Dental tissue changes in juvenile and adult mice with osteogenesis imperfecta.

Osteogenesis imperfecta (OI), a disorder of type I collagen, causes skeletal deformities as well as defects in dental tissues, which lead to increased enamel wear and smaller teeth with shorter roots. Mice with OI exhibit similar microstructural dentin changes, including reduced dentin tubule density and dentin cross-sectional area. However, the effects of these mutations on gross dental morphology and dental tissue volumes have never been characterized in the osteogenesis imperfecta murine (OIM) mouse model. Here we compare mineralized dental tissue measurements of OIM mice and unaffected wild type (WT) littermates at the juvenile and adult stages. The maxillary and mandibular incisors and first molars were isolated from microCT scans, and tissue volumes and root length were measured. OIM mice have smaller teeth with shorter roots relative to WT controls. Maxillary incisor volumes differed significantly between OIM and WT mice at both the juvenile and young adult stage, perhaps due to shortening of the maxilla itself in OIM mice. Additionally, adult OIM mice have significantly less crown enamel volume than do juveniles, potentially due to loss through wear. Thus, OIM mice demonstrate a dental phenotype similar to humans with OI, with decreased tooth size, decreased root length, and accelerated enamel wear. Further investigation of dental development in the OIM mouse may have important implications for the development and treatment of dental issues in OI patients.

Neurocranial growth in the OIM mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a disorder of type I collagen characterized by abnormal bone formation. The OI craniofacial phenotype includes midfacial underdevelopment, as well as neurocranial changes (e.g., macrocephaly and platybasia) that may also affect underlying nervous tissues. This study aims to better understand how OI affects the integrated development of the neurocranium and the brain. Juvenile and adult mice with OI (OIM) and unaffected wild type (WT) littermates were imaged using in vivo micro-computed tomography (microCT). Virtual endocast models were used to measure brain volume, and 3D landmarks were collected from the cranium and brain endocasts. Geometric morphometric analyses were used to compare brain shape and integration between the genotypes. OIM mice had increased brain volumes (relative to cranial centroid size) only at the juvenile stage. No significant difference was seen in cranial base angle (CBA) between OIM and WT mice. However, CBA was higher in juvenile than in adult OIM mice. Brain shape was significantly different between OIM and WT mice at both stages, with OIM mice having more globular brains than WT mice. Neurocranial and brain morphology were strongly integrated within both genotypes, while adult OIM mice tended to have lower levels of skull-brain integration than WT mice. These results suggest that neurocranial dysmorphologies in OI may be more severe at earlier stages of postnatal development. Decreased skull-brain integration in adult mice suggests that compensatory mechanisms may exist during postnatal growth to maintain neurological function despite significant changes in neurocranial morphology.

Morphological variability in the inner ear of mice with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is known to cause hearing loss in ~60% of the affected human population. While OI-related pathologies have been studied in the middle ear, the development of cochlear pathologies is less well understood. In this study, we examine OI-related pathologies of the cochlea in a mouse model of OI to (1) document variation between OI and unaffected mice, and (2) assess the intrusion of the otic capsule onto the cochlea by analyzing differences in duct volumes. Juvenile and adult OIM C57BL/6mice were compared to unaffected wildtype (WT) mice using three-dimensional models of the cochlea generated from high resolution micro-CT scans. Two-tailed Mann-Whitney U tests were then used to investigate duct volume differences both within and between the OI and WT samples. Areas of higher ossification were observed at the cochlear base in the OI sample. OI mice also had significant intraindividual differences in duct volume between right and left ears (4%-15%), an effect not observed in WT mice. WT and OI duct volumes showed a large degree of overlap, although the OIM volumes were more variable. Our findings indicate that OIM mice are likely to exhibit more asymmetry and variation in cochlear volume despite minor differences in sample cochlear volumes, possibly due to bony capsule intrusion. This suggests a potential mechanism of hearing loss, and a high potential for cochlear and otic capsule alteration in OIM mice.

Indications of musculoskeletal health in deceased male individuals with lower-limb amputations: comparison to non-amputee and diabetic controls.

Individuals with lower-limb amputations, many of whom have type 2 diabetes, experience impaired musculoskeletal health. This study: (1) compared residual and intact limbs of diabetic and non-diabetic post-mortem individuals with amputation to identify structures vulnerable to injury, and (2) compared findings to diabetic and healthy control groups to differentiate influences of amputation and diabetes on musculoskeletal health. Postmortem CT scans of three groups, ten individuals each, were included: (1) individuals with transtibial or transfemoral amputations, half with diabetes (2) diabetic controls, and (3) healthy controls. Hip and knee joint spaces, cross-sectional thigh muscle and fat areas, and cross-sectional bone properties (e.g. area, thickness, geometry) were measured. Wilcoxon Signed-Rank and Kruskal-Wallis tests assessed statistical significance. Asymmetry percentages between limbs assessed clinical significance. Residual limbs of individuals with amputation, particularly those with diabetes, had significantly less thigh muscle area and thinner distal femoral cortical bone compared to intact limbs. Compared to control groups, individuals with amputation had significantly narrower joint spaces, less thigh muscle area bilaterally, and thinner proximal femoral cortical bone in the residual limb. Diabetic individuals with amputation had the most clinically significant asymmetry. Findings tended to align with those of living individuals. However, lack of available medical information and small sample sizes reduced the anticipated clinical utility. Larger sample sizes of living individuals are needed to assess generalizability of findings. Quantifying musculoskeletal properties and differentiating influences of amputation and diabetes could eventually help direct rehabilitation techniques.

A review of musculoskeletal adaptations in individuals following major lower-limb amputation.

Structural musculoskeletal adaptations following amputation, such as bone mineral density (BMD) or muscle architecture, are often overlooked despite their established contributions to gait rehabilitation and the development of adverse secondary physical conditions. The purpose of this review is to provide a summary of the existing literature investigating musculoskeletal adaptations in individuals with major lower-limb amputations to inform clinical practice and provide directions for future research. Google Scholar, PubMed, and Scopus were searched for original peer-reviewed studies that included individuals with transtibial or transfemoral amputations. Summary data of twenty-seven articles indicated reduced BMD and increased muscle atrophy in amputees compared to controls, and in the amputated limb compared to intact and control limbs. Specifically, BMD was reduced in T-scores and Z-scores, femoral neck, and proximal tibia. Muscle atrophy was evidenced by decreased thigh cross-sectional area, decreased quadriceps thickness, and increased amounts of thigh fat. Overall, amputees have impaired musculoskeletal health. Future studies should include dysvascular etiologies to address their effects on musculoskeletal health and functional mobility. Moreover, clinicians can use these findings to screen increased risks of adverse sequelae such as fractures, osteopenia/porosis, and muscular atrophy, as well as target specific rehabilitation exercises to reduce these risks.

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.

Craniofacial allometry in the OIM-/- mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a skeletal disorder characterized by the impaired synthesis of type I collagen (Col1). This study tests the hypothesis that the craniofacial phenotype of severe OI is linked to an overall reduction in body size. 3D landmark data were collected from µCT scans of adult OIM-/- and wild-type (WT) mice and used to calculate centroid sizes (CS) and interlandmark distances (ILDs). To remove the effect of body size, ILDs were scaled against craniomandibular lengths and CS. Mann-Whitney U tests were used to compare CS and absolute/scaled ILDs between genotypes. OIM-/- mice are smaller than their WT littermates in body mass, craniomandibular CS, and absolute ILDs including skull, basicranial, palatal, mandibular, and toothrow lengths. When linear distances are scaled to CS, OIM-/- mice have a relatively short midface, short nasal bones, tall mandibular corpora, and long mandibular toothrows. Results underscore the importance of size and scaling in morphometric analyses. The deleterious effect of Col1 mutations on global skeletal dimensions combined with localized morphometric changes may underlie the facial phenotype seen in human patients with severe OI. Attempts to identify these localized changes should first account for the restricted growth and small body sizes present in individuals with OI.

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.

Limitations of a morphological criterion of adaptive inference in the fossil record.

Experimental analyses directly inform how an anatomical feature or complex functions during an organism's lifetime, which serves to increase the efficacy of comparative studies of living and fossil taxa. In the mammalian skull, food material properties and feeding behaviour have a pronounced influence on the development of the masticatory apparatus. Diet-related variation in loading magnitude and frequency induce a cascade of changes at the gross, tissue, cellular, protein and genetic levels, with such modelling and remodelling maintaining the integrity of oral structures vis-à-vis routine masticatory stresses. Ongoing integrative research using rabbit and rat models of long-term masticatory plasticity offers unique insight into the limitations of functional interpretations of fossilised remains. Given the general restriction of the palaeontological record to bony elements, we argue that failure to account for the disparity in the hierarchical network of responses of hard versus soft tissues may overestimate the magnitude of the adaptive divergence that is inferred from phenotypic differences. Second, we note that the developmental onset and duration of a loading stimulus associated with a given feeding behaviour can impart large effects on patterns of intraspecific variation that can mirror differences observed among taxa. Indeed, plasticity data are relevant to understanding evolutionary transformations because rabbits raised on different diets exhibit levels of morphological disparity comparable to those found between closely related primate species that vary in diet. Lastly, pronounced variation in joint form, and even joint function, can also characterise adult conspecifics that differ solely in age. In sum, our analyses emphasise the importance of a multi-site and hierarchical approach to understanding determinants of morphological variation, one which incorporates critical data on performance.

XROMM analysis of tooth occlusion and temporomandibular joint kinematics during feeding in juvenile miniature pigs.

Like humans, domestic pigs are omnivorous and thus are a common model for human masticatory function. Prior attempts to characterize food-tooth interactions and jaw movements associated with mastication have been limited to aspects of the oral apparatus that are visible externally (with videography) and/or to 2D movements of oral structures (with monoplanar videofluoroscopy). We used XROMM, a 3D technique that combines CT-based morphology with biplanar videofluoroscopy, to quantify mandibular kinematics, tooth occlusion and mandibular condylar displacements within the temporomandibular joint (TMJ) during feeding. We observed that the pig TMJ moved detectably in only three of six possible degrees of freedom during mastication: two rotations, pitch and yaw; and one translation, protraction-retraction. Asymmetrical yaw around a dorsoventral axis produced the observed alternating left-right chewing cycles responsible for food reduction. Furthermore, the relative motions of the upper and lower premolars contained a substantial mesiodistal component in addition to the buccolingual component, resulting in an oblique (rather than a strictly transverse) power stroke. This research demonstrates the capacity of XROMM to explore the kinematic underpinnings of key masticatory movements, such as the occlusal power stroke, by integrating tooth, joint and rigid body jaw movements. XROMM also allowed us to test kinematic hypotheses based on skeletal anatomy with actual kinematics observed during naturalistic feeding behaviors. We observed that the soft tissue structures of the TMJ appear to play a significant role in limiting the range of motion of a joint, and thus analyses based solely on osseous morphology may over-estimate joint mobility.

Masticatory loading, function, and plasticity: a microanatomical analysis of mammalian circumorbital soft-tissue structures.

In contrast to experimental evidence regarding the postorbital bar, postorbital septum, and browridge, there is exceedingly little evidence regarding the load-bearing nature of soft-tissue structures of the mammalian circumorbital region. This hinders our understanding of pronounced transformations during primate origins, in which euprimates evolved a postorbital bar from an ancestor with the primitive mammalian condition where only soft tissues spanned the lateral orbital margin between frontal bone and zygomatic arch. To address this significant gap, we investigated the postorbital microanatomy of rabbits subjected to long-term variation in diet-induced masticatory stresses. Rabbits exhibit a masticatory complex and feeding behaviors similar to primates, yet retain a more primitive mammalian circumorbital region. Three cohorts were obtained as weanlings and raised on different diets until adult. Following euthanasia, postorbital soft tissues were dissected away, fixed, and decalcified. These soft tissues were divided into inferior, intermediate, and superior units and then dehydrated, embedded, and sectioned. H&E staining was used to characterize overall architecture. Collagen orientation and complexity were evaluated via picrosirius-red staining. Safranin-O identified proteoglycan content with additional immunostaining performed to assess Type-II collagen expression. Surprisingly, the ligament along the lateral orbital wall was composed of elastic fibrocartilage. A more degraded organization of collagen fibers in this postorbital fibrocartilage is correlated with increased masticatory forces due to a more fracture-resistant diet. Furthermore, the lack of marked changes in the extracellular composition of the lateral orbital wall related to tissue viscoelasticity suggests it is unlikely that long-term exposure to elevated masticatory stresses underlies the development of a bony postorbital bar.

Evidence for the influence of diet on cranial form and robusticity.

The evolutionary significance of cranial form and robusticity in early Homo has been variously attributed to allometry, encephalization, metabolic factors, locomotor activity, and masticatory forces. However, the influence of such factors is variably understood. To evaluate the effect of masticatory loading on neurocranial form, sibling groups of weanling white rabbits were divided into two cohorts of 10 individuals each and raised on either a soft diet or a hard/tough diet for 16 weeks until subadulthood. Micro-CT was used to quantify and visualize morphological variation between treatment groups. Results reveal trends (P < 0.10) for greater outer table thickness of the frontal bones, zygomatic height, and cranial globularity in rabbits raised on a hard/tough diet. Furthermore, analyses of three-dimensional coordinate landmark data indicate that the basicrania of hard/tough diet rabbits exhibit more robust middle cranial fossae and pterygoid plates, as well as altered overall morphology of the caudal cranial fossa. Thus, long term increases in masticatory loads may result in thickening of the bones of the neurocranial vault and/or altering the curvature of the walls. Differences in cranial regions not directly associated with the generation or resistance of masticatory forces (i.e., frontal bone, basicranium) may be indirectly correlated with diet-induced variation in maxillomandibular morphology. These findings also suggest that long-term variation in masticatory forces associated with differences in dietary properties can contribute to the complex and multifactorial development of neurocranial morphology.

Septa and processes: convergent evolution of the orbit in haplorhine primates and strigiform birds.

According to the "nocturnal visual predation hypothesis" (NVPH), the convergent eyes and orbits of primates result from selection for improved stereoscopic depth perception to facilitate manual capture of prey at night. Within primates, haplorhines share additional derived orbital morphologies, including a postorbital septum and greater orbital convergence than any other mammalian clade. While the homology and function of the haplorhine septum remain controversial, experimental data suggest that septa evolved to inhibit mechanical disturbance of the orbital contents by the anterior temporalis muscle during mastication. According to this "insulation hypothesis," haplorhines are particularly susceptible to disruption of the orbital contents because they have large and highly convergent eyes and orbits. However, comparative tests of the insulation hypothesis have been hindered by the morphological uniqueness of the haplorhine septum among mammals. Among birds, owls (Strigiformes) exhibit an expanded postorbital process that may be functionally analogous to the haplorhine septum. Here we present a comparative analysis of orbital morphology in 103 avian species that tests two hypotheses: (1) large, convergent orbits are associated with nocturnal visual predation, and (2) the strigiform postorbital process and haplorhine postorbital septum similarly function to insulate the eyes from contractions of mandibular adductors. Strigiforms, as nocturnal visual predators, possess relatively large orbits and exhibit the highest degree of orbital convergence in our sample. Notably, orbital convergence does not scale with orbit size in birds as in mammals. Owls are also unique among the birds examined in possessing extensive, plate-like postorbital processes that largely isolate the orbits from the temporal fossae. Furthermore, dissections of four owl species demonstrate that the expanded strigiform postorbital process deflects the path of mandibular adductors around the eye's inferolateral margin. These findings provide further comparative support for both the NVPH and the insulation hypothesis.

Phenotypic plasticity and function of the hard palate in growing rabbits.

Morphological variation related to differential loading is well known for many craniomandibular elements. Yet, the function of the hard palate, and in particular the manner in which cortical and trabecular bone of the palate respond to masticatory loads, remains more ambiguous. Here, experimental data are presented that address the naturalistic influence of biomechanical loading on the postweaning development and structure of the hard palate. A rabbit model was used to test the hypothesis that variation in the morphology of the hard palate is linked to variation in masticatory stresses. Rabbit siblings were divided as weanlings into soft and hard/tough dietary treatment groups of 10 subjects each and were raised for 15 weeks until subadulthood. MicroCT analyses indicate that rabbits subjected to elevated masticatory loading developed hard palates with significantly greater bone area, greater cortical bone thickness along the oral lamina, and thicker anterior palates. Such diet-induced levels of palatal plasticity are comparable to those for other masticatory elements, which likely reflect osteogenic responses for maintaining the functional integrity of the palate vis-à-vis elevated stresses during unilateral mastication. These data support a role for mechanical loading in the determination of palatal morphology, especially its internal structure, in living and fossil mammals such as the hominin Paranthropus. Furthermore, these findings have potential implications for the evolution of the mammalian secondary hard palate as well as for clinical considerations of human oral pathologies.

Using "Mighty Mouse" to understand masticatory plasticity: myostatin-deficient mice and musculoskeletal function.

Knockout mice lacking myostatin (Mstn), a negative regulator of the growth of skeletal muscle, develop significant increases in the relative mass of masticatory muscles as well as the ability to generate higher maximal muscle forces. Wild-type and Mstn-deficient mice were compared to investigate the postnatal influence of elevated masticatory loads due to increased jaw-adductor and bite forces on the biomineralization of mandibular articular and cortical bone, the internal structure of the jaw joints, and the composition of temporomandibular joint (TMJ) articular cartilage. To provide an interspecific perspective on the long-term responses of mammalian jaw joints to altered loading conditions, the findings on mice were compared to similar data for growing rabbits subjected to long-term dietary manipulation. Statistically significant differences in joint proportions and bone mineral density between normal and Mstn-deficient mice, which are similar to those observed between rabbit loading cohorts, underscore the need for a comprehensive analysis of masticatory tissue plasticity vis-à-vis altered mechanical loads, one in which variation in external and internal structure are considered. Differences in the expression of proteoglycans and type-II collagen in TMJ articular cartilage between the mouse and rabbit comparisons suggest that the duration and magnitude of the loading stimulus will significantly affect patterns of adaptive and degradative responses. These data on mammals subjected to long-term loading conditions offer novel insights regarding variation in ontogeny, life history, and the ecomorphology of the feeding apparatus.