regions (anterior, posterior, posterior-deep, and posterior-intermediate) to assay fiber phenotype proportions. In controls, the proportion of muscle fibers containing MyHC IIb increased in the posterior-intermediate and posterior-deep regions between 7 and 14 days (ANOVA, p < 0.05). With bite opening, the increase in proportion of MyHC IIb fibers in the bite opening group (exp-14) did not occur when compared to the control group (ctl-14) (p < 0.05). In addition, after 14 days of bite opening, the proportion of fibers positive for MyHC IIa was greater in the anterior region with bite opening compared to control masseters. Fiber diameter remained unchanged in both groups (experimental and control) and over time (p > 0.10). These data are consistent with a selective plasticity of the expression of MyHC IIb in the deep regions of the male masseter muscle.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Vo Nguyen.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Widmer, Charles G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024472:00001


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1 INCREASED VERTICAL DIMENSION EFFECTS ON MASSETER MUSCLE PHENOTYPE DURING MATURATION By VO DANH MANH NGUYEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Vo Danh Manh Nguyen

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3 To my familyI am grateful for the love they have provided me.

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4 ACKNOWLEDGMENTS I thank m y parents, brother, and uncle for their help and loving support. I would not be where I am without their belief in me. I thank my girlfriend for being by my side and having the patience to make this journey enjoyable. I would also like to thank the members of my committee Drs. Calogero Dolce, Joyce Morris-Wiman, and Charles Widmer for their guidance and mentorship throughout this project.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES.........................................................................................................................7 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INTRODUCTION..................................................................................................................10 2 MATERIALS AND METHODS........................................................................................... 12 Animals...................................................................................................................................12 Groups.....................................................................................................................................12 Bite Opening...........................................................................................................................12 MyHC (myosin heavy chain) Immunolabeling...................................................................... 13 Muscle Fiber Sampling and Assessment................................................................................ 14 Statistical Analyses........................................................................................................... ......15 3 RESULTS...............................................................................................................................18 Animal Weight.................................................................................................................. ......18 Masseter Muscle Fiber Numbers............................................................................................ 18 Myosin Heavy Chain Phenotype............................................................................................18 Differences Between Control Groups Over Time........................................................... 18 Effects of a VDO (vertical dimens ion of occlusion) Increase ......................................... 19 Cross-Sectional Diameter................................................................................................ 20 4 DISCUSSION.........................................................................................................................28 LIST OF REFERENCES...............................................................................................................32 BIOGRAPHICAL SKETCH.........................................................................................................35

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6 LIST OF TABLES Table page 3-1 Mixed model analysis of va riance for percent fiber type .................................................. 21 3-2 Mixed model analysis of variance for fiber diam eter........................................................ 26

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7 LIST OF FIGURES 2-1 Retraction for placement of bite opening composite......................................................... 16 2-2 Representative images of transverse sections of the masseter immunostained for MyHC ( myosin heavy chain) IIa and IIb showing the regions sampled........................... 17 3-1 The posterior-deep region.................................................................................................. 22 3-2 The posterior-int erm ediate region...................................................................................... 23 3-3 The anterior region........................................................................................................ .....24 3-4 The posterior region....................................................................................................... ....25 3-5 Minimum fiber diameter (mean + st. error) for the four control/experimental groups...... 27

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INCREASED VERTICAL DIMENSION EFFECTS ON MASSETER MUSCLE PHENOTYPE DURING MATURATION By Vo Danh Manh Nguyen May 2009 Chair: Charles G. Widmer Major: Dental Science Select orthodontic cases are facilitated by in creasing the vertical dimension of occlusion (VDO). In animal studies that increased VDO by 20% or greater, masseter myosin heavy chain (MyHC) message shifts in relativ e expression from fast to slow However, it is unclear what changes in MyHC protein expression may occur w ith a clinically releva nt vertical dimension increase. Six experimental CD-1 male mice (age: 6 weeks) underwent a 10% bite opening to replicate the clinical condition using composite on the maxillary molars and were compared to six age-matched control male mice. The mice were sacrificed at day 7 and 14 after bite opening. A representative masseter transverse cryosection from each animal was examined in selected sampling regions (anterior, posterior, posterior-d eep, and posterior-interme diate) to assay fiber phenotype proportions. In controls the proportion of muscle fibers containing MyHC IIb increased in the posterior-intermediate and posterior-deep regions between 7 and 14 days (ANOVA, p<0.05). With bite opening, the increase in proportion of MyHC IIb fibers in the bite opening group (exp-14) did not occur when compared to the control group (ctl-14) (p<0.05). In addition, after 14 days of bite opening, the pr oportion of fibers positive for MyHC IIa was greater in the anterior region with bite opening compared to control masseters. Fiber diameter remained unchanged in both groups (experimental and control) and over time (p > 0.10). These

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9 data are consistent with a selective plasticity of the expression of MyHC IIb in the deep regions of the male masseter muscle.

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10 CHAPTER 1 INTRODUCTION The treatm ent of select orthodont ic cases is facilitated by incr easing the vertical dimension of occlusion (VDO). The VDO is defined as the le ngth of the face when the teeth are in contact and the mandible is in centric relation or the teeth are in centric relation.1 Orthodontic treatment modalities that open the VDO are implemented to al leviate crossbites, to allow the placement of mandibular brackets in deep bite cases, to disclude the dentition facilitating tooth movement, or to help level the curve of Spee.2 The increase in VDO stretches jaw closing muscles and, thus, may have a more direct effect on jaw closers compared to ot her muscles in the oral region.3 Studies using adult animal models have examined the effect of elongating limb muscle on their contractile properties.4-7 However, it is unclear whether or not jaw musculature responds to stretch in the same manner with vertical opening. To complicate the assessment of the effects of an increase in VDO, many patients treated by an increase in VDO in ort hodontics are adolescents and are still growing and it is unclear wh at effect growth has on this response. A muscles contractile propert ies are in part governed by th e protein, myosin. Myosin is made up of a pair of heavy and pair of light ch ains. Muscle speed of contraction depends on the myosin heavy chain (MyHC) isoforms, and th e major types of adult MyHC isoforms in mammalian skeletal muscles can be categorized as slow (MyHC I/ ) or fast (MyHC II).8, 9 Fast fiber types can further be separated into MyHC IIa, IIx/d, or IIb based on their contraction speeds.10-13 During muscle fiber development, early MyHC isoforms (embryonic and neonatal/perinatal/fetal) are redu ced or eliminated completely and replaced by adult forms. Within these adult forms, muscles that functi on with slow, prolonged contractions will have a predominant proportion of slow MyHC fibers wh ile muscles that have quick, short duration, phasic contractions will have a higher proportion of fast MyHC fibers. These slow and fast

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11 muscles are pre-programmed; howev er, the final phenotypes of these muscles can vary due to the plasticity of muscle to functional demands muscle stretch, and hormonal influences.14-17 Muscle stretching can alter MyHC phenotype wi thin muscle fibers. The MyHC IIa gene has been shown to be upregulated with muscle fiber stretch in rat plantaris and gastrocnemius muscles.18, 19 Passive stretching of these muscles led to an increase in MyHC IIa messenger RNA (mRNA). A similar result was observed in C2C12 mouse myoblasts cells. Mechanical stretching of the myoblasts shifted the MyHC mRNA towa rds a MyHC IIa expression. The stretch caused an initial increase in IIb mR NA but continued stretching cau sed an increase in IIa mRNA.20 These findings demonstrate the close relati onship between MyHC plasticity and function. The effect of bite opening (or muscle stre tch) on masseter MyHC fiber phenotype has not been examined in depth. Only a few studies ha ve evaluated VDO and mass eter MyHC plasticity 21-23; however, in all studies, the au thors opened the bite beyond an orthodontic clin ical relevance (20 50%). In humans the average maximum opening is about 45 50 mm and orthodontic procedures open the anterior bite by about 3 4 mm, which is 10% or less of the maximum bite opening at the incisors.24-27 A smaller, yet clinically appli cable, bite opening has not been investigated. The purpose of our study was to examine the eff ects of a clinically re levant bite opening on the muscle fiber MyHC content in the adoles cent male mouse masseter during maturation. Two hypotheses were tested: (1) the myosin heavy chain phenotype of the masseter muscle fibers will change to a slower (MyHC IIa) phenotype in response to the new, increased VDO; and (2) increasing the VDO will increase the mean diamet er of muscle fibers containing MyHC IIa.

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12 CHAPTER 2 MATERIALS AND METHODS Animals CD-1 m ale mice (Charles Rivers Laboratories) were maintained in the animal facilities at the University of Florida Health Science Cent er. Mice were maintained on a 12 h light/ dark cycle with food and water ad libitum. Twelve mice, six weeks of age, were used in the study. Sample size estimation was based on power-sampl e size calculations usi ng a power of 0.9, alpha of 0.05, and effect size of at l east 1.5 (from previous data). Animal testing procedures and general handling complied with the ethical guidelines and standards established by the Institutional Animal Care & Us e Committee at the University of Florida, and all procedures complied with the Guide for Care and Use of Laboratory Animals. Groups The m ice were divided into two groups: six mice were assigned to the control group and six mice were assigned to the treatment group. Both control (ctl) and experimental (exp) groups experienced the same anesthesia ( ketamine: 70 80 mg/kg body weight and xylazine: 5 10 mg/kg body weight) The experimental group was subjected to an increase of vertical dimension using composite placed over the maxillary molars for 7 or 14 days while the control group only had a sham manipulation consisting of mandibul ar retraction without composite placement. Weight was monitored at the beginning a nd end of the study. Mice were sacrificed after 7 days (ctl-7, exp-7) and 14 days (ctl14, exp-14) and masseter muscles we re harvested bilaterally, flash frozen, and stored at -20C until sectioned. Bite Opening The m aximum bite opening of a six week old mouse was measured to be 10 mm using a caliper. Therefore, a 1 mm vertical separator wa s placed between the incisors to produce a 10%

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13 vertical opening. The oral cav ity was exposed using custom-f abricated 0.018 mm orthodontic wires (Figure 2-1). Using endodontic cotton pellets, the right and left maxillary molars were dried after a water rinse and etch ed for 45 s (etch gel 40%). The etchant was then removed with cotton, rinsed, and dried and bonding agent was applied with a microbrush, followed by light curing. Flowable composite was applied and adjusted as needed with a scalar. The composite was set using a 20 s light cure. The control mice had the same manipulations but no composite was placed. All animals received 0.05 mg/kg buprenorphine h ydrochloride for the first 48 h to manage post-operative discomfort. MyHC Immunolabeling One m asseter from each animal was cr yosectioned in the transverse plane Serial 14 m transverse sections of masseter were placed consecutively on sets of contiguous slides to allow the direct comparison of different antibody la beling. Slides were stored at -20C until immunostained. Sections were washed in PBS (phosphate buffered saline) and incubated for 20 min in 1% BSA (bovine serum albumin) with 50 g/ml goat-anti-mouse IgG (for mouse monoclonal primary antibodies) or 1% normal goat serum (for polyclonal primary antibodies) to block non-specific binding. Sections were incuba ted overnight at 4C in primary antibodies: mouse anti-IIa MyHC (myosin heavy chain) IgG (clone SC71, ATCC, Schiaffino et al8); mouse anti-IIb MyHC IgM (clone B FF3, ATCC, Schiaffino et al8); and rat anti collagen IV (Biodesign). The sections were washed in PBS prior to inc ubation in an appropriate secondary antibody (goat anti-mouse IgG conjugated to Alexa 488; goat antimouse IgM conjugated to Alexa 546; or goat anti-rat IgG conjugated to Alexa 350) for 1 h at room temperature. After extensive rinsing, sections were mounted in glycerol to which an anti-bleaching agent had been added and viewed on a Nikon FXA photomicroscope. Control slides were incubated in se condary antibody only. The immunolabeled transverse muscle section just superior to the aponeurosis of the superficial

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14 layer of the masseter was acquired using a digitiz ing camera (Zeiss Mrc5); the individual images comprising the section assembled into collages (Photoshop CS2) and analyzed using Image Pro Plus software (Media Cybernetics). Muscle Fiber Sampling and Assessment To obtain a representative sa m pling of the masseter secti on, four regions (anterior, posterior, posterior-intermediate, posterior-deep) were chosen, based on the spatial distributions of MyHC isoforms and anatomy of the muscle (Figure 2-2).28 The anterior region represented an area with a high proportion of MyHC IIa fibers, while the posterior region has a high proportion of MyHC IIb fibers. The posterior-intermediate and posterior-deep regions have been identified as areas of fiber type differences between the male and female. Each region was sampled using two 450 x 450 m areas of interest. Thus, a total of eight areas of interest representing the four regions were evaluate d. All fiber counts and fiber identifications were completed by a single investigator who was blinded to the source of the images to minimize bias. Each area of intere st image was thresholded to remove background fluorescence and the muscle fibers were outlin ed and then designated as IIa, IIb, IIa/b combination, or unlabeled. The fiber counts from the two areas of inte rest were pooled and proportions of each fiber type for a designate d region were then calculated. The minimum diameter of each muscle fiber in the two areas of interest for each region was also pooled to characterize fibers for each re gion. A test-retest of fiber type assessment was calculated to determine the reliabil ity of identification. A total count of muscle fibers was obtained fr om assessment of all fibers identified on a transverse cryosection of the ma sseter of three control mice (age 7 weeks). The total count was used to compare the number of fibers obtaine d by the sampling procedure to assess the sampling process.

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15 Statistical Analyses Myosin heavy chain phenotype pr oportions (IIa and IIb) and mu scle fiber diameters were analyzed for differences across time (7 and 14 da ys), occlusal condition (increased vertical dimension of occlusion and sham manipulated) and location (4 regions) utilizing a mixed model ANOVA (Analysis of Variance) and a probabil ity level of p<0.05. When the ANOVA was found to be significant, multiple comparisons were conducted using the Least Significant Difference (LSD) test.

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16 Figure 2-1. Retraction for placement of bite ope ning composite. Intraoral access to the maxillary molars was accomplished using custom-fabricat ed separators to maintain vertical opening and cheek retraction.

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17 Figure 2-2. Representative images of transver se sections of the masseter immunostained for MyHC (myosin heavy chain) IIa and IIb showing the regions sampled. Squares delineate two standardized areas of intere st per region that were sampled for the anterior, posterior, posterior-i ntermediate, and posterior-deep regions. A) IIa fibers B) IIb fibers. Anterior Posterior Post.-Deep Post.-Intermed. B Anterior Posterior Post.-Deep Post.-Intermed. A

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18 CHAPTER 3 RESULTS Animal Weight All m ice gained weight during the period of study (7 or 14 days). Mean mouse weight gain (+ st. error) after 7 days was 4.3 g (+ 0.3) and after 14 days was 6.8 g (+ 1.0). The weight gain after 14 days was significantly greater compared to one week (unpair ed t-test, p < 0.03). Masseter Muscle Fiber Numbers The m ean (+ st. error) number of muscle fibers assessed for each muscle transverse section was 673 + 14 fibers. The average number of muscle fibe rs within the entire transverse section of three control masseter muscles at 7 weeks of age was found to be 4726 + 215. Therefore, the sampling technique assessed approximately 14% of the total fiber population which was an adequate sample to evaluate regional fiber char acteristics. A MyHC (myo sin heavy chain) type IIa/b dual expression in muscle fibers was identified in our study but was found in less than 1% of the muscle fibers. Due to the limited numbers of these fibers, we did not report on them. Testretest reliability was found to be 8% for all fi ber types (MyHC IIa, IIb and IIa/IIb) with most of the discrepancies (> 6%) attributed to the MyHC IIa/I Ib identification. Myosin Heavy Chain Phenotype Differences Between Control Groups Over Time After calculation of the ANOVA (Analysis of Variance) for differences in the proportion of MyHC IIa and IIb, a statis tically significant interaction w as observed among all factors (regions, MyHC, group, and time; Table 3-1). Po st hoc comparisons were calculated for all combinations of factors for each region of the transverse section (Least Significant Difference test; significant differences show n in Figures 3-1A through 3-4A).

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19 Statistically significant differences were observed in the proportion of fiber types in the posterior-deep region between contro l (ctl) groups at 7 and 14 days (ctl-7 and ctl-14 groups). The proportion of IIa fibers decreased from 30% to 5% and the proportion of IIb fibers increased from 28% to 50% (Figure 3-1A, ct l-7 vs. ctl-14). A similar statistically significant change in the proportion of fiber types was found in the posteri or-intermediate region. The proportion of IIa fibers decreased from 39% to 5% and the propor tion of IIb fibers increased from 11% to 39% (Figure 3-2A, ctl-7 vs. ctl-14). No statistically significant changes in fiber type proportions were detected in the anterior and posterior regions. Effects of a VDO Increase To evaluate the effects of an increase in VDO (vertical dim ension of occlusion) on MyHC fiber type proportions, statistical comparisons of the control and experimental (exp) groups were made after 7 days and 14 days of bite opening (7 days: ctl-7 vs. exp-7; 14 days: ctl-14 vs. exp14). No significant differences were found in the proportions of MyHC IIa or IIb fibers after 7 days of bite opening (Figure 3-1A through 34A: ctl-7 vs. exp-7). However, significant differences in MyHC IIa and IIb fiber proportions were observed after 14 days of bite opening in specific regions of the transverse section of the masseter muscle (LSD test (p < 0.05); Figure 31A through 3-4A: ctl-14 vs. exp-14). In the posterior-deep region, a significant diffe rence between the cont rol and experimental groups was found in the proportion of MyHC IIb fi bers (49% vs. 13% ) while no difference was observed in the proportion of MyHC IIa fibers (F igure 3-1A: ctl-14 vs. ex p-14). A representative image of the posterior-deep region of the masse ter is shown for both 14 day control (Figure 31B) and experimental groups (Figure 3-1C) and a significant reduction in the number of IIb fibers can be observed in the experimental group.

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20 An effect of bite opening was also found in the posterior-intermediate region as seen in Figure 3-2A. The proportion of MyHC IIb fibers (39% vs. 2%) and MyHC IIa fibers (5% vs. 39%) differed significantly between the control and experimental groups respectively (Figure 32A, ctl-14 vs. exp-14). A represen tative image of the posterior-intermediate region from the ctl14 group is shown in Figure 3-2B The region is mainly compos ed of IIb fibers and a few scattered IIa fibers. An obvious re duction in the proportion of MyHC IIb fibers after bite opening and the proportional increase of MyHC IIa fibers can be observed in th e exp-14 group (Figure 32C). In the anterior region, no MyHC IIb fibers were identified. However, a significant reduction in the proportion of MyHC IIa fibers (55%-29%) between cont rol and experimental groups was observed after 14 days of bite opening (Figure 3-3A). These differences in the density of MyHC IIa labeled fibers can be readily detected in sections from the anterior regions of control and experimental massete rs as shown in Figure 3-3B, C. In the posterior region, no statistically significant differences were found in the proportion of IIa or IIb fibers between 14 day control to experimental gro ups (Figure 3-4A: ctl-14 vs. exp14). The posterior region contains predominantly MyHC IIb fibers (Figure 3-4B) with a very small proportion of IIa fibers. Bite opening did no t have an obvious eff ect on the proportion of muscle fiber MyHC IIa or IIb phenotype s in this region (Figures 3-4B,C). Cross-Sectional Diameter The m inimum cross-sectional diameter of the My HC IIa and IIb muscle fibers did not vary across the control or experimental groups (Tab le 3-2). However, a statistically significant difference was observed between the sizes of the IIa fibers in comparison with the IIb fibers with the IIa fibers being consiste ntly smaller (Figure 3-5).

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21 Table 3-1. Mixed Model Analysis of Variance for Percent Fiber Type Effect SS Degrees of Freedom F-value p-value Between Group (Control vs. VDO) 3231 2.01 0.19 Time (day 7 vs. day 14) 2641 1.64 0.24 Group x Time 401 0.25 0.63 Within Regions (ant, post, postintermediate, post-deep) 2674 3 6.28 0.0027* Regions x Group 6773 1.59 0.22 Regions x Time 1313 0.31 0.82 Regions x Group x Time 5083 1.19 0.33 MyHC (IIa vs. IIb) 1391 0.44 0.53 MyHC x Group 2921 0.92 0.37 MyHC x Time 17351 5.44 0.048* MyHC x Group x Time 6261 1.96 0.20 Regions x MyHC 304463 57.09 0.0000* Regions x MyHC x Group 26123 4.90 0.009* Regions x MyHC x Time 7893 1.48 0.25 Regions x MyHC x Group x Time 27543 5.16 0.0067* *indicates statistically significant using ANO VA, LSD test, p< 0.05

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22 Posterior-Deep 0 20 40 60 80 Ctl-7 Exp-7Ctl-14Exp-14 GroupPercent Labeled Fibers IIA IIB Figure 3-1. The posterior-deep regi on. A) Percent labele d MyHC IIa and IIb fibers in the four groups: control-7 days (ctl-7), experimental-7 days (exp-7), control-14 days (ctl-14) and experimental-14 days (e xp-14). Statistically significant differences are denoted between groups by horizontal lines (LSD test, *p < 0.05). Re presentative images of the posterior-deep masseter region immunostained for MyHC IIa and IIb are shown for 14 day B) control and C) experimental groups. These images are composites of images immunostained for MyHC IIa (green ) and IIb (red) which have been overlaid to allow an analysis of the proportions of each fiber phenotype in each group at 14 days. A * Posterior-Deep Ctl-14 B Posterior-Deep Exp-14 C

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23 Posterior-Intermediate 0 20 40 60 80 Ctl-7 Exp-7Ctl-14Exp-14 GroupPercent Labeled Fibers IIA IIB Figure 3-2. The posterior-intermedi ate region. A) Percent labeled My HC IIa and IIb fibers in the four groups: control-7 days (ctl -7), experimental-7 days (e xp-7), control-14 days (ctl14) and experimental-14 days (exp-14). St atistically significant differences were denoted between groups by horizontal line s (LSD test, *p < 0.05). Representative images of the posterior-intermediate ma sseter region immunostained for MyHC IIa and IIb are shown for 14 day B) control and C) experimental groups. These images are composites of images immunostained fo r MyHC IIa (green) and IIb (red) which have been overlaid to allow an analysis of the proportions of each fiber phenotype in each group at 14 days. A * Posterior-Intermed. Ctl-14 B Posterior-Intermed. Exp-14 C

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24 Anterior 0 20 40 60 80 Ctl-7Exp-7Ctl-14Exp-14 GroupPercent Labeled Fibers IIA IIB Figure 3-3. The anterior region. A) Percent labeled MyHC IIa and IIb fibers in the four groups: control-7 days (ctl-7), expe rimental-7 days (exp-7), control-14 days (ctl-14) and experimental-14 days (exp-14) Statistically significant differences were denoted between groups by horizontal lines (LSD test, *p < 0.05). Re presentative images of the anterior masseter region immunostaine d for MyHC IIa and IIb are shown for 14 day B) control and C) experimental groups. These images are composites of images immunostained for MyHC IIa (green) and IIb (red) which have been overlaid to allow an analysis of the proportions of each fi ber phenotype in each group at 14 days. A Anterior Ctl-14 A Anterior Exp-14 B

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25 Posterior 0 20 40 60 80 Ctl-7 Exp-7Ctl-14Exp-14 GroupPercent Labeled Fibers IIA IIB Figure 3-4. The posterior region. A) Percent labeled MyHC IIa and IIb fibers in the four groups: control-7 days (ctl-7), expe rimental-7 days (exp-7), control-14 days (ctl-14) and experimental-14 days (exp-14) Statistically significant differences were denoted between groups by horizontal lines (LSD test, *p < 0.05). Re presentative images of the posterior masseter region immunostaine d for MyHC IIa and IIb are shown for 14 day B) control and C) experimental groups. These images are composites of images immunostained for MyHC IIa (green) and IIb (red) which have been overlaid to allow an analysis of the proportions of each fi ber phenotype in each group at 14 days. A Posterior Ctl-14 B Posterior Exp-14 C

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26 Table 3-2. Mixed Model Analysis of Variance for Fiber Diameter Effect SS Degrees of Freedom F-value p-value Between Group (Control vs. VDO) 4.08 1 0.065 0.81 Time (day 7 vs. day 14) 0.99 1 0.016 0.90 Group x Time 16.6 1 0.26 0.62 Within Regions (ant, post, post-intermediate, post-deep) 18.57 2 0.55 0.59 Regions x Group 76.6 2 2.25 0.14 Regions x Time 30.6 2 0.90 0.43 Regions x Group x Time 43.9 2 1.29 0.30 MyHC (IIa vs. IIb) 1499 1 65.5 0.00004* MyHC x Group 0.02 1 0.0007 0.98 MyHC x Time 0.94 1 0.04 0.84 MyHC x Group x Time 44.5 1 1.95 0.20 Regions x MyHC 11.09 2 0.58 0.57 Regions x MyHC x Group 5.55 2 0.29 0.75 Regions x MyHC x Time 4.93 2 0.26 0.78 Regions x MyHC x Group x Time 18.61 2 0.97 0.40 *indicates statistically significant using ANO VA, LSD test, p< 0.05

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27 Minimum Fiber Diameter 0 5 10 15 20 25 30 35 40 Ctl-7 Exp-7Ctl-14Exp-14 GroupDiameter (microns ) IIA IIB Figure 3-5. Minimum fiber diameter (mean + st. error) for the four control/experimental groups. No statistically significant differences we re found between groups for each fiber type (ANOVA, p > 0.1). However, masseter muscle fibers that contain MyHC IIa were statistically smaller than type IIb fibers (ANOVA, LSD test, *p < 0.05). *

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28 CHAPTER 4 DISCUSSION Using m ale adolescent mice to model the age of orthodontic patients, this study assessed the plasticity of the masseter muscle to a sma ll, short term vertical bite opening. The rapid decrease in the proportion of MyHC (myosin hea vy chain) IIb protein in the experimental, bite opening group over a 14 day period in this study was consistent with other reports in the literature that have evaluated MyHC responses in fast contra cting muscles. Chronic, low frequency electrical stimulation of the rat tibialis anterior muscle reached a significant change in MyHC IIb protein levels after 8 days.29 In cranial nerve innervated extraocular muscle, a surgical shortening of the muscle caused a significant decrease in MyHC IIb protein 3 days after surgery.30 The IIb protein has a half life of 14.5 days in limb muscle29 and the degradation of this protein is necessary to allow the insertion of new protein (for exam ple, MyHC IIa or IIx) in the sarcomere. One advantage of this study was the examina tion of regions of the masseter muscle to assess potential local plastic changes. In fact, the masseter posterior-intermediate and posteriordeep regions were found to be responsive to a small increase in VDO (vertical dimension of occlusion) with a relative propor tional decrease in MyHC IIb fi bers and a rela tive increase in MyHC IIa fibers when compared to controls at 14 days. Interestingly, a different response was found in the anterior region with a relative increase in the proportion of MyHC IIa fibers while no change was found in the posterior region of the muscle. It has been shown previously in the rabbit masseter that this muscle is multipennate and has multiple functional compartments that are activated at different times in th e chewing cycle depe nding on the task.31 In addition, the orientation of muscle fibers va ries throughout the muscle and those fibers with a more vertical orientation would be more highly affected by a muscle stretch than oblique fibers.

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29 A possible explanation for the maintenance of a relatively slower phenotype with bite opening in the posterior-intermedi ate and posterior-deep regions may be due to fiber stretch.15, 16 Muscle stretch of the rabbit ex tensor digitorum longus muscle causes a MyHC transition to a slower phenotype.16 The transition to a slower phenotype f ound in fast limb muscle is consistent with masseter muscle. In rat masseter studies th at used an approximate 20% increase in the VDO, a relative decrease in MyHC IIb and increase in MyHC IIa message was found.22, 23 These changes in MyHC gene expressi on were consistent with the pr otein changes observed in this study. In addition to stretch, a second possible expl anation for the maintenance of a slower phenotype in our bite opening group is an increase in muscle function. Although, the experimental mice were eating and gaining we ight, they may have changed their chewing patterns and masticatory muscle activity. Motor units in the posterior-intermediate and posteriordeep areas may have been preferentially recru ited to chew or brux due to composite placement on maxillary molars. Increased regional muscle ac tivation has been shown in adult rat tibialis anterior muscle to promote a slower phenotype.32 The slower phenotype associated with increased muscle activity was also shown w ith Japanese Waltzing mice, a line of mice characterized by mutations affec ting the vestibular apparatus l eading to increased locomotor activity when compared to normal mice.33 The change in motor activation strategies may have also affected the anterior masseter by reduced acti vation of this region. The anterior region of the masseter muscle is typically recruited during incising34 and the ability to incise would theoretically be reduced with the increase in vertical dimension due to the increase in gape. The reduced activation would preferentially favor the pr esence of faster contra cting fibers and would result in the relative reduction in the proportion of MyHC IIa fibers. Since no MyHC IIb positive

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30 fibers were identified in the anterior regi on, the faster phenotype could be MyHC IIx, a phenotype that was not ev aluated in this study. In the control mice, a transition to a faster phenotype represented by increases in the proportion of MyHC IIb and decrease in the prop ortion of MyHC IIa occu rred in the posteriordeep and posterior-intermediate regions of the masseter muscle at 14 days compared to 7 days. MyHC IIb positive fibers are found in higher proportions in the male mouse masseter compared to the female 28, 35 and sexual dimorphism has been observed in these two regions. The increase in the proportion of MyHC IIb fibers in the posterior-deep and posteri or-intermediate regions may be linked to hormones such as testosterone. Testosterone has been demonstrated to play a role in the transition of MyHC fiber types to a faster phenotype during masseter maturation in male mouse as well as in rabbit and guinea pig.17, 36, 37 The average diameter of IIb fibers has been obs erved to be greater than IIa fibers both in limb muscle and mouse masseter.28, 38 Therefore, one would predic t that the increases in the proportion of IIb fibers observed at 14 day in the posterior-deep and posterior-intermediate regions of control masseter would be accompan ied by changes in average fiber diameter. However, no significant differences in fiber diameter were obser ved; the fiber size remained consistent across all groups. On the other hand our inability to detect change in the muscle fiber size may be due to the short duration of the study. The limitations of this study incl ude the lack of an unmanipulat ed control and the inability to identify type IIx fibers. Our controls unde rwent the same surgical procedures as the experimental animals including mouth retraction. The stretch of the ma sseter produced by mouth retraction could possibly have either damaged the masseter or influenced muscle fiber phenotype. No differences were observed in fibe r phenotype between cont rol and experimental

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31 animals at 7 days. This lack of b ite opening effects coul d be the result of th e insensitivity of our method of detection (MyHC immunostaining), be the result of a normal delay in muscle phenotype switching in male mice at this age, or be due to adverse eff ects of mouth retraction. Additionally, the lack of a means to monitor MyHC IIx limits our interpretation of the results. We do not know if an immunonegative fiber is MyHC IIx, I, embryonic or neonatal. Therefore, any changes in proportions of immunonegative fibers could be due to increases in IIx (towards a faster phenotype) or due to increases in I (towards a slower phenotype). In summary, our study demonstrated that in control male mouse masseter two weeks of maturation was accompanied by transition towards a faster (IIb) phenotype. This maturation shift towards a faster phenotype in th e posterior-deep and posterior-int ermediate regions did not occur in masseters from male mice in which bite opening was increased by a 10%. However, the interpretations of the results of this study are limited by the lack of an unmanipulated control and methodological restrictions. In addition, the long term effects of bite opening are currently unclear and require further examination.

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32 LIST OF REFERENCES 1. Anonym ous. The glossary of prosthodontic terms. J Prosthet Dent 2005;94:10-92. 2. Muratore F, Varvara G, Tripodi D, de Simone R, Pascetta C, Festa F. Cerec 3 for orthodontics: A tool for treating deep bite. Int J Comput Dent. 2002;5:25-31. 3. Lindauer SJ, Gay T, Rendell J. Effect of jaw opening on masticatory muscle EMG-force characteristics. J Dent Res. 1993;72:51-5. 4. Tanaka H, Tanaka M, Sugi H. The effect of sarcomere length and stretching on the rate of ATP splitting in glycerinated rabbit psoas muscle fibers. J Biochem (Tokyo) 1979;86:1587-93. 5. Seider MJ, Kapp R, Chen CP, Booth FW. The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation. Biochem J 1980;188:247-54. 6. Matano T, Tamai K, Kurokawa T. Adaptation of skeletal muscle in limb lengthening: a light diffraction study on the sarcomere length in situ. J Orthop Res. 1994;12:193-6. 7. Coutinho EL, Gomes AR, Franca CN, Oishi J, Salvini TF. Effect of pa ssive stretching on the immobilized soleus muscle fiber morphology. Braz J Med Biol Res 2004;37:1853-61. 8. Schiaffino S, Reggiani C. Myosin isof orms in mammalian skeletal muscle. J Appl Physiol 1994;77:493-501. 9. Pette D, Staron RS. Myosin isoforms, muscle fiber types, and transitions. Microsc Res Tech 2000;50:500-9. 10. Sher J, Cardasis C. Skeletal musc le fiber types in the adult mouse. Acta Neurol Scand 1976;54:45-56. 11. Termin A, Staron RS, Pette D. Myosin heavy ch ain isoforms in histochemically defined fiber types of rat muscle. Histochemistry 1989;92:453-7. 12. Reiser PJ, Moss RL, Giulian GG, Greaser ML. S hortening velocity and myosin heavy chains of developing rabbit muscle fibers. J Biol Chem 1985;260:14403-5. 13. Morris TJ, Brandon CA, Horton MJ, Carlson DS, Sciote JJ. Maximum shortening velocity and myosin heavy-chain isoform expre ssion in human masseter muscle fibers. J Dent Res 2001;80:1845-8. 14. Agbulut O, Noirez P, Beaumont F, Butler-Browne G. Myosin heavy chain isoforms in postnatal muscle development of mice. Biol Cell 2003;95:399-406. 15. Morgan MJ, Loughna PT. Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression. FEBS Lett 1989;255:427-30.

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33 16. Yang H, Alnaqeeb M, Simpson H, Goldspink G. Changes in muscle fibre type, muscle mass and IGF-I gene expression in rabbit skeletal muscle subjected to stretch. J Anat. 1997;190:613-22. 17. Reader M, Schwartz G, English AW. Brief e xposure to testosterone is sufficient to induce sex differences in the rabbit masseter muscle. Cells Tissues Organs. 2001;169:210-7. 18. Loughna PT, Izumo S, Goldspink G, Nadal-Ginard B. Disuse and passive stretch cause rapid alterations in expression of developmental a nd adult contractile protein genes in skeletal muscle. Development 1990;109:217-23. 19. Loughna PT, Gibbs L, Bayol S, Brownson C. Ch anges in adult muscle phenotype in response to disuse and passive stretch. Biochem Soc Trans 1996;24:284S. 20. Sakiyama K, Abe S, Tamatsu Y, Ide Y. Effect s of stretching stress on the muscle contraction proteins of skeletal muscle myoblasts. Biomed Res. 2005;26:61-8. 21. Faulkner JA, McCully KK, Carlson DS, McNama ra JA,Jr. Contractile properties of the muscles of mastication of rhesus monkeys (Macaca mulatta) following increase in muscle length. Arch Oral Biol 1982;27:841-5. 22. Ohnuki Y, Saeki Y, Yamane A, Yanagisawa K. Quantitative changes in the mRNA for contractile proteins and metabolic enzymes in masseter muscle of bite-opened rats. Arch Oral Biol 2000;45:1025-32. 23. Arai C, Ohnuki Y, Umeki D, Saeki Y. Effe cts of bite-opening and cyclosporin A on the mRNA levels of myosin heavy chain and the muscle mass in rat masseter. Jpn J Physiol 2005;55:173-9. 24. Hellsing E, Hellsing G, Eliasson S. Effects of fixed anterior biteplane therapy--a radiographic study. Am J Orthod Dentofacial Orthop 1996;110:61-8. 25. Manns A, Miralles R, Palazzi C. EMG, bite force, and elongation of the masseter muscle under isometric voluntary contractions a nd variations of vertical dimension. J Prosthet Dent 1979;42:674-82. 26. Manns A, Miralles R, Guerrero F. The changes in electrical activity of the postural muscles of the mandible upon varying the vertical dimension. J Prosthet Dent. 1981;45:438-45. 27. Proffit WR, Fields HW, editors. Contemporary Orthodontics St. Louis, MO: Mosby; 2000. 28. Widmer CG, Morris-Wiman JA, Nekula C. Sp atial distribution of myosin heavy-chain isoforms in mouse masseter. J Dent Res 2002;81:33-8. 29. Termin A, Pette D. Changes in myosin h eavy-chain isoform synthesis of chronically stimulated rat fast-twitch muscle. Eur J Biochem 1992;204:569-73.

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34 30. Park SC, Kim YT, Kim SA, Oh SY. Changes in muscle fiber size and in the composition of myosin heavy chain isoforms of rabbit extraocular rectus muscle following recession surgery. Jpn J Ophthalmol 2008;52:386-92. 31. Widmer CG, Carrasco DI, English AW. Di fferential activation of neuromuscular compartments in the rabbit masseter musc le during different oral behaviors. Exp Brain Res. 2003;150:297-307. 32. Kirschbaum BJ, Schneider S, Izumo S, Mahda vi V, Nadal-Ginard B, Pette D. Rapid and reversible changes in myosin heavy chai n expression in response to increased neuromuscular activity of rat fast-twitch muscle. FEBS Lett 1990;268:75-8. 33. Asmussen G, Schmalbruch I, Soukup T, Pette D. Contractile properties, fiber types, and myosin isoforms in fast and slow muscles of hyperactive Japanese waltzing mice. Exp Neurol 2003;184:758-66. 34. Weijs WA, Dantuma R. Functional anatomy of the masticatory apparatus in the rabbit. Neth J Zool 1981;31:99-147. 35. Eason JM, Schwartz GA, Pavlath GK, English AW. Sexually dimorphic expression of myosin heavy chains in the adult mouse masseter. J Appl Physiol. 2000;89:251-8. 36. Lyons GE, Kelly AM, Rubinstein NA. Testoste rone-induced changes in contractile protein isoforms in the sexually dimorphic te mporalis muscle of the guinea pig. J Biol Chem 1986;261:13278-84. 37. English AW, Schwartz G. Development of sex differences in the rabbit masseter muscle is not restricted to a critical period. J Appl Physiol 2002;92:1214-22. 38. Rivero JL, Talmadge RJ, Edgerton VR. Fibre size and metabolic properties of myosin heavy chain-based fibre types in rat skeletal muscle. J Muscle Res Cell Motil. 1998;19:733-42.

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BIOGRAPHICAL SKETCH Vo Danh Manh Nguyen was born in Hanover, Germ any. He graduated from the University of Florida in 2001 with a Bachelor of Scien ce degree in food science and human nutrition. He received his Doctor of Dental Medicine degree in 2006 from the Un iversity of Florida College of Dentistry. He continued at the University of Flor ida and earned his Master of Science in dental science degree and Certifi cate in Orthodontics in 2009.