In Homo sapiens, early brain growth is important in determining the size and shape of the cranium (Weidenreich, 1941; Moss, 1969; Moss and Salentijn, 1969; Enlow and Hans, 1996; Mooney et al., 2002; Richtsmeier et al., 2006). However, there is a period of stasis following the cessation of neurocranial expansion, which precedes cranial suture remodeling and fusion (Meindl and Lovejoy, 1985; Cohen and MacLean, 2000; Cohen, 2005). Cranial sutures, classified as ligaments or syndesmoses (i.e., two bones held together by fibrous connective tissue), are osteogenic environments when morphologically described as patent (Cohen and MacLean, 2000; Cohen, 2005; Byron, 2006). It has been observed that the quasi-static strain of the expanding brain or severe biomechanical tensile forces (i.e., such as untreated hydrocephalus or distraction osteogenesis) can in some extreme cases affect calvarial suture morphology (Mooney and Richtmeier, in press). Nevertheless, neurocranial expansion is not temporally related to human ectocranial suture activity, osteoblastic, or osteoclastic activity that results in bone formation across the suture and is thus unlikely to influence eventual suture morphology and fusion (Todd and Lyon, 1925a, b, c; Meindl and Lovejoy, 1985).
Although quasi-static strains result from the ontogenetically expanding brain, cyclic strains generally result from the direct influence of mastication, masseter, temporalis, and pterygoid muscle activities (Mao, 2002; Herring, 2008). These cyclic strains may have a high degree of influence on later ectocranial suture morphology and fusion (Herring, 1993; Byron et al., 2004, 2006; Herring, 2008). Sutures respond through compensatory bone growth and remodeling at the osteogenic fronts in response to both tensile (two forces act along a straight line in opposite directions) and compressive (two forces that act along a straight line in the same direction) forces (Herring and Teng, 2000; Mao, 2002; Alaqeel et al., 2006; Downey and Siegel, 2006; Herring, 2008). In general, sutures adapting under compressive forces show more interdigitation, thicker bones, and narrower sutures. In contrast, sutures adapting under tensile forces and cyclic strain show greater widening and more osteogenic front elongation (Fong et al., 2003; Wu et al., 2007; Herring, 2008). It has been demonstrated that the cranial sutures, throughout ontogeny, respond to strain by increasing complexities, waveform patterns, and interdigitations and maintain a relatively constant width by bony adaption to strain before osseous fusion (Yu et al., 2009). Thus, strain magnitudes would have to increase throughout ontogeny to have additive effects on bony morphology.
It has been demonstrated in several animal models that eruption of the adult dentition is an indicator of a developed masticatory apparatus (Krogman, 1930; Swindler, 2002) and corresponds to an increase in bite force-producing capabilities of the masticatory musculature (Dechow and Carlson, 1990; McCollum et al., 2006). The increased masticatory forces observed in the adult ontogeny have been shown to correlate with an increase in tension across cranial sutures (Herring and Teng, 2000; Sun et al., 2004; McCollum et al., 2006; Shibazaki et al., 2007). In contrast, compressive forces may significantly dissipate with sutural remodeling (Sun et al., 2004; Shibazaki et al., 2007). Furthermore, data from a hypermuscular murine GDF-8 (myostatin) knockout model have shown an age-dependent positive functional relationship between masticatory muscle size and sutural complexity including increased sutural interdigitation and alterations in craniofacial morphology and mandible shape compared with wild-type mice (Byron et al., 2004, 2006; Vecchione et al., 2007, 2010).
Recent research suggests that there is a similar pattern of ectocranial suture closure between Pan troglodytes and H. sapiens (Cray et al., 2008). P. troglodytes, like H. sapiens, were found to display early closure in the sagittal suture, with more delayed changes in coronal and lambdoid sutures. In addition, like Homo and Gorilla gorilla, P. troglodytes exhibit suture activity commencement (bony bridging) and termination (osseous obliteration) of the lateral-anterior sutures in an anterior-to-posterior sequence (Cray et al., 2008). It is possible that a predetermination of suture fusion timing exists as evidenced by the heritability of craniofacial and suture morphologies and patterns of fusion (Coussens and van Daal, 2005; Wang et al., 2006a). These species may also share common genetic growth factor releases (i.e., Tgfβ, FGFs, and MSX) during their ontogeny that result in the patterning of osseous fusion, which can dampen biomechanical strains across the cranial sutures (Opperman and Ogle, 2002; Rawlins and Opperman, 2008).
The timing of bony bridging and fusion of the calvarial sutures has been described as proceeding after adult dental eruption in humans (Todd and Lyon, 1925a, b, c; Meindl and Lovejoy, 1985). Because masticatory forces continue further into the life cycle than neural expansion, the resulting biomechanical masticatory forces may have a greater influence on ectocranial suture morphology in humans. However, no investigations have been conducted concerning these timing of ectocranial suture activity in other hominoids. This study was designed to test these relationships in P. troglodytes. In particular, we will investigate suture morphology (commencement and termination of osseous activity at the suture site) and its relationships to both cranial volume (as a proxy for brain growth) and dental eruption (as an indicator of adulthood and a proxy for masticatory influences). These data will allow us to test our hypothesis that in Pan, similar to humans, there exists a period of stasis between the cessation of brain growth and later suture adaptation and synostosis. In addition, if sutural adaptations are observed to extend later in ontogeny after brain growth is completed, then they likely result from biomechanical forces of the masticatory apparatus.
One hundred and fifty-five P. troglodytes skulls, housed at the Cleveland Museum of Natural History, were examined at 10 ectocranial suture sites by the primary author. Seven of the suture sites were classified as cranial vault sutures: midlambdoid, lambda, obelion, anterior sagittal, bregma, midcoronal, and pterion; three of the sutures were classified as lateral-anterior sutures (two from the cranial vault were classified as both): midcoronal, pterion, sphenofrontal inferior sphenotemporal, and superior sphenotemporal, Fig. 1 (Meindl and Lovejoy, 1985; Cray et al., 2008). Each suture site was examined for osseous bridging: (1) commencement—defined as the earliest onset of bone formation activity within the fibrous joint (scored as 0 if not commenced or 1 if commenced); or (2) termination/obliteration—defined as the osseous obliteration or synostosis, i.e., the fibrous joint is replaced by bone (scored as 0 if not completely obliterated or 1 if obliterated; Fig. 2). The scores were then added to obtain a score for the sum of active suture sites for commencement (0–7 for vault and 0–5 for lateral anterior analyses) and the sum of terminated suture sites for termination (0–7 for vault and 0–5 for lateral anterior analyses) (Meindl and Lovejoy, 1985; Cray et al., 2008). In this study, the primary author collected suture site data on each skull twice, on separate days. Intraobserver reliability was 98.5% (that is over 98% of the suture site observations across study specimens were the same at each of the two observation times).
The direct determination of cranial volume (in cubic centimeters) and sex determinations were provided by the Cleveland Museum of Natural History as part of their database of information on skeletal specimens. Cranial volume determination followed the methodology of Simmons, including sand, water, and anthropometric techniques (Simmons, 1942).
Dental eruption status, a proxy for ontogeny and masticatory muscle influence, was also scored for each skull and assigned to the following categories according to sequence of permanent dental eruption in accordance with Krogman (1930):
2first permanent molar eruption
3permanent incisors and premolars eruption
4second permanent molar eruption
5permanent canines and last molars eruption
6wear of age.
To determine if cranial volume measures differ by the sum of suture scores (commencement and termination for vault and lateral anterior suture sites) by sex, a two-way ANOVA was conducted for each analysis to assess the significance of the interaction term. This analysis elucidated whether there were sex-specific patterns in neurocranial expansion as related to suture morphology.
To assess the statistical relationship between suture activity and cranial volume, a linear regression model, using a single predictor, was used to assess the relationship between cranial volume and the sums of cranial vault, and lateral anterior suture commencement and termination (0–7 for vault and 0–5 for lateral anterior) were treated as the independent variables, respectively. Cranial volume was treated as the dependent variable or predicted variable (Vittinghoff et al., 2005). This analysis allowed for the determination of whether neurocranial expansion was related to suture activity, bridging, and synostosis in Pan ontogeny.
To assess the statistical relationship between the sum of active suture sites score (an ordinal variable) and dental status (an ordinal variable), a Kendall's tau (τ) was calculated (Ferguson, 1976). Sexual dimorphism is assessed for the sum of active and terminated suture sites for vault and lateral anterior sutures, as well as dental status. This analysis allowed for the determination of whether suture activity was correlated with dental eruption as a proxy for ontogeny and masticatory influence on morphology. All analyses were conducted using SPSS 15 (Chicago, IL).
Demographics were provided by the Cleveland Museum of Natural History as part of their database of information on skeletal specimens. Of the 155 skulls that were analyzed, 72 were classified as subadults and 83 adults. Of the subadults, 31 were classified as unknown sex, 10 males, and 21 females. Of the adult specimens, one was classified as unknown sex, 31 males, and 51 females.
The sample was culled (N = 96) to include only those skulls that had data for cranial volume analysis. A 2 × 8 between-subjects analysis of variance was performed on cranial volume as a function of sex (male, female) and vault suture commencement and termination score (0–7), respectively. The assumption of normality was met for all groups, P > 0.05 for each analysis. The assumption of homogeneity of variance was violated for each analysis (commencement F(13,60) = 2.50, P < 0.05; termination F(14,60) = 1.918, P < 0.05). This is most likely an artifact of unequal samples in each group. However, the increase in Type I errors, wrongly rejecting the null hypothesis, is noted. The interaction terms demonstrate that there is no significant pattern of difference on cranial volume as a function of sex and vault suture commencement score, F(5,60) = 0.602, P = 0.698, or as a function of sex and vault suture termination score, F(6,60) = 1.800, P = 0.114.
A 2 × 6 between-subjects analysis of variance was performed on cranial volume as a function of sex (male, female) and lateral anterior suture commencement and termination score (0–5), respectively. The assumption of normality was met for all groups, P > 0.05 for each analysis. The assumption of homogeneity of variance was met for each analysis (commencement F(10,64) = 1.759, P = 0.087; termination F(10,63) = 1.759, P = 0.081). The interaction terms demonstrate that there is no significant pattern of difference on cranial volume as a function of sex and lateral anterior commencement score, F(4,64) = 1.069, P = 0.379, or as a function of sex and vault termination score, F(5,63) = 0.429, P = 0.827.
The resulting regression model for cranial volume predicted by the sum of vault suture commencement score was significant (r2 = 0.278, P < 0.001), cranial volume = 326.99 + 8.670*vault suture commencement score. The resulting regression model for cranial volume predicted by the sum of vault suture termination score was significant (r2 = 0.153, P < 0.001), cranial volume = 353.06 + 5.204*vault suture termination score. The resulting regression model for cranial volume predicted by the sum of lateral anterior suture commencement score was significant (r2 = 0.213, P < 0.001), cranial volume = 331.72 + 10.435*lateral anterior suture commencement score. The resulting regression model for cranial volume predicted by the sum of lateral anterior suture termination score was significant (r2 = 0.090, P < 0.01), cranial volume = 358.47 + 5.523*lateral anterior suture termination score. Figure 3 represents the resulting regression models with constants included.
Data were culled to 136 specimens including only those skulls that had dentition to observe. Sex was not significantly correlated with dental stage (τ = −0.052, P = 0.553), sum of vault suture commencement score (τ = −0.065, P = 0.467), sum of lateral anterior suture commencement score (τ = −0.051, P = 0.568), or sum of vault suture termination score (τ = 0.036, P = 0.693), and lateral anterior suture termination score (τ = −0.048, P = 0.577). Figures 4 and 5 exhibit the vault and lateral anterior suture scores as a function of dental stages. Mean vault and lateral anterior commencement appear as early as deciduous dentition to permanent incisor and canine eruption, but it is at third molar eruption where it appears most suture sites have commenced bony bridging. Mean vault and lateral anterior termination does not begin until third molar eruption and proceeds after.
Dental status was significantly correlated with sum of vault suture commencement score (τ = 0.715, P < 0.001), sum of lateral anterior suture commencement score (τ = 0.720, P < 0.001), or sum of vault suture termination score (τ = 0.635, P < 0.001), and sum of lateral anterior suture termination score (τ = 0.587, P < 0.001). All analyses exhibited highly significant positive correlations, suggesting a strong association between advanced dental status and osseous suture bridging and synostosis.
Data from this study support our hypothesis that suture patency continues further into adulthood than third molar eruption. This timing difference should allow for a greater masticatory influence on the resulting suture morphology than earlier neurocranial expansion. This corroborates data previously reported for humans (Meindl and Lovejoy, 1985; Cohen and MacLean, 2000). Cranial volumes reported here do exhibit a positive relationship with suture scores for both vault and lateral-anterior commencement and termination. However, the r2 values are very low suggesting that cranial volume is explaining little of the variation seen in osseous suture activity and is a poor predictive model. In addition, mean cranial capacities for each suture score reflect a mature (full size) cranial capacity for P. troglodytes (Tobias reports these to be 292–500 cc for males and 282–460 for females) (Tobias, 1971). This suggests that these specimens had completed neurocranial expansion before any evidence of sutural bridging. Thus, it would appear that, like humans, the expanding neurocranium is more important to the determination of cranial shape and early suture morphology in Pan and has little influence on later sutural bridging and synostosis patterns (Weidenreich, 1941; Moss, 1969; Moss and Salentijn, 1969; Enlow and Hans, 1996; Mooney et al., 2002; Richtsmeier et al., 2006).
In contrast, the data presented for suture scores and dental eruption status exhibit a significant positive relationship. Although some suture sites appear to commence activity earlier, Pan exhibits most of its ectocranial suture bridging and synostosis after permanent canine and third molar eruption. This is very similar to the timing that was observed in human dry skull studies (Todd and Lyon, 1925a, b, c; Krogman, 1930; Meindl and Lovejoy, 1985; Cray et al., 2008). This is clear evidence of a trend toward the appearance of bony bridging later in hominoid adulthood. The appearance of bony bridging later in hominoid ontogeny also allows for a discussion of the greater influence on suture morphology by the developed masticatory apparatus. Definitive or mature sutures have evolved to serve a number of important functions including distributing and absorbing biomechanical stresses from trauma or masticatory strain (Cohen and MacLean, 2000; Herring, 2008). Occlusal bite forces appear to absolutely increase in adult primates to be nearly double that observed for juveniles (Dechow and Carlson, 1990). Further, experimental animal modeling has shown increased masticatory forces in adults to translate to increase tension placed across the cranial sutures (Herring and Teng, 2000; Sun et al., 2004).
Changes in suture morphology reflect adaptation for energy absorption to the tensile and compressive forces of mastication as well as impact forces, pulsation of blood vessels, and any changes in intracranial pressure throughout ontogeny (Cohen and MacLean, 2000; Herring, 2008). The most apparent morphological changes are bony interdigitation across the suture. Experimentally, it has been shown that an increase in jaw muscle force activity results in greater interdigitation (Herring and Teng, 2000; Mao, 2002; Byron et al., 2004; Henderson et al., 2004; Byron, 2006; Shibazaki et al., 2007; Herring, 2008). These interdigitated sutures still exhibit some flexibility and only synostosis obliterates this feature (Herring, 2008). The ontogenic changes in suture morphology have also been suggested to reflect the optimization (perhaps selection) of cranial form as the animal grows, i.e., wide open sutures for brain growth and interdigation later in ontogeny to resist masticatory forces (Shibazaki et al., 2007). However, although biomechanical forces are implicated in changes in suture morphology, there is little evidence to suggest it directly causes the final suture fusion (Herring, 2008).
Chimpanzees display on an average 85–95% of brain growth before first molar eruption (Tobias, 1971; Herndon et al., 1999; McCollum et al., 2006). Our data suggest that there is a delay between the completion of neurocranial expansion and the onset of osseous bridging across the cranial sutures. This relationship is not surprising given the abundant data demonstrating that brain growth determines cranial vault growth and morphology (functional capsular matrix). In contrast, the developed masticatory apparatus is temporally related to changes in suture morphology. Further, biomechanical forces from mastication have also been shown to affect ectocranial morphology including sutural interdigitation and the position of the temporal lines on the cranial vault (Moss and Young, 1960; Riesenfeld, 1967; Byron et al., 2004; Sun et al., 2004; Richtsmeier et al., 2006; Sardi et al., 2007; Vecchione et al., 2007, 2010; Mooney and Richtmeier, in press). Extremely strong associations were noted in our sample between suture scores and dental eruption status. This finding corroborates previous research demonstrating that masticatory musculature and associated biomechanical forces are more likely to affect suture morphology than neurocranial expansion (Herring, 1993; Fong et al., 2003; Byron et al., 2004, 2006, 2008; Vecchione et al., 2007, 2010; Wu et al., 2007; Herring, 2008).
Pan and Homo likely share similar ontogenetic factors contributing to sutural organization and osseous fusion, including biomechanical strains. At the cellular level, sutural strain magnitudes have been shown to increase with age and bone deposition rates, which are consistent with a strain model influencing osteoblast activity at the sutural fronts (Henderson et al., 2004). At the tissue level, these species may share similar collagenous fiber organization within the cranial sutures. Histological evidence demonstrates that collagenous fibers in the suture have a more random organization before the onset of fusion. In contrast, during osseous fusion, connective tissue cells and fibers decrease in concentration in the sutural area creating an organized trabecular orientation. At this time, collagen fibers increase in tensile strength and decrease in extensibility, creating an organized orthogonal collagen lattice immediately preceding and during fusion, suggesting suture fusion may be one way to dampen strains across the sutures (Anderson et al., 2006; Wang et al., 2006a, b; Warren et al., 2008).
It is also likely that these species share age-related changes in perisutural growth factor concentration gradients, especially members of the transforming growth factor-beta (Tgf-β) family. The Tgf-βs also interact with many other growth factors and genes (i.e., FGFs and MSX) in the sutural ligament and target osteoprogenitor cells at the sutural fronts causing them to produce excess collagen and bone resulting in suture fusion (Opperman and Ogle, 2002; Poisson et al., 2004; Rawlins and Opperman, 2008; Rawlins et al., 2008). It may also be that the biomechanical strains exert shared activation of ETs-2, a protooncogene of the CAM-kinase pathway implicated in antiossification positioned between the Tgf-β and FGF signaling pathways, to maintain suture homeostais in these species (Yu et al., 2009).
Like humans, it appears that ectocranial suture activity in P. troglodytes does not exhibit a strong relationship with the expansion of the neurocranium. These data show a period of stasis (i.e., a time lag) between the attainment of adult brain size and osseous ectocranial suture bridging and synostosis. These data also show that similar to human, Pan exhibits an increase in ectocranial suture bridging following full dental eruption. Thus, it is unlikely that growth from the expanding brain (Weidenreich, 1941; Moss, 1969; Moss and Salentijn, 1969; Enlow and Hans, 1996; Mooney et al., 2002; Richtsmeier et al., 2006) provides influence on ectocranial suture synostosis in humans and P. troglodytes. Instead, sutural adaptations seen in older individuals suggest that masticatory forces influence both ectocranial suture morphology and suture synostosis (Herring, 1993; Fong et al., 2003; Byron et al., 2004, 2006, 2008; Byron, 2006; Vecchione et al., 2007, 2010; Wu et al., 2007; Herring, 2008).
H. sapiens and P. troglodytes exhibit similar ectocranial suture synostosis patterns. Results suggest that, as also found in Homo, suture synostosis pattern in P. troglodytes exhibits a similar period of stasis after neurocranial expansion cessation and the onset of suture synostosis activity and does not have a strong relationship with brain development. Instead, suture synostosis extends further into ontogeny and may be more influenced by masticatory influences. Most osseous activity was observed in the sutures occurred after full adult dental eruption. These findings suggest that masticatory forces may influence both ectocranial suture morphology and synostosis more than brain expansion in Pan, as noted similarly in Homo. Future research should continue to explore the relationship of ectocranial suture activity among and between hominoids, and primates in general, and the causal mechanism of suture synostosis.
The authors thank Lymann Jellema of the Cleveland Museum of Natural History for access to the study specimens.