Investigation of direction- and age-dependent prestretch in mouse cranial dura mater.

Cranial dura mater is a dense interwoven vascularized connective tissue that helps regulate neurocranial remodeling by responding to strains from the growing brain. Previous ex vivo experimentation has failed to account for the role of prestretch in the mechanical behavior of the dura. Here we aim to estimate the prestretch in mouse cranial dura mater and determine its dependency on direction and age. We performed transverse and longitudinal incisions in parietal dura excised from newborn (day ∼ 4) and mature (12 weeks) mice and calculated the ex vivo normalized incision opening (measured width over length). Then, similar incisions were simulated under isotropic stretching within Abaqus/Standard. Finally, prestretch was estimated by comparing the ex vivo and in silico normalized openings. There were no significant differences between the neonatal and adult mice when comparing cuts in the same direction, but adult mice were found to have significantly greater stretch in the anterior-posterior direction than in the medial-lateral direction, while neonatal dura was essentially isotropic. Additionally, our simulations show that increasing curvature impacts the incision opening, indicating that flat in silico models may overestimate prestretch.

Prolonged cyclical loading induces Haversian remodeling in mandibles of growing rabbits.

Bone adaptation to mechanical loading happens predominantly via modeling and remodeling, but the latter is poorly understood. Haversian remodeling (cortical bone replacement resulting in secondary osteons) is thought to occur in regions of low strain as part of bone maintenance or high strain in response to microdamage. However, analyses of remodeling in primates have revealed an unappreciated association with the number of daily load cycles. We tested this relationship by raising 30 male domestic rabbits (Oryctolagus cuniculus) on disparate diets from weaning to adulthood (48 weeks), facilitating a naturalistic perspective on mandibular bone adaptation. A control group consumed only rabbit pellets and an 'overuse' group ate hay in addition to pellets. To process hay, which is tougher and stiffer, rabbits increase chewing investment and duration without increasing bite force (i.e. corpus mean peak strain is similar for the two foods). Corpus remodeling in overuse rabbits was ∼1.5 times that of controls, measured as osteon population density and percentage Haversian bone. In the same subjects, there was a significant increase in overuse corpus bone formation (ratio of cortical area to cranial length), consistent with previous reports on the same dietary manipulation and bone formation in rabbits. This is the first evidence that both modeling and remodeling are simultaneously driven by the number of load cycles, independent of strain magnitude. This novel finding provides unique data on the feeding apparatus, challenges traditional thought on Haversian remodeling, and highlights the need for experimental studies of skeletal adaptation that examine mechanical factors beyond strain magnitude.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

More Challenging Diets Sustain Feeding Performance: Applications Toward the Captive Rearing of Wildlife.

The rescue and rehabilitation of young fauna is of substantial importance to conservation. However, it has been suggested that incongruous diets offered in captive environments may alter craniofacial morphology and hinder the success of reintroduced animals. Despite these claims, to what extent dietary variation throughout ontogeny impacts intrapopulation cranial biomechanics has not yet been tested. Here, finite element models were generated from the adult crania of 40 rats (n = 10 per group) that were reared on 4 different diet regimes and stress magnitudes compared during incisor bite simulations. The diets consisted of (1) exclusively hard pellets from weaning, (2) exclusively soft ground pellet meal from weaning, (3) a juvenile switch from pellets to meal, and (4) a juvenile switch from meal to pellets. We hypothesized that a diet of exclusively soft meal would result in the weakest adult skulls, represented by significantly greater stress magnitudes at the muzzle, palate, and zygomatic arch. Our hypothesis was supported at the muzzle and palate, indicating that a diet limited to soft food inhibits bone deposition throughout ontogeny. This finding presents a strong case for a more variable and challenging diet during development. However, rather than the "soft" diet group resulting in the weakest zygomatic arch as predicted, this region instead showed the highest stress among rats that switched as juveniles from hard pellets to soft meal. We attribute this to a potential reduction in number and activity of osteoblasts, as demonstrated in studies of sudden and prolonged disuse of bone. A shift to softer foods in captivity, during rehabilitation after injury in the wild for example, can therefore be detrimental to healthy development of the skull in some growing animals, potentially increasing the risk of injury and impacting the ability to access full ranges of wild foods upon release. We suggest captive diet plans consider not just nutritional requirements but also food mechanical properties when rearing wildlife to adulthood for reintroduction.

Bone remodeling and cyclical loading in maxillae of New Zealand white rabbits (Oryctolagus cuniculus).

Mammalian feeding behaviors are altered when mechanically challenging (e.g., tough, stiff) foods require large bite forces or prolonged mastication. Bony responses to high bite forces are well-documented for the mammalian skull, but osteogenesis due to cyclical loading, caused by repetitive chewing, is more poorly understood. Previous studies demonstrate that cyclical loading results in greater bone formation in the rabbit masticatory apparatus and in substantial Haversian remodeling in primate postcrania. Here we assess the relationship between cyclical loading and remodeling in the rabbit maxilla. Twenty male New Zealand white rabbits (Oryctolagus cuniculus) were raised on either an overuse or control diet (10 per group) for 48 weeks, beginning at weaning onset. The control group was raised on a diet of rabbit pellets (E = 29 MPa, R = 1031 J/m2 ), whereas the overuse group ate rabbit pellets and hay, which has high stiffness (E = 3336 MPa) and toughness (R = 2760 J/m2 ) properties. Hay requires greater chewing investment (475 chews/g) and longer chewing durations (568 s/g) than pellets (161 chews/g and 173 s/g), therefore causing cyclical loading of the jaws. Remodeling was measured as osteon population density (OPD), percent Haversian bone (%HAV), and osteon cross-sectional area (On.Ar). The only significant difference found was greater On.Ar in the alveolar region of the maxilla (p < 0.001) in the overuse group. The hypothesis that cyclical loading engenders Haversian remodeling in the developing maxilla is not supported. The continuation of modeling throughout the experimental duration may negate the need for remodeling as newly laid bone tends to be more compliant and resistant to crack propagation.

Ascites-induced compression alters the peritoneal microenvironment and promotes metastatic success in ovarian cancer.

The majority of women with recurrent ovarian cancer (OvCa) develop malignant ascites with volumes that can reach > 2 L. The resulting elevation in intraperitoneal pressure (IPP), from normal values of 5 mmHg to as high as 22 mmHg, causes striking changes in the loading environment in the peritoneal cavity. The effect of ascites-induced changes in IPP on OvCa progression is largely unknown. Herein we model the functional consequences of ascites-induced compression on ovarian tumor cells and components of the peritoneal microenvironment using a panel of in vitro, ex vivo and in vivo assays. Results show that OvCa cell adhesion to the peritoneum was increased under compression. Moreover, compressive loads stimulated remodeling of peritoneal mesothelial cell surface ultrastructure via induction of tunneling nanotubes (TNT). TNT-mediated interaction between peritoneal mesothelial cells and OvCa cells was enhanced under compression and was accompanied by transport of mitochondria from mesothelial cells to OvCa cells. Additionally, peritoneal collagen fibers adopted a more linear anisotropic alignment under compression, a collagen signature commonly correlated with enhanced invasion in solid tumors. Collectively, these findings elucidate a new role for ascites-induced compression in promoting metastatic OvCa progression.

Masticatory Loading and Ossification of the Mandibular Symphysis during Anthropoid Origins.

An ossified or 'fused' mandibular symphysis characterizes the origins of the Anthropoidea, a primate suborder that includes humans. Longstanding debate about the adaptive significance of variation in this jaw joint centers on whether a bony symphysis is stronger than an unfused one spanned by cartilage and ligaments. To provide essential information regarding mechanical performance, intact adult symphyses from representative primates and scandentians were loaded ex vivo to simulate stresses during biting and chewing - dorsoventral (DV) shear and lateral transverse bending ('wishboning'). The anthropoid symphysis requires significantly more force to induce structural failure vs. strepsirrhines and scandentians with unfused joints. In wishboning, symphyseal breakage always occurs at the midline in taxa with unfused conditions, further indicating that an ossified symphysis is stronger than an unfused joint. Greater non-midline fractures among anthropoids suggest that fusion imposes unique constraints on masticatory function elsewhere along the mandible, a phenomenon likely to characterize the evolution of fusion and jaw form throughout Mammalia.

Internal architecture of the mandibular condyle of rabbits is related to dietary resistance during growth.

Although there is considerable evidence that bone responds to the loading environment in which it develops, few analyses have examined phenotypic plasticity or bone functional adaptation in the masticatory apparatus. Prior work suggests that masticatory morphology is sensitive to differences in food mechanical properties during development; however, the importance of the timing/duration of loading and variation in naturalistic diets is less clear. Here, we examined microstructural and macrostructural differences in the mandibular condyle in four groups of white rabbits (Oryctolagus cuniculus) raised for a year on diets that varied in mechanical properties and timing of the introduction of mechanically challenging foods, simulating seasonal variation in diet. We employed sliding semilandmarks to locate multiple volumes of interest deep to the mandibular condyle articular surface, and compared bone volume fraction, trabecular thickness and spacing, and condylar size/shape among experimental groups. The results reveal a shared pattern of bony architecture across the articular surface of all treatment groups, while also demonstrating significant among-group differences. Rabbits raised on mechanically challenging diets have significantly increased bone volume fraction relative to controls fed a less challenging diet. The post-weaning timing of the introduction of mechanically challenging foods also influences architectural properties, suggesting that bone plasticity can extend well into adulthood and that bony responses to changes in loading may be rapid. These findings demonstrate that bony architecture of the mandibular condyle in rabbits responds to variation in mechanical loading during an organism's lifetime and has the potential to track dietary variation within and among species.

Ontogeny of Orbit Orientation in Primates.

Orbit orientation in primates has been linked to adaptive factors related to activity pattern and size-related variation in structural influences on orbit position. Although differences in circumorbital form between anthropoids and strepsirrhines appear to be related to interspecific disparities in levels of orbital convergence and orbital frontation, there is considerable overlap in convergence between suborders. Unfortunately, putative links between convergence and frontation across primates, and consequent arguments about primate and anthropoid origins, are likely to be influenced by allometry, the size range of a respective sample, and adaptive influences on encephalization and activity patterns. Such a multifarious system is less amenable to interspecific treatment across higher-level clades. An ontogenetic perspective is one way to evaluate transformations from one character state to another, especially as they pertain to allometric effects on phenotypic variation. We characterized the ontogeny of orbital convergence and frontation in 13 anthropoid and strepsirrhine species. In each suborder, correlation and regression analyses were used to test hypotheses regarding the allometric bases of variation in orbital orientation. Growth trajectories were analyzed intra- and inter-specifically. Frontation decreased postnatally in all taxa due to the negative scaling of brain vs. skull size. Further, interspecific variation in relative levels of frontation was linked to corresponding ontogenetic transpositions in encephalization that differed within both suborders. In strepsirrhines, postnatal increases in convergence were largely due to the negative allometry of orbit vs. skull size. In contrast, convergence in anthropoids varied little during growth, being unrelated to ontogenetic variation in either relative orbit or interorbit size. Anat Rec, 302:2093-2104, 2019. © 2019 American Association for Anatomy.

Non-sutural basicranium-derived cells undergo a unique mineralization pathway via a cartilage intermediate in vitro.

The basicranium serves as a key interface in the mammalian skull, interacting with the calvarium, facial skeleton and vertebral column. Despite its critical function, little is known about basicranial bone formation, particularly on a cellular level. The goal of this study was therefore to cultivate a better understanding of basicranial development by isolating and characterizing the osteogenic potential of cells from the neonatal murine cranial base. Osteoblast-like basicranial cells were isolated, seeded in multicellular aggregates (designated micromasses), and cultured in osteogenic medium in the presence or absence of bone morphogenetic protein-6 (BMP6). A minimal osteogenic response was observed in control osteogenic medium, while BMP6 treatment induced a chondrogenic response followed by up-regulation of osteogenic markers and extensive mineralization. This response appears to be distinct from prior analyses of the calvarium and long bones, as basicranial cells did not mineralize under standard osteogenic conditions, but rather required BMP6 to stimulate mineralization, which occurred via an endochondral-like process. These findings suggest that this site may be unique compared to other cranial elements as well as the limb skeleton, and we propose that the distinct characteristics of these cells may be a function of the distinct properties of the basicranium: endochondral ossification, dual embryology, and complex loading environment.

Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. EOC dissemination is predominantly via direct extension of cells and multicellular aggregates (MCAs) into the peritoneal cavity, which adhere to and induce retraction of peritoneal mesothelium and proliferate in the submesothelial matrix to generate metastatic lesions. Metastasis is facilitated by the accumulation of malignant ascites (500 ml to >2 l), resulting in physical discomfort and abdominal distension, and leading to poor prognosis. Although intraperitoneal fluid pressure is normally subatmospheric, an average intraperitoneal pressure of 30 cmH2O (22.1 mmHg) has been reported in women with EOC. In this study, to enable experimental evaluation of the impact of high intraperitoneal pressure on EOC progression, two new in vitro model systems were developed. Initial experiments evaluated EOC MCAs in pressure vessels connected to an Instron to apply short-term compressive force. A Flexcell Compression Plus system was then used to enable longer-term compression of MCAs in custom-designed hydrogel carriers. Results show changes in the expression of genes related to epithelial-mesenchymal transition as well as altered dispersal of compressed MCAs on collagen gels. These new model systems have utility for future analyses of compression-induced mechanotransduction and the resulting impact on cellular responses related to intraperitoneal metastatic dissemination.This article has an associated First Person interview with the first authors of the paper.

Aging Increases Susceptibility to Ovarian Cancer Metastasis in Murine Allograft Models and Alters Immune Composition of Peritoneal Adipose Tissue.

Ovarian cancer, the most deadly gynecological malignancy in U.S. women, metastasizes uniquely, spreading through the peritoneal cavity and often generating widespread metastatic sites before diagnosis. The vast majority of ovarian cancer cases occur in women over 40 and the median age at diagnosis is 63. Additionally, elderly women receive poorer prognoses when diagnosed with ovarian cancer. Despite age being a significant risk factor for the development of this cancer, there are little published data which address the impact of aging on ovarian cancer metastasis. Here we report that the aged host is more susceptible to metastatic success using two murine syngeneic allograft models of ovarian cancer metastasis. This age-related increase in metastatic tumor burden corresponds with an increase in tumor infiltrating lymphocytes (TILs) in tumor-bearing mice and alteration of B cell-related pathways in gonadal adipose tissue. Based on this work, further studies elucidating the status of B cell TILs in mouse models of metastasis and human tumors in the context of aging are warranted.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders.

Multiparity activates interferon pathways in peritoneal adipose tissue and decreases susceptibility to ovarian cancer metastasis in a murine allograft model.

Ovarian cancer is the fifth leading cause of cancer deaths in U.S. women and the deadliest gynecologic malignancy. This lethality is largely due to the fact that most cases are diagnosed at metastatic stages of the disease when the prognosis is poor. Epidemiologic studies consistently demonstrate that parous women have a reduced risk of developing ovarian cancer, with a greater number of births affording greater protection; however little is known about the impact of parity on ovarian cancer metastasis. Here we report that multiparous mice are less susceptible to ovarian cancer metastasis in an age-matched syngeneic murine allograft model. Interferon pathways were found to be upregulated in healthy adipose tissue of multiparous mice, suggesting a possible mechanism for the multiparous-related protective effect against metastasis. This protective effect was found to be lost with age. Based on this work, future studies exploring therapeutic strategies which harness the multiparity-associated protective effect demonstrated here are warranted.

Dietary variation and mechanical properties of articular cartilage in the temporomandibular joint: implications for the role of plasticity in mechanobiology and pathobiology.

Due to their nature as tissue composites, skeletal joints pose an additional challenge in terms of evaluating the functional significance of morphological variation in their bony and cartilaginous components in response to altered loading conditions. Arguably, this complexity requires more direct means of investigating joint plasticity and performance than typically employed to analyze macro- and micro-anatomical phenomena. To address a significant gap in our understanding of the plasticity of the mammalian temporomandibular joint (TMJ), we investigated the histology and mechanical properties of condylar articular cartilage in rabbits subjected to long-term variation in diet-induced masticatory stresses, specifically cyclical loading. Three cohorts of male weanlings were raised for six months on different diets until adulthood. Following euthanasia, the TMJ condyles on one side were dissected away, fixed, decalcified, dehydrated, embedded and sectioned. Safranin O staining was employed to identify variation in proteoglycan content, which in turn was used to predict patterns of articular cartilage stiffness in contralateral condylar specimens for each treatment group. Hematoxylin and eosin staining was used to quantify diet-induced changes in chondrocyte hypertrophy and cellularity. Mechanical tests document significant decreases in articular cartilage stiffness corresponding to patterns of extracellular matrix relative protein abundance in rabbits subjected to greater cyclical loading. This indicates that TMJs routinely subjected to higher masticatory stresses due to a challenging diet eventually develop postnatal decreases in the ability to counter compressive loads during postcanine biting and chewing. These findings provide novel information regarding TMJ performance, with broader implications about the costs and benefits of phenotypic plasticity as well as implications for how such biological processes affect connective tissue mechanobiology and pathobiology.

Intracranial and hierarchical perspective on dietary plasticity in mammals.

The effect of dietary properties on craniofacial form has been the focus of numerous functional studies, with increasingly more work dedicated to the importance of phenotypic plasticity. As bone is a dynamic tissue, morphological variation related to differential loading is well established for many masticatory structures. However, the adaptive osteogenic response of several cranial sites across multiple levels of bony organization remains to be investigated. Here, rabbits were obtained at weaning and raised for 48 weeks until adulthood in order to address the naturalistic influence of altered loading on the long-term development of masticatory and non-masticatory elements. Longitudinal data from micro-computed tomography (µCT) scans were used to test the hypothesis that variation in cortical bone formation and biomineralization in masticatory structures is linked to increased stresses during oral processing of mechanically challenging foods. It was also hypothesized that similar parameters for neurocranial structures would be minimally affected by varying loads as this area is characterized by low strains during mastication and reduced hard-tissue mechanosensitivity. Hypotheses were supported regarding bone formation for maxillomandibular and neurocranial elements, though biomineralization trends of masticatory structures did not mirror macroscale findings. Varying osteogenic responses in masticatory elements suggest that physiological adaptation, and corresponding variation in skeletal performance, may reside differentially at one level of bony architecture, potentially affecting the accuracy of behavioral and in silico reconstructions. Together, these findings underscore the complexity of bone adaptation and highlight functional and developmental variation in determinants of skull form.

Facing the facts: The Runx2 gene is associated with variation in facial morphology in primates.

The phylogenetic and adaptive factors that cause variation in primate facial form-including differences among the major primate clades and variation related to feeding and/or social behavior-are relatively well understood. However, comparatively little is known about the genetic mechanisms that underlie diversity in facial form in primates. Because it is essential for osteoblastic differentiation and skeletal development, the runt-related transcription factor 2 (Runx2) is one gene that may play a role in these genetic mechanisms. Specifically, polymorphisms in the QA ratio (determined by the ratio of the number of polyglutamines to polyalanines in one functional domain of Runx2) have been shown to be correlated with variation in facial length and orientation in other mammal groups. However, to date, the relationship between variation in this gene and variation in facial form in primates has not been explicitly tested. To test the hypothesis that the QA ratio is correlated with facial form in primates, the current study quantified the QA ratio, facial length, and facial angle in a sample of 33 primate species and tested for correlation using phylogenetic generalized least squares. The results indicate that the QA ratio of the Runx2 gene is positively correlated with variation in relative facial length in anthropoid primates. However, no correlation was found in strepsirrhines, and there was no correlation between facial angle and the QA ratio in any groups. These results suggest that, in primates, the QA ratio of the Runx2 gene may play a role in modulating facial size, but not facial orientation. This study therefore provides important clues about the genetic and developmental mechanisms that may underlie variation in facial form in primates.

The fracture toughness of small animal cortical bone measured using arc-shaped tension specimens: Effects of bisphosphonate and deproteinization treatments.

Small animal models, and especially transgenic models, have become widespread in the study of bone mechanobiology and metabolic bone disease, but test methods for measuring fracture toughness on multiple replicates or at multiple locations within a single small animal bone are lacking. Therefore, the objective of this study was to develop a method to measure cortical bone fracture toughness in multiple specimens and locations along the diaphysis of small animal bones. Arc-shaped tension specimens were prepared from the mid-diaphysis of rabbit ulnae and loaded to failure to measure the radial fracture toughness in multiple replicates per bone. The test specimen dimensions, crack length, and maximum load met requirements for measuring the plane strain fracture toughness. Experimental groups included a control group, bisphosphonate treatment group, and an ex vivo deproteinization treatment following bisphosphonate treatment (5 rabbits/group and 15 specimens/group). The fracture toughness of ulnar cortical bone from rabbits treated with zoledronic acid for six months exhibited no difference compared with the control group. Partially deproteinized specimens exhibited significantly lower fracture toughness compared with both the control and bisphosphonate treatment groups. The deproteinization treatment increased tissue mineral density (TMD) and resulted in a negative linear correlation between the measured fracture toughness and TMD. Fracture toughness measurements were repeatable with a coefficient of variation of 12-16% within experimental groups. Retrospective power analysis of the control and deproteinization treatment groups indicated a minimum detectable difference of 0.1MPa·m1/2. Therefore, the overall results of this study suggest that arc-shaped tension specimens offer an advantageous new method for measuring the fracture toughness in small animal bones.

Ontogenetic and functional modularity in the rodent mandible.

The material properties of diets consumed by juvenile individuals are known to affect adult morphological outcomes. However, much of the current experimental knowledge regarding dietary effects on masticatory form is derived from studies in which individuals are fed a non-variable diet for the duration of their postweaning growth period. Thus, it remains unclear how intra-individual variation in diet, due to ontogenetic variation in feeding behaviors or seasonal resource fluctuations, affects the resulting adult morphology. Furthermore, the mandible is composed of multiple developmental and functional subunits, and the extent to which growth and plasticity among these modules is correlated may be misestimated by studies that examine non-variable masticatory function in adults only. To address these gaps in our current knowledge, this study raised Sprague Dawley rats (n=42) in four dietary cohorts from weaning to skeletal maturity. Two cohorts were fed a stable ("annual") diet of either solid or powdered pellets. The other two cohorts were fed a variable ("seasonal") diet consisting of solid/powdered pellets for the first half of the study, followed by a shift to the opposite diet. Results of longitudinal morphometric analyses indicate that variation in the mandibular corpus is more prominent at immature ontogenetic stages, likely due to processes of dental eruption and the growth of tooth roots. Furthermore, adult morphology is influenced by both masticatory function and the ontogenetic timing of this function, e.g., the consumption of a mechanically resistant diet. The morphology of the coronoid process was found to separate cohorts on the basis of their early weanling diet, suggesting that the coronoid process/temporalis muscle module may have an early plasticity window related to high growth rates during this life stage. Conversely, the morphology of the angular process was found to be influenced by the consumption of a mechanically resistant diet at any point during the growth period, but with a tendency to reflect the most recent diet. The prolonged plasticity window of the angular process/pterygomasseteric muscle module may be related to delayed ossification and muscular maturation within this module. The research presented here highlights the importance of more naturalistic models of mammalian feeding, and underscores the need for a better understanding of the processes of both morphological and behavioral maturation that follow weaning.

Bone up: craniomandibular development and hard-tissue biomineralization in neonate mice.

The presence of regional variation in the osteogenic abilities of cranial bones underscores the fact that the mechanobiology of the mammalian skull is more complex than previously recognized. However, the relationship between patterns of cranial bone formation and biomineralization remains incompletely understood. In four strains of mice, micro-computed tomography was used to measure tissue mineral density during perinatal development in three skull regions (calvarium, basicranium, mandible) noted for variation in loading environment, embryological origin, and ossification mode. Biomineralization levels increased during perinatal ontogeny in the mandible and calvarium, but did not increase in the basicranium. Tissue mineral density levels also varied intracranially, with density in the mandible being highest, in the basicranium intermediate, and in the calvarium lowest. Perinatal increases in, and elevated levels of, mandibular biomineralization appear related to the impending postweaning need to resist elevated masticatory stresses. Similarly, perinatal increases in calvarial biomineralization may be linked to ongoing brain expansion, which is known to stimulate sutural bone formation in this region. The lack of perinatal increase in basicranial biomineralization could be a result of earlier developmental maturity in the cranial base relative to other skull regions due to its role in supporting the brain's mass throughout ontogeny. These results suggest that biomineralization levels and age-related trajectories throughout the skull are influenced by the functional environment and ontogenetic processes affecting each region, e.g., onset of masticatory loads in the mandible, whereas variation in embryology and ossification mode may only have secondary effects on patterns of biomineralization. Knowledge of perinatal variation in tissue mineral density, and of normal cranial bone formation early in development, may benefit clinical therapies aiming to correct developmental defects and traumatic injuries in the skull, and more generally characterize loading environments and skeletal adaptations in mammals by highlighting the need for multi-level analyses for evaluating functional performance of cranial bone.