Biomineralization and adaptive plasticity of the temporomandibular joint in myostatin knockout mice

Archives of Oral Biology (2006) 51, 37—49

www.intl.elsevierhealth.com/journals/arob

Biomineralization and adaptive plasticity of the temporomandibular joint in myostatin knockout mice Elisabeth K. Nicholson a, Stuart R. Stock b, Mark W. Hamrick c, Matthew J. Ravosa a,* a

Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611-3008, USA b IBNAM, Northwestern University Feinberg School of Medicine, Chicago, IL, USA c Department of Cell Biology and Anatomy, Medical College of Georgia, Augusta, GA, USA Accepted 27 May 2005

KEYWORDS Temporomandibular joint (TMJ); Myostatin knockout mouse; MicroCT; Masticatory stress; Functional adaptation; Condylar form and function

Summary Mice lacking myostatin (GDF-8), a negative regulator of skeletal muscle growth, show a significant increase in muscle mass versus normal mice. We compared wild-type and myostatin deficient mice to assess the postnatal effect of elevated masticatory loads due to increased jaw-adductor muscle activity and greater bite forces on mandibular condyle morphology. Microcomputed tomography (microCT) was used to provide details of internal condylar morphology and quantify bone density in three condylar regions. Biomineralization levels, as well as external mandibular dimensions, were used to characterize within-slice, within-joint, within-group and between-group variation. Dimensions of the mandible and mandibular condyle were similar between the myostatin knockout and normal mice. Knockout mice exhibited significantly more biomineralization on the outer surface of the condylar subchondral bone and along the condylar neck, most notably on the buccal side of the condylar neck. The buccal side of the inner aspect of the condyle was significantly less biomineralized in knockout mice, both for the pooled data and for the posterior and anterior condylar slices. Whilst normal mice had symmetric subchondral bone surfaces, those of knockout mice were asymmetric, with a lower, less convex surface on the buccal side versus the lingual side. This appears related to the ontogenetic effects of increased masticatory stress in the mandibles of knockout mice as compared to normal mice. Significant differences in biomineralization between normal and myostatin knockout mice, coupled with the lack of significant differences in certain external dimensions, underscores a need for information on the external and internal morphology of mineralized tissues vis-a `-vis altered or excessive mechanical loads. # 2005 Published by Elsevier Ltd.

* Corresponding author. Tel.: +1 312 503 0492; fax: +1 312 503 7912. E-mail address: [email protected] (M.J. Ravosa). 0003–9969/$ — see front matter # 2005 Published by Elsevier Ltd. doi:10.1016/j.archoralbio.2005.05.008

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Introduction The initiation and progression of temporomandibular joint (TMJ) disease has been attributed to biomechanical factors including loss of posterior teeth, unilateral chewing, bruxism and masticatory dysfunction that lead to relative or absolute overloading of joint elements.1 Repetitive or cyclical loading also has been implicated in the etiology of TMJ disease.2 In addition to excessive, repetitive and/ or altered biomechanical loading, failure of the mechanical integrity of TMJ tissues is linked to a reduced postnatal ability of the composite tissues — bone, cartilage and synovium — to adapt to applied stresses. A number of studies suggest that the postnatal ontogeny of masticatory elements such as the TMJ is sensitive to dynamic variation in jaw-loading patterns.3—6 Indeed, it has been posited that mechanical loads are essential to maintain normal form and function of the TMJ, and growth responses of the mandibular condyle following alteration of local mechanical conditions (both increased and decreased loads) can lead to hyperplastic or hypoplastic changes in condylar soft- and hard-tissues.5 This relationship is fully established following weaning, when juvenile mammals ingest adult foods and develop ‘‘adult’’ jaw-adductor activity patterns7—10 with corresponding bony and soft-tissue responses to corresponding ‘‘adult’’ loading regimes.11—15 For instance, growing monkeys raised on a ‘‘hard’’ or resistant diet exhibit greater cortical bone remodelling as well as greater mandibular depth and cortical bone thickness.3 Compared to the TMJ of ‘‘soft-diet’’ macaques, hard-diet macaques also develop a higher density of connective tissue and subchondral bone as well as thicker condylar articular cartilage.4 Similar postnatal patterns characterize condylar measures and articular cartilage thickness in growing rats fed differing diets.5,16—19 These findings support the hypothesis that dynamic forces affect the ageing and function of the mandibular condyle and associated fibrocartilage disc, with integrated anatomical and molecular changes in such masticatory elements designed to maintain a sufficient safety factor to routine physiological loads.6,20—23 As the initiation and/or progression of TMJ disease appears linked to excessive, repetitive and/or altered biomechanical loading of the joint, we have used a new mouse model, mice lacking myostatin (GDF-8), to further clarify the functional relationship between the morphology of the mandibular condyle and jaw-adductor and bite forces. Myostatin is a negative regulator of skeletal muscle growth, and knockout mice lacking myostatin have approxi-

E.K. Nicholson et al.

mately twice the skeletal muscle mass of normal mice at both 2 and 10 months of age.24 In the masticatory complex, myostatin knockout mice exhibit masseter muscles 56% larger in mass and temporalis muscles 61% larger in mass (due to larger muscle fiber cross-sections and more muscle cells24,25), and presumably in physiological crosssection and muscle attachment size, than similarly sized normal mice. These morphological differences have behavioural consequences, as GDF-8 deficient mice have been shown to produce relatively greater bite forces than normal mice.25 The effect of myostatin deficiency appears be dose-dependent, as mice heterozygous for the disrupted GDF-8 sequence have muscle masses intermediate between those of normal mice and mice homozygous for the myostatin mutation.26 In situ hybridization data show that myostatin is first expressed in mouse embryos in the myotome compartment of somites, and myostatin transcripts can still be detected in adults.24,27 Myostatin knockout mice do not differ from normal mice (relative to body mass) in metabolic rate, food consumption or body temperature.26 Recent studies demonstrate that mice homozygous for the myostatin mutation have greater bone density than normal mice in both the spine and hindlimb as well as relatively larger muscle attachment areas.28,29 The increased bone mineral density of the myostatin deficient animals appears due to increased relative muscle forces since the myostatin receptor, the type IIB activin receptor, is not expressed at significant levels in skeletal tissues.30 Here, we compared myostatin knockout (GDF-8 deficient) to normal (control) mice to test the hypothesis that condylar morphology and masticatory muscle attachment size are affected, respectively, by dynamic increases in bite-force magnitudes and elevated jaw-adductor forces. In addition to measurement of the gross external proportions of the TMJ and mandible, we employ microcomputed tomography (microCT) to investigate regional variation in condylar biomineralization within and between experimental groups.

Materials and methods Sample Twenty-three mandibles (22 left mandibles and 1 right mandible) from male mice were studied. Of these, 11 were wild-type and 12 were homozygous for the disrupted GDF-8 sequence. The mice were fed ad libitum a diet of Harlan TekLad rodent chow and sacrificed at 6 months of age.

TMJ Adaptation in myostatin knockout mice

Measurements Masticatory-related variation in condylar bone proportions and mineral levels was evaluated using microCT of fixed mandibular tissues (cf. 15,31—33). Although the consensus is that microCT provides a ‘gold standard’ for bone microarchitecture analysis,34 only a few have gone beyond simple segmentation of the 3D data sets into bone and non-bone volume elements and interpreted the linear attenuation coefficient (m) in terms of local mineral density.31—33 Using a Scanco Medical MicroCT 40 system, values of m were measured within three reconstructed slices parallel to the coronal plane (anterior, middle and posterior) of the TMJ condyle (Fig. 1). The microfocus X-ray tube was operated at 70 kV and 57 mA, and the beam passed through a 0.13 mm thick Be window on the X-ray tube and through a 0.50 mm thick Al filter before encountering the sample. With this cone beam system, data were collected with the longest integration time (0.300 s per view) and in the highest sensitivity mode (1000 projections over 1808, 2048 samples per projection). Reconstruction was with 8 mm voxels (volume elements). In order to compare measured values of m for the TMJ with values expected for bone, one first has to consider the characteristics of the X-rays incident on the sample. Any X-ray tube produces a spectrum of X-rays modified by any filters or windows between the X-ray source and the sample. This quantity is generally not well known for a given tube, and one should note that each wavelength is absorbed differently by a sample. In practice, it is generally adequate to determine an effective X-ray energy for the tube operated at a specific voltage and base

Figure 1 Three-dimensional reconstruction of a mandible showing the location and orientation of a slice through the condyle. Three slices were examined from each specimen.

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comparisons on tabulated values of the attenuation coefficients at this energy.33 A sample of aluminum of known composition (and roughly the same linear attenuation coefficients, 2.92 < m < 2.96 cm 1, as the TMJ, 2.6 < m < 3.3 cm 1, at 70 keV) was used to determine the effective energy. Using the NIST tabulation of mass attenuation coefficients,35 the effective energy for the Scanco MicroCT 40 operated at 70 keV is 30 keV. Slices were collected in 3 groups of 40, 1 group each from the anterior, middle and posterior sections of the condyle, and one representative slice was chosen from each group. From each of the representative slices, m was recorded at equidistant points along the outer surface of the subchondral bone (n = 5), inner aspect of the subchondral bone (n = 4) and condylar neck cortical bone (two per side) (Fig. 2). In addition, the width of the condyle and the height of the condylar articular cartilage at three locations on the condyle (above the locations of points outer2—4) were measured on each slice. These data were used to characterize within-slice, within-joint, within-group and between-group variation in condylar biomineralization. This approach

Figure 2 Sites of linear attenuation coefficients (m, cm 1). The image is from a control mouse and uses the default colour table for the Scanco MicroCT 40; from lowest to highest values of m: blue, black, dark grey, light grey, white. The black region above the condylar surface is articular cartilage (m  0.65 cm 1 which is characteristic of unmineralized soft-tissue imaged with this accelerating voltage), and the surrounding blue region is air within the specimen tube (m < 0.1 cm 1). Buccal–—left and lingual–— right. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Mean (standard deviation) and p-values for measurements of control and GDF-8 deficient mice.

Condylar width (mm) Lingual cartilage height (mm) Middle cartilage height (mm) Buccal cartilage height (mm) Condylar widthb (mm) Condyle contour length(mm) Contour/width b Area of cartilageb (mm2) Area/width b Body mass (g) Masseter mass (g) Mandibular mass (g) Mandibular length (mm) Mandibular height (mm) Mandibular width (mm) Tooth row length (mm) Condylar width (mm) Condylar length (mm) Masseter insertion (mm) Temporalis insertion (mm)

Control (n = 11a)

GDF-8 deficient (n = 12a)

p-Value (Mann—Whitney U-test)

0.90 (0.08) 0.12 (0.06) 0.16 (0.10) 0.14 (0.07) 77.62 (7.21) 109.40 (11.58) 1.41 (0.11) 1003.22 (565.81) 12.93 (7.11) 43.19 (6.45) 0.12 (0.02) 0.074 (0.010) 13.27 (0.58) 3.87 (0.23) 1.54 (0.19) 3.40 (0.24) 0.81 (0.05) 1.81 (0.14) 3.85 (0.34) 3.93 (0.33)

0.87 (0.07) 0.14 (0.07) 0.18 (0.07) 0.20 (0.08) 74.93 (5.61) 103.63 (11.18) 1.38 (0.13) 1036.12 (467.10) 13.79 (6.17) 47.79 (4.45) 0.20 (0.03) 0.075 (0.008) 12.89 (0.38) 3.88 (0.25) 1.57 (0.09) 3.61 (0.38) 0.86 (0.08) 1.72 (0.24) 4.22 (0.38) 4.18 (0.18)

0.143 0.322 0.394 0.002 0.266 0.086 0.285 0.895 0.746 0.131 <0.001 0.630 0.149 1.000 0.412 0.083 0.359 0.312 0.037 0.142

a

Due to some broken specimens, n = 8 for control and n = 7 for GDF-8 deficient masseter insertion site; n = 5 for control and n = 4 for GDF-8 deficient temporalis insertion sites. b Measured from a figure at 86 magnification. All other measurements are at a 1:1 scale.

appears quite novel as relatively few quantitative microCT studies on joints have appeared in the literature.15,36,37 Using the software Image/J, the contour length of the condylar surface and the area of the articular cartilage were measured from each representative slice. The width of the condyle also was measured in order to calculate the ratio of contour length to condylar width and the ratio of cartilage area to condyle width, to control for condylar size. In an image of each slice at 86 magnification, each dimension was measured to the closest voxel (8 mm); the most medial and lateral points on the condyle were the endpoints. The contours were traced by hand in the Microsoft Word drawing function, then scaled to the same condylar width and superimposed on one another in order to compare the shapes of the condylar surfaces. The contours as well as the locations of the attenuation coefficients on the right mandible in the sample were reflected to correspond to the data from the other left mandibles. At the time of collection, the masseter and body masses were recorded for each specimen. Each mandible was weighed to the nearest 0.0001 g, and on each mandible the following were measured with digital calipers to the nearest 0.01 mm: mandibular length (measured from the most posterior point of the condyle to the most anterior point of

the symphysis), corpus height (measured midway along the tooth row from the top of the teeth to the bottom of the corpus), corpus width (buccolingual width, measured midway along the line used to measure corpus height), tooth row length, condylar width and condylar length. Estimates of temporalis and masseter insertion sites (defined as back of condyle to gonial angle for masseter and back of condyle to tip of coronoid process for temporalis) also were measured; only a subset of eight normal and seven myostatin knockout mandibles were measured as other specimens were damaged prior to commencing this study and had missing or broken gonial angles and/or coronoid processes. The three measures of condylar width (from the microCT scan, the magnified image in Image/J and the mandible itself) are included in Table 1. Each of the three measures of condylar width show similar results for both normal and myostatin knockout mice.

Statistical analyses The mean, median, range and standard deviation were used to characterize within-slice by group, within-joint by group and between-group patterns of variation in condylar biomineralization, proportions and contours. Because no significant differences exist in Mann—Whitney U-test ( p < 0.05) of body mass, mandibular length and mandibular mass

TMJ Adaptation in myostatin knockout mice

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between cohorts (Table 1), condylar and corpus proportions of both groups were compared without first correcting for size. Due to the small sample sizes, non-parametric ANOVAs (Kruskal—Wallis Htest and Mann—Whitney U-test) were used to assess within-joint variation and between-group differences. Using the linear attenuation coefficients for all 13 condylar sites (Fig. 2), discriminant function analyses were performed separately for samples from each of the three slices (anterior, middle and posterior) to more fully characterize patterns of variation in biomineralization between knockout and normal mice. In providing a multivariate assessment of differences in condylar values for each group versus one another, such analyses of bone density values facilitate the identification of condylar slices correctly classified as belonging to its designated group. The quantitative and qualitative parameters and analyses defined above were used to test the following predictions–—as compared to normal mice, the

myostatin knockout mice will exhibit: (1) relatively larger condyles as well as other relatively larger masticatory proportions (e.g. larger muscle attachment sites); (2) relatively thicker articular cartilage; (3) elevated biomineralization of the outer surface of subchondral bone, inner subchondral bone and articular neck cortical bone.

Results Metric dimensions None of the condylar measurements taken on the three slices (condylar width, articular cartilage height, contour length and area of articular cartilage) varied anteroposteriorly in either the normal or the myostatin knockout mice (Kruskal—Wallis Htest, p > 0.05). The myostatin knockout mice had a slightly higher (but not significantly different) mean body

Table 2 Measurements of control and GDF-8 deficient mice by location on condyle. Control (n = 11)

GDF-8 deficient (n = 12)

p-Value (Mann—Whitney U-test)

Posterior slice Condylar width (mm) Lingual cartilage height (mm) Middle cartilage height (mm) Buccal cartilage height (mm) Condylar width (mm) Condyle contour length (mm) Contour/width Area of cartilage (mm2) Area/width

0.90 0.12 0.13 0.15 76.45 109.07 1.43 1049.68 13.84

(0.08) (0.06) (0.07) (0.08) (6.76) (13.39) (0.14) (580.00) (7.60)

0.88 0.15 0.16 0.22 74.91 105.21 1.41 1119.35 15.01

(0.05) (0.07) (0.06) (0.09) (4.60) (9.47) (0.14) (466.45) (6.33)

0.391 0.211 0.391 0.091 0.580 0.712 0.854 0.712 0.806

Middle slice Condylar width (mm) Lingual cartilage height (mm) Middle cartilage height (mm) Buccal cartilage height (mm) Condylar width (mm) Condyle contour length (mm) Contour/width Area of cartilage (mm2) Area/width

0.91 0.12 0.15 0.13 79.07 109.70 1.39 1040.00 13.11

(0.09) (0.06) (0.09) (0.07) (7.60) (11.71) (0.09) (618.71) (7.39)

0.89 0.15 0.16 0.22 76.30 104.98 1.38 1101.29 14.35

(0.06) (0.08) (0.07) (0.08) (4.75) (8.74) (0.09) (524.52) (6.73)

0.580 0.695 0.805 0.013 0.479 0.325 0.854 0.806 0.667

Anterior slice Condylar width (mm) Lingual cartilage height (mm) Middle cartilage height (mm) Buccal cartilage height (mm) Condylar width (mm) Condyle contour length (mm) Contour/width Area of cartilage (mm2) Area/width

0.90 0.12 0.11 0.13 77.32 109.45 1.42 919.35 11.81

(0.09) (0.07) (0.06) (0.07) (7.70) (10.62) (0.10) (543.23) (6.83)

0.85 0.12 0.14 0.17 73.58 100.71 1.37 889.03 11.99

(0.09) (0.05) (0.08) (0.08) (7.24) (13.16) (0.15) (407.10) (5.51)

0.339 0.806 0.356 0.207 0.442 0.140 0.218 0.712 0.951

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mass than the control mice; however, their masseters were significantly larger by 67% (Table 1). The masseter insertion site was also significantly larger in the knockout mice. The temporalis insertion site was similarly larger in the knockout mice, though not to a significant degree, possibly because of the small sample size for this measurement (Table 1). The knockout mice had thicker cartilage on the buccal side only, with articular cartilage depth at the central and lingual regions being of similar proportions in both groups (Table 1). None of the other metric dimensions differed significantly between myostatin knockout and normal mice (Mann—Whitney U-test, p > 0.05). This also was true when measurements from each slice (posterior, middle and anterior) were compared separately (Table 2). Condylar width was larger in normal mice than in knockout mice in each of the three slices when measured directly from the microCT scan or in Image/J, but was larger in myostatin knockout mice when measured by hand; neither of these two differences in condylar width was significant (Mann— Whitney U-test, p > 0.05; Table 1).

Contour shapes Normal mice had fairly symmetrical contours at all three slices on the condyle (Figs. 3 and 4). Amongst the myostatin knockout mice, there was one (# 053) that had contours that were consistently higher and more curved than those of the other knockout mice. When this specimen is excluded, the knockout mice have contours that are very similar to each other and that are non-symmetrical, with a lower, less convex surface on the buccal side than on the lingual side. Thus, the contours of the myostatin knockout mice approximated those of the normal mice on the lingual side, but were much lower on the buccal side (Fig. 4). This difference was observed at each of the three slices on the condyle. On the posterior slice, two of the myostatin knockout mice have condyles that are flattened across the entire width of the condyle. Although the shape of the condylar surfaces differs between groups, the length of the contours and the ratio of contour length to condyle width were not significantly different between groups (Table 1).

Linear attenuation coefficients In normal mice, the attenuation coefficients from the outer condyle, the inner condyle and the condylar neck were significantly different from each other, with the largest values in the inner condyle and the smallest values in the outer condyle (three

Figure 3 Shape of condylar surface of control and GDF-8 deficient mice at three locations on the condyle: (a) posterior slice, (b) middle slice and (c) anterior slice. Solid black line–—control; red dashed line–—GDF-8 deficient. Lingual–—left and buccal–—right. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

pairwise Mann—Whitney U-test, p < 0.001; Fig. 5). In the myostatin knockout mice, the attenuation coefficients from the outer and inner condyle and from the outer condyle and condylar neck were significantly different from each other (Mann—Whitney U-test, p < 0.001), but the attenuation coefficients from the inner condyle and condylar neck were not (Mann—Whitney U-test, p > 0.05); this was true for each of the three slices on the condyle and when all the data were pooled. These results reflect patterns found in individual mice: all mice had the lowest attenuation values in the outer surface of the condyle, and most (all but 1 of the control mice, and 5 out of the 12 myostatin knockout mice) had the largest attenuation values on the inner condyle. Bone density differed buccolingually only in the myostatin knockout mice when values from the three slices were combined (Table 3). In the knockout mice, there was no significant difference between lingual and buccal values from the outer condyle (Mann— Whitney U-test, p > 0.05); however, the midline

TMJ Adaptation in myostatin knockout mice

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Figure 4 Example of condylar shape in control and GDF-8 deficient mice. Although the subchondral bone surface is asymmetrical in the GDF-8 deficient mouse, the surface of the articular cartilage cap is symmetrically convex. Thus, the thicker articular cartilage along the buccal side (*) is correlated with a reduction in underlying subchondral bone. Middle slice; lingual–—left and buccal–—right.

values were significantly higher than both the lingual and buccal values (Mann—Whitney U-test, p = 0.047 and 0.020, respectively). A significant difference was not found when comparing lingual and buccal values from each slice. Like the knockout mice, normal mice also had higher attenuation values in the middle of the outer surface of the condyle compared to either side, though not to a significant degree. The myostatin knockout mice had higher attenuation coefficients on the lingual side compared to the buccal side of the inner condyle, but this difference was not significant at the middle and anterior slices (Table 4). The attenuation coefficients on the buccal side of the condylar neck were higher than on the lingual side in myostatin knockout mice; the opposite is true for the normal mice. This pattern characterizes all slices, but was only statistically significant in knockout mice.

None of the attenuation values differed significantly in myostatin knockout mice amongst the anterior, middle and posterior slices. In normal mice, only inner1 differed (Kruskal—Wallis H-test, p = 0.015) due to lower values in the middle slice. Mann—Whitney U-test indicated that the linear attenuations coefficients from the condylar surface and the condylar neck were significantly larger in the myostatin knockout mice than in the normal mice (Table 5). These differences were largely due to differences at subsets of these sites (outer1 and 3, neck3 and 4) and varied across slices: the attenuation values from the condylar neck were significantly larger in knockout mice at the posterior and middle slices, and the values from the outer condyle were larger in knockout mice only in the middle slice (Table 6). When all attenuation coefficients from the inner condyle were combined, there

Table 3 Linear attenuation coefficients (m, cm 1) at lingual and buccal positions. Lingual mean

Midline mean

Buccal mean

p-Value (Kruskal—Wallis H-test)

Control Outer Inner Neck

2.76 (0.33) 3.31 (0.30) 3.12 (0.26)

2.89 (0.34)

2.81 (0.34) 3.27 (0.31) 3.11 (0.24)

0.314 0.422 0.624

GDF-8 deficient Outer Inner Neck

2.90 (0.34) 3.31 (0.28) 3.20 (0.27)

3.05 (0.32)

2.90 (0.37) 3.20 (0.31) 3.31 (0.26)

0.057 0.028 0.008

Lingual: outer1 and 2; inner1 and 2; neck1 and 2. Midline: outer3. Buccal: outer4 and 5; inner3 and 4; neck3 and 4.

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Figure 5

Boxplots of linear attenuation coefficients (m, cm 1) in control and GDF-8 deficient mice.

was no significant difference between myostatin knockout and normal mice; however, myostatin knockout mice had significantly lower values for inner4.

When the data were divided into subsets of buccal, midline and lingual points, and knockout and normal mice were compared, the myostatin knockout mice had significantly larger values on the buc-

Table 4 Linear attenuation coefficients (m, cm 1) at lingual and buccal positions by location on condyle. Lingual mean

Midline mean

Buccal mean

p-Value (Kruskal—Wallis H-test)

Control Posterior slice Outer Inner Neck

2.83 (0.32) 3.39 (0.27) 3.14 (0.31)

2.96 (0.38)

2.84 (0.34) 3.27 (0.29) 3.07 (0.22)

0.819 0.164 0.285

Middle slice Outer Inner Neck

2.76 (0.30) 3.25 (0.38) 3.11 (0.20)

2.95 (0.37)

2.67 (0.32) 3.26 (0.32) 3.10 (0.24)

0.031 0.646 0.617

Anterior slice Outer Inner Neck

2.73 (0.37) 3.28 (0.25) 3.23 (0.22)

2.83 (0.21)

2.90 (0.34) 3.33 (0.28) 3.11 (0.25)

0.584 0.570 0.160

GDF-8 deficient Posterior slice Outer Inner Neck

2.93 (0.38) 3.35 (0.32) 3.23 (0.25)

3.07 (0.25)

2.96 (0.33) 3.16 (0.36) 3.26 (0.31)

0.407 0.037 0.536

Middle slice Outer Inner Neck

2.89 (0.39) 3.32 (0.25) 3.21 (0.28)

3.01 (0.32)

2.93 (0.33) 3.29 (0.28) 3.32 (0.23)

0.608 0.516 0.187

Anterior slice Outer Inner Neck

2.88 (0.26) 3.28 (0.27) 3.17 (0.29)

3.06 (0.40)

2.82 (0.44) 3.16 (0.28) 3.34 (0.25)

0.187 0.158 0.018

TMJ Adaptation in myostatin knockout mice

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Table 5 Mean (standard deviation) and p-values for linear attenuation coefficients (m, cm 1) in control and GDF-8 deficient mice. Outer Inner Neck

Control

GDF-8 deficient

p-Value (Mann—Whitney U-test)

2.81 (0.34) 3.29 (0.30) 3.11 (0.25)

2.93 (0.35) 3.26 (0.30) 3.26 (0.27)

<0.001 0.667 <0.001

cal, midline and lingual portions of the outer condyle (Mann—Whitney U-test, p < 0.05) and on the buccal side of the condylar neck (Mann—Whitney Utest, p < 0.001). There were no significant differences between groups for the lingual side of the condylar neck or for either the buccal or lingual portions of the inner condylar subchondral bone. Attenuation values from the neck were significantly larger in knockout mice than in normal mice in the posterior and middle slices, as were those from the outer condylar surface in the middle slice (Table 6). At the posterior slice, the mean attenuation values were larger in the myostatin knockout mice than in the normal mice for all sites on the outer condyle and on the condylar neck (not significantly, although when all values from the outer condyle are combined, p < 0.1); the values were larger in the normal mice on the inner condyle although only significantly so at the most buccal site. On the middle slice, myostatin knockout mice had significantly higher values for the outer condyle and the condylar neck. On the anterior slice, myostatin knockout mice had higher values for the outer condylar sites and for the sites on the buccal side of the neck (not to a significant degree, though pvalues were rather low: p < 0.13), but had significantly lower mean attenuation coefficient at a position at the buccal side of the inner condyle (inner4; p < 0.05). Discriminant function analyses highlighted significant variation in condylar biomineralization

between myostatin deficient mice and normal mice. Using linear attenuation coefficient values for all 13 condylar sites (Fig. 2), 100% of 11 normal mice were correctly identified at all 3 condylar slices in a sample including 12 myostatin knockouts. Combining all three slices, an average 92% of the condylar slices for myostatin deficient mice were classified correctly (33 of 36), and no individual specimen exhibited a misidentified slice more than once. For instance, at the anterior slice, 11 of 12 knockout mice (92%) were identified correctly, with only one condyle misclassified as having a wild-type pattern of bone density (# 095). At the middle slice, all 12 myostatin deficient mice were classified appropriately. At the posterior slice, 10 of 12 knockout mice (83%) were identified correctly, with two of these condyles misclassified as exhibiting a normal condition (#s 085 and 087). Thus, multivariate patterns of biomineralization indicate that, myostatin and, especially, normal mice cluster much more closely with members of their own group than with one another. Several measures loaded heavily on the first (and only) discriminant function (Table 7). At the anterior slice, inner2 and outer3 loaded strongly positively, whereas inner1 and neck3 loaded highly negatively. At the middle slice, neck4 and inner1 loaded positively whilst neck2 and inner2 loaded negatively. At the posterior slice, outer1 and inner1 loaded positively, whereas outer3 and neck4 loaded strongly negatively. In considering all three slices, outer1

Table 6 Linear attenuation coefficients (m, cm 1) in control and GDF-8 deficient mice by location on condyle. Control

GDF-8 deficient

p-Value (Mann—Whitney U-test)

Posterior slice Outer Inner Neck

2.86 (0.34) 3.33 (0.28) 3.10 (0.27)

2.97 (0.34) 3.25 (0.35) 3.25 (0.28)

0.087 0.428 0.022

Middle slice Outer Inner Neck

2.75 (0.34) 3.24 (0.35) 3.07 (0.24)

2.93 (0.35) 3.30 (0.26) 3.27 (0.26)

0.004 0.262 <0.001

Anterior slice Outer Inner Neck

2.82 (0.33) 3.31 (0.26) 3.17 (0.24)

2.89 (0.37) 3.22 (0.28) 3.26 (0.28)

0.107 0.339 0.120

46

E.K. Nicholson et al.

Table 7 Combined-sample ranked canonical discriminant function scores for each location (m, cm 1), standardized by within variances for each slice. Posterior slice Outer1 Inner1 Inner2 Inner4 Neck3 Outer5 Inner3 Neck2 Outer4 Neck1 Outer2 Neck4 Outer3

Score 1.199 0.915 0.807 0.775 0.416 0.163 0.425 0.506 0.700 0.821 0.834 1.418 1.498

Middle slice neck4 inner1 outer1 neck1 outer2 neck3 outer3 inner4 outer4 outer5 inner3 inner2 neck 2

was always amongst the top three positive variable scores, whilst neck2 and inner3 consistently exhibited negative variable scores. Discriminant function scores also demonstrated buccolingual patterns of covariation along the condylar head as well as superoinferior patterns of covariation along the condylar neck (Table 7). At the anterior slice: outer1—3 loaded positively like inner2, whereas outer4 and 5 loaded negatively similar to inner3 and 4; both inferior neck points showed positive loadings (neck1 and 4) whilst superior neck points evinced negative scores (neck2 and 3). At the middle slice, outer1—3 exhibited positive loadings much as inner1, whereas outer4 and 5 scores were negative similar to inner2—4; most neck points, especially inferior variables, loaded positively (neck1, 3 and 4). At the posterior slice, lateral-most outer points were positive (outer1 and 5) whilst central points loaded negatively (outer2—4); most inner points loaded positively (inner1 and 2, 4); and the majority of neck points exhibited negative loadings (neck1 and 2, 4). In sum, there was covariation by side for outer and inner discriminant function scores from the anterior and middle slices. However, posterior slices indicated a different pattern–—positive inner loadings combined with negative central outer scores and positive buccal- and lingual-most outer variable scores.

Discussion and conclusions The size of the mandibles, and of any functionally important bony masticatory elements, except for the size of the masseter insertion site, did not differ significantly between the myostatin knockout and the normal mice. In this regard, our findings are seemingly at odds with earlier studies of monkeys,

Score 1.904 1.382 1.146 1.135 0.926 0.266 0.097 0.402 0.490 0.552 0.818 0.963 1.008

Anterior slice inner2 outer3 outer1 neck1 neck4 outer2 outer4 inner3 inner4 outer5 neck2 neck3 inner1

Score 2.148 2.039 0.963 0.685 0.543 0.191 0.016 0.510 0.590 0.983 1.235 1.751 1.813

rabbits and rats indicating the presence of some differences in masticatory proportions between conspecifics raised on a hard- or over-use diet versus a soft- or under-use diet. One obvious difference with our study is the comparison between an overuse model (myostatin deficient mice) and control mice both fed a similar, normal diet. It is altogether likely that an under-use model of mouse mastication would result in greater differences with myostatin knockout mice. It is further important to note that many of the differences noted in over-use/underuse comparisons are non-significant and small in magnitude. Controlling for size in a study of rat masticatory elements, 8 of 11 comparisons differed by no more than 4%.5 This suggests that external proportions may not be the most appropriate level at which to detect and evaluate adaptive plasticity of masticatory elements. The size of a bony muscle attachment site is affected by the strength of the muscle,38—40 the direction of the muscle force relative to the surface of the bone,41—43 and frequency of muscle use.44 Myostatin knockout mice produce relatively greater bite forces25 and possess larger masseter (Table 1) and temporalis25 muscles. Accordingly, myostatin knockout mice had larger masseter and temporalis attachment sites (Table 1), much as is the case for muscle attachment proportions in the locomotor skeleton. Myostatin knockout mice had thicker articular cartilage than normal mice only at the buccal side of the condyle (Table 1). Compensatory increases in articular cartilage amongst myostatin knockout mice would be expected as greater bite forces produce elevated condylar reaction forces during routine masticatory behaviours.45,46 In contrast, most external mandibular proportions in GDF-8 deficient mice were slightly smaller than in the wildtype mice. If the significantly more biomineralized

TMJ Adaptation in myostatin knockout mice

condyles of knockout mice are an indication of greater bone density throughout the mandible, the combination of larger mandibles in normal mice and greater bone density in knockout mice could result in the near-equivalent mandibular mass observed in both groups. Based on the effective energy of the Scanco MicroCT 40, the NIST table35 gives m/r = 3.0 cm2/ g. From Currey’s47 values of cortical bone density, 1.9—2.0 g/cm3, one expects to measure 2.5 < m < 2.7 cm 1 in the TMJ. The TMJ appears, therefore, to be somewhat more mineralized than the cortical bone described in the NIST tabulation. The difference is hardly surprising, given that bone is a complex mixture of mineral and collagen at the nanometre-level and of dense material and empty space (or at least soft-tissue like blood vessels, osteocytes, etc.) with absorptivities much less than mineralized portions of the bone). Differences in mineral level documented for different bones by Currey47 (which affect both the density and the mass attenuation coefficient) can easily account for the slight apparent shift between tabulation and TMJ. In general, in both normal and myostatin knockout mice, the bone density of the subchondral bone of the inner condyle was greater than that of the cortical bone of the condylar neck, which was in turn larger than that of the outer condyle subchondral bone. The outer condyle and the condylar neck were more biomineralized in knockout mice than in normal mice, specifically the midline of the outer condyle and the buccal condylar neck (Table 5). Assuming similar masticatory behaviours as noted for rats and primates,46,48—50 such regions would be differentially loaded during biting and chewing.46 As in the knockout mice, the midline point of the outer condyle of normal mice had a higher attenuation coefficient than did the lingual and buccal points, but the differences were not significant in the normal mice (Table 3). If a greater muscle mass in myostatin knockout mice produces greater jaw-adductor forces, higher bite forces, and in turn elevated condylar reaction forces, the results can be viewed as the cumulative ontogenetic effects of increased masticatory stress in the mandibles of knockout mice as compared to normal mice. Increased biomineralization in the buccal side of the condylar neck is expected if the buccal side of the condylar surface experiences greater compressive loads than the lingual side during biting and chewing (due to mandibular eversion46,48—51). The resulting lateral compression of the TMJ would produce bending of the condylar neck in the coronal plane and thus compression of the lateral/buccal aspect of the neck as well as compression of the lateral aspect of the condyle. The

47

difference in compressive loads between normal and knockout mice is more pronounced in the posterior part of the condyle, leading to significant differences in biomineralization levels of the condylar neck in the posterior and middle slices, but not in the anterior slice (Table 6). Discriminant function analyses were able to correctly classify all normal mice, and misclassified only 3 out of 36 knockout mice, supporting the prediction that greater TMJ loading in the knockout mice significantly alters condylar bone density patterns. Both groups were correctly classified at the middle slice, the region likely to be loaded most frequently during both jaw opening and closing. In both the anterior and posterior slices, there was moderate covariation by side of outer and inner scores. This did not hold true for the posterior slice, in which most inner scores were positive, whilst central outer scores (scores for outer2—4) were negative. The pattern of covariation supports the hypothesis that the buccal side of the condyle is differentially loaded during mastication. The finding of lower levels of biomineralization in the inner subchondral bone at the buccal side of the condyle in myostatin knockout mice is seemingly contradictory. One possibility is that the greater biomineralization of the outer subchondral bone, and thicker articular cartilage at the buccal side, adequately resists elevated masticatory stresses and thus obviates a need for increased density of the inner subchondral bone. It is worth noting, however, that extremely high stress levels in bone can be deleterious, inducing osteoclastic activity and a consequent decrease in bone density.52 This interpretation is consistent with the unilateral disparity in subchondral bone surface contours. Because the condylar surfaces of the myostatin knockout mice were lower than the surfaces of the normal mice on the buccal side whilst all mice had symmetrical articular cartilage caps, cartilage along the buccal side of the condyle tended to be vertically thicker in myostatin knockout mice than in normal mice (Table 1 and Fig. 5). The posterior part of the condyle in knockout mice was particularly affected, with two mice exhibiting almost flat condylar surfaces, lower than normal mice on both the buccal and lingual sides (Fig. 4). The development of increased cartilage would be expected if the buccal side or the posterior part of the condyle routinely experienced elevated compressive stress,45 a finding mirrored by research on dietary manipulation and articular cartilage thickness in macaques.4 The finding of significantly higher levels of biomineralization of the mandibular condyle in myostatin deficient mice is consistent with earlier work

48 on macaques4 and with findings for the spine and hindlimb of this knockout mouse model.28,29 The lack of significant differences in external dimensions between myostatin deficient and normal mice highlights the need for studies of internal anatomy of mineralized structures, particularly those that can characterize regional variation in soft- and hardtissues. Indeed, whilst the deposition of additional material is considered a typical means by which bones and joints respond to elevated and altered loads, our research highlights the role of plasticity in tissue material properties as another component of this adaptive process (i.e. greater stiffness and perhaps lower bone-strain levels due to increased biomineralization). In this regard, the application of microCT to the study of TMJ adaptive plasticity has been of great utility, especially given the rarity of such analyses of cranial and postcranial joints as well as the dearth of information on the dynamic postnatal effects of loading variation on bone mineral density patterns.

Acknowledgements Funding for this research was provided by the National Institutes of Health (AR049717-01A2). G. Wise, D. Daegling and an anonymous reviewer are thanked for helpful comments and suggestions.

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