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Craniofacial Morphology in Myostatin-deficient Mice
1 Department of Plastic Surgery, Correspondence: * corresponding author, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA, lisa.vecchione{at}chp.edu.
GDF-8 (myostatin) is a negative growth regulator of skeletal muscle, and myostatin-deficient mice are hypermuscular. Muscle size and force production are thought to influence growth of the craniofacial skeleton. To test this relationship, we compared masticatory muscle size and craniofacial dimensions in myostatin-deficient and wild-type CD-1 control mice. Myostatin-deficient mice had significantly (p < 0.01) greater body (by 18%) and masseter muscle weight (by 83%), compared with wild-type controls. Significant differences (p < 0.05) were noted for cranial vault length, maxillary length, mandibular body length, and mandibular shape index. Significant correlations were noted between masseter muscle weight and mandibular body length (r = 0.68; p < 0.01), cranial vault length (r = –0.57; p < 0.05), and the mandibular shape index (r = –0.56; p < 0.05). Masticatory hypermuscularity resulted in significantly altered craniofacial morphology, probably through altered biomechanical stress. These findings emphasize the important role that masticatory muscle function plays in the ontogeny of the cranial vault, the maxilla, and, most notably, the mandible.
Key Words: morphology • myostatin • mice • craniofacial growth
Craniofacial morphology is influenced by genetic and environmental factors. Muscle function is believed to affect bone shape and size. Masticatory muscle function is an environmental factor that helps to determine craniofacial morphology. The role of muscle function on skeletal growth and development has long been an issue of debate. Bone remodels in response to physical forces, and this remodeling can be described mathematically. Bony trabeculae are arranged in accordance with stress trajectories, and bones remodel to equilibrate stress (Wolff, 1892). Wolff’s Law, however, can neither predict the particular effects of mechanical loading on living bone mathematically nor provide a mechanism to explain how these effects come about (Frost, 1994). Other researchers caution that Wolff’s Law, which states that bone adapts to mechanical loading produced by muscle forces, should be renamed "bone functional adaptation". The concept of bone functional adaptation suggests that physical environment can alter an organism’s physiology. Bone adapts to mechanical strain by depositing new bone (Ruff et al., 2006). The degree to which muscle function affects craniofacial form is a complex topic. There is a large range of variations in human skull morphology, which is mutltifactorial. Hypermuscularity may be one factor involved. According to Collins’ "hard chewing hypothesis", the distinctive shape of the Inuit (i.e., Eskimo) skull is related to vigorous chewing. The Inuit skull is adapted to produce and dissipate large vertical and biting forces (Hylander, 1977). The Inuit skull is characterized by a large mandible, larger muscle attachments, and palatal and mandibular tori. The masseter muscles are also positioned more anteriorly, which may help generate larger forces. Myostatin (or growth and differentiation factor 8, GDF-8) has recently been identified as a highly conserved member of the TGF-β superfamily and an important growth factor in mammalian skeletal muscle. Inactivation of the myostatin gene in mice (GDF8–/– mice) results in the development of greater numbers of skeletal muscle fibers during early growth periods, and an increase in total muscle fiber number and mean muscle fiber area in adulthood (McPherron et al., 1997). Subsequent to this finding, cows bred to produce unusually large amounts of muscle mass, the Belgian Blue and Piedmontese strains, were investigated and found to have myostatin gene mutations (Grobet et al., 1997). In normal development, myostatin acts as a negative regulator of skeletal muscle, so studies determining the biologic effects of its inactivation are important in our understanding of the effects of hypermuscularity on skeletal growth and development. Myostatin-deficient mice have been shown to have increased muscle attachment sites, increased muscle strength, increased bite forces (Byron et al., 2004), and temporalis muscles with more glycolytic muscle fibers (Byron et al., 2006). Most recently, a myostatin deficiency has been demonstrated in a human neonate presenting with hypertrophic skeletal muscle bellies (Schuelke et al., 2004). This child’s mother is an elite athlete who came from a family known to have exceptional athletic abilities. To our knowledge, the craniofacial growth of the myostatin-deficient child has yet to be studied. Identifying how myostatin deficiencies, either from complete inactivity or from variations in activity from genetic differences such as single-nucleotide polymorphisms, affect craniofacial growth will help to determine how variations in muscle size affect craniofacial growth. The present study examined the relationship between increased masticatory muscle size and subsequent craniofacial morphology in myostatin-deficient mice (GDF-8–/–) compared with wild-type controls.
Sixteen (9 wild-type control and 7 GDF-8–/– myostatin-deficient), six-month-old, male CD-1 mice were used. Myostatin-deficient mice were produced by deletion of the C-terminal region of the myostatin gene in embryonic stem cells (McPherron et al., 1997). All mice were housed together and given food (Harlan Teklad hard rodent chow, Madison, WI, USA) and water ad libitum. Mice were killed at 6 mos of age, at skeletal maturity (Byron et al., 2004), by CO2 overdose according to a protocol approved by the Medical College of Georgia, and body weights were immediately recorded. Mouse skulls were disarticulated and weighed. Masseter muscles were then dissected and weighed to the nearest 0.001 g. Skulls were fixed in 10% neutral buffered formalin for 24 hrs, and then transferred to 70% ethanol for radiographic analysis. Lateral and dorsoventral radiographs were taken with the use of a Faxitron MX-20 (Faxitron X-Ray Corporation, Wheeling, IL, USA) at 35 kV for 250 sec at 5X magnification with X-OMAT V diagnostic film (Kodak, Rochester, NY, USA). Radiographs were scanned on an AGFA DuoScan equipped with AGFA FotoLook 3.2 software (Wilmington, MA, USA). Twenty-nine cephalometric landmarks were identified, and 21 craniofacial measurements were made with Image J software (NIH, Bethesda, MD, USA). Lateral cephalometric landmarks were identified as follows: Pa, the most superior point of the parietal bone; Na, the most anterior point of the nasal bone; Rh, rhinion, the most anterior point of the midpalatal suture; Os, occipitosphenale; Fp, frontoparietal suture; Ne, the most anterior inferior point on the premaxilla; Ps, pre-sphenoid; A, junction of the lingual/superior surface of the mandibular alveolar process with the lingual surface of the mandibular incisor; M1, junction of the lingual/superior surface of the mandibular alveolar process with the mesial surface of the mandibular first molar; M2, distal of the mandibular second molar; Ra, ramus point; Go, the most posterior point of the mandibular angular process; Gn, the most inferior point of contour of the angular process of the mandible; Co, the most posterosuperior point of the condylar process; I, the most prominent point between the incisal edges of the lower incisors; Op, opisthion, the most posterior rim of the foramen magnum; X, the most posterior point of the occipital bone. Dorso-ventral cephalometric landmarks were identified as follows: Rh, rhinion, the most anterior point of the midpalatal suture; Op, opisthion, the most posterior rim of the foramen magnum; Cw1, calvarium left; Cw2, calvarium right; Zp1, the left posterior junction of the zygomatic arch and temporal bone; Zp2, the right posterior junction of the zygomatic arch and temporal bone; Mp1, left midpoint zygomatic arch; Mp2, right midpoint zygomatic arch; Ms1, the left anterior junction of the zygomatic arch and the maxillary process; Ms2, the right anterior junction of the zygomatic arch and the maxillary process; Bs1, the right midpoint of the basi-occipital basi-sphenoid synchondrosis; and Bs2, the left midpoint of the basi-occipital basi-sphenoid synchondrosis. Mean somatic and cephalometric data were calculated and compared between groups, by means of an unpaired Student’s t test. The relationships between masseter muscle mass and various cephalometric landmarks were assessed by a Pearson Product Moment Correlation. All data were analyzed by SPSS 12.0 for Windows (SPSS, Inc., Chicago, IL, USA). Differences were considered significant if p < 0.05.
Myostatin-deficient mice were dramatically more robust than wild-type controls at 6 mos of age. The myostatin-deficient mice had significantly (p < 0.01) greater body weight (by 18%) at 6 mos of age, compared with wild-type controls (Fig. 1  ). Myostatin-deficient mice also had significantly (p < 0.001) greater masseter muscle weight (by 83%) at 6 mos of age, compared with wild-type controls (Fig. 1).
Qualitatively, as seen on dorsoventral and lateral head radiographs, myostatin knock-out mice were more brachycephalic, in both the cranial vault and the maxilla, compared with wild-type controls (Fig. 2 ). Myostatin-deficient mice also showed significantly remodeled mandibular rami, mandibular bodies, and coronoid processes, resulting in a "rocker-shaped" mandibular morphology compared with that of wild-type controls (Fig. 2).
Cephalometric analysis revealed quantitative differences in all 21 measurements between the two groups. Significant group mean differences were noted for the cranial vault length (Pa-Na) and maxillary length (Ms1-Ms2-Rh) measurements (Fig. 3 ). Myostatin-deficient mice had significantly shorter crania (p < 0.05) and maxillae (p < 0.001) compared with wild-type controls (Fig. 3). Significant group mean differences were also noted for mandibular body length (Ra-I) and the mandibular shape index [(Co-Go/Ra-I)*100] (Fig. 4). Myostatin-deficient mice had significantly longer mandibles (p < 0.01) and significantly smaller mandibular height-to-length indices (p < 0.05) compared with wild-type controls (Fig. 4). No significant mean group differences were noted for the other measures.
The relationship between masseter muscle mass and various cephalometric measurements for both groups were assessed by a Pearson Product Moment Correlation. A significant positive correlation coefficient was noted between masseter muscle weight and mandibular body (Ra-I) length (r = 0.68; p < 0.01). Significant negative correlation coefficients were noted between masseter muscle weight and cranial vault length (Pa-Na) (r = –0.57; p < 0.05) and the mandibular shape index [(Co-Go/Ra-I)*100] (r = –0.56; p < 0.05).
Cephalometric findings from the present study revealed that mice deficient for the myostatin gene (GDF8–/– mice) had dramatically altered crania and mandibles by 6 mos of age compared with wild-type controls. Significant associations noted between masseter muscle weight and several craniofacial dimensions suggest that hypermuscularity and increased bite force probably played a significant role in producing these deformities. Similar associations have been suggested by others (Ringqvist, 1974; Houghton, 1978; Byron et al., 2004; Rowlerson et al., 2005). Differences in the mechanics of biting are thought to influence vertical facial dimension and sagittal jaw growth. The etiology of typical "long face" open-bite malocclusions and "short face" deep-bite malocclusions in humans (Schendel et al., 1976; Opdebeeck and Bell, 1978) is presumed to originate from the anatomic position of jaw-closing muscles and the subsequent force vectors acting on the skeleton during crucial periods of facial growth, tooth eruption, and dento-alveolar displacement (Sassouni, 1969). Another important clinical consideration is the extent to which human jaw-closing muscle composition is related to differences in craniofacial form. Early histochemical studies, which classified jaw-closing muscle fibers into either fast-contracting type II fibers or slow-contracting type I fibers, found associations between differences in fiber-type composition of masseter muscle and bite force (Ringqvist, 1973, 1974). These studies, however, could be considered as only preliminary, since the variability in fiber diameters between subjects was high, and the number of subjects studied was small. More recently, a more comprehensive study of human masseter muscle fiber type composition, determined by surgical biopsy (Rowlerson et al., 2005), demonstrated that an increase in facial vertical jaw dimension is correlated with a decrease in the cross-sectional size of type II fibers. Interestingly, in mysostatin-deficient mice, increases in temporalis bite force were also found to accompany decreases in type II fiber diameter, as well as an overall increase in the proportion of type II myofibers (Byron et al., 2006). The mechanism by which jaw-closing muscles have an influence on craniofacial form comes from their ability to apply force, either compressive or tensile, to bony joints during periods of active growth. Calvarial and facial sutures serve as joints, but are also responsible for growth by surface deposition at the sutural ligament. These are thought to be "growth sites", rather than "growth centers" like the epiphyseal plate of long bones, since compressive force on sutures retards surface deposition of bone, and tensile force enhances deposition (Kiliaridis, 1995). For the mandible, however, growth in length is produced largely by ossification of cartilaginous tissue produced by the mandibular condyle. Here also there may be some possibility of growth modification from altered force, but not nearly as much as with sutures. Mandibular condylar cartilage is histomorphologically and biochemically different from cartilage found in the epiphyseal cartilage of long bones (Meikle, 1973; McNamara and Carlson, 1979), and can act as either a growth site or a growth center. The variations in muscle forces acting on mandibular cartilage and craniofacial sutures will eventually modify overall growth patterns. The myostatin-deficient mouse has significantly greater masticatory muscle mass and greater bite forces (Byron et al., 2004, 2006) than do normal mice. Muscle mass is controlled in two ways: Systemically acting growth promoters, such as growth hormone, act to increase insulin-like growth factors (IGFs) produced in the liver, which in turn have anabolic effects on muscle tissue. Muscle also produces its own growth factors, such as myostatin, which acts in a paracrine fashion as a negative regulator of muscle growth (McPherron et al., 1997). There is also direct interaction between myostatin and IGF, since myostatin knockout mice show alterations in IGF-II and IGF-IR expression (Kocamis et al., 2002). Further, increases in myostatin expression have been shown to correlate with an atrophy of type II muscle fibers (Reardon et al., 2001). Given the association between vertical facial dimension and the cross-sectional size of type II fibers in human masseter muscle, the myostatin-deficient mouse represents a good animal model for determining the associations between muscle variations and craniofacial growth. Significant differences in craniofacial dimensions were also found, which most likely resulted from both hypermuscularity and increased bite force in the myostatin-deficient mouse. The cranial vault and maxillary skeleton were both shorter in the myostatin-deficient mouse than in the wild-type. Myostatin-deficient mice have been found to have sutures with increased anatomic complexity, apparently to dissipate increased mechanical loads (Byron et al., 2004). Both suture complexity and loading probably were key factors leading to the overall shortening of the facial height and calvarial vault length dimensions. Increased mandibular body length and decreased mandibular shape index resulted in a longer mandible and deeper dental bite. This confirms previous findings in humans (Rowlerson et al., 2005), that increased muscle fiber size resulted in deep bite malocclusions. Further, the overall shape of the myostatin-deficient mouse mandible, in comparison with the wild-type control mandible, resembled a unique human jaw morphology described previously in Polynesian populations (Houghton, 1978; Schendel et al., 1980; Kean and Houghton, 1990). The effect of hypermuscularity in Polynesians is thought to have resulted in a characteristic mandibular shape described as a "rocker mandible" (Marshall and Snow, 1956). A rocker mandible lacks an antegonial notch, and the angle of the mandible is convex, which results in rocking when the mandible is placed on a flat surface. These features of the Polynesian mandible occur at puberty (Houghton, 1977), when bone shape is altered by local muscular forces (Enlow, 1975). The Inuit (i.e., Eskimo) skull is also characterized by a large mandible, larger muscle attachments, and palatal and mandibular tori. It has also been suggested that the distinctive shape of the Inuit skull is a result of vigorous chewing (Collins’ "hard chewing hypothesis"; Hylander, 1977). The Inuit skull is adapted to generate and dissipate large vertical and biting forces, and the masseter muscles are also positioned more anteriorly, which may help generate larger bite forces. These observations also suggest that hypermuscularity is an important determinant of craniofacial morphology. This study demonstrates that myostatin deficiency, and subsequent masticatory muscle hypermuscularity, resulted in significantly altered craniofacial morphology, probably through altered biomechanical stress. These findings reiterate the important role that masticatory muscle function plays on the ontogeny of the cranial vault, maxilla, and, most notably, the mandible.
This investigation was supported by research grant AR 049717 from the National Institutes of Health, Bethesda, MD 20892, USA. Received for publication January 28, 2007. Revision received June 1, 2007. Accepted for publication June 7, 2007.
Journal of Dental Research, Vol. 86, No. 11,
1068-1072 (2007) This article has been cited by other articles:
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