Mutation of the galectin-3 glycan-binding domain (Lgals3-R200S) enhances cortical bone expansion in male mice and trabecular bone mass in female mice.

We previously observed that genomic loss of galectin-3 (Gal-3; encoded by Lgals3) in mice has a significant protective effect on age-related bone loss. Gal-3 has both intracellular and extracellular functionality, and we wanted to assess whether the affect we observed in the Lgals3 knockout (KO) mice could be attributed to the ability of Gal-3 to bind glycoproteins. Mutation of a highly conserved arginine to a serine in human Gal-3 (LGALS3-R186S) blocks glycan binding and secretion. We generated mice with the equivalent mutation (Lgals3-R200S) and observed a subsequent reduction in Gal-3 secretion from mouse embryonic fibroblasts and in circulating blood. When examining bone structure in aged mice, we noticed some similarities to the Lgals3-KO mice and some differences. First, we observed greater bone mass in Lgals3-R200S mutant mice, as was previously observed in Lgals3-KO mice. Like Lgals3-KO mice, significantly increased trabecular bone mass was only observed in female Lgals3-R200S mice. These results suggest that the greater bone mass observed is driven by the loss of extracellular Gal-3 functionality. However, the results from our cortical bone expansion data showed a sex-dependent difference, with only male Lgals3-KO mice having an increased response, contrasting with our earlier study. These notable sex differences suggest a potential role for sex hormones, most likely androgen signaling, being involved. In summary, our results suggest that targeting extracellular Gal-3 function may be a suitable treatment for age-related loss of bone mass.

Cortical bone development, maintenance and porosity: genetic alterations in humans and mice influencing chondrocytes, osteoclasts, osteoblasts and osteocytes.

Cortical bone structure is a crucial determinant of bone strength, yet for many years studies of novel genes and cell signalling pathways regulating bone strength have focused on the control of trabecular bone mass. Here we focus on mechanisms responsible for cortical bone development, growth, and degeneration, and describe some recently described genetic-driven modifications in humans and mice that reveal how these processes may be controlled. We start with embryonic osteogenesis of preliminary bone structures preceding the cortex and describe how this structure consolidates then matures to a dense, vascularised cortex containing an increasing proportion of lamellar bone. These processes include modelling-induced, and load-dependent, asymmetric cortical expansion, which enables the cortex's transition from a highly porous woven structure to a consolidated and thickened highly mineralised lamellar bone structure, infiltrated by vascular channels. Sex-specific differences emerge during this process. With aging, the process of consolidation reverses: cortical pores enlarge, leading to greater cortical porosity, trabecularisation and loss of bone strength. Each process requires co-ordination between bone formation, bone mineralisation, vascularisation, and bone resorption, with a need for locational-, spatial- and cell-specific signalling pathways to mediate this co-ordination. We will discuss these processes, and a number of cell-signalling pathways identified in both murine and human genetic studies to regulate cortical bone mass, including signalling through gp130, STAT3, PTHR1, WNT16, NOTCH, NOTUM and sFRP4.

Voluntary Wheel Running Partially Compensates for the Effects of Global Estrogen Receptor-α Knockout on Cortical Bone in Young Male Mice.

Estrogen receptor-α knockout (ERKO) in female, but not male, mice results in an impaired osteogenic response to exercise, but the mechanisms behind this ability in males are unknown. We explored the main and interactive effects of ERKO and exercise on cortical geometry, trabecular microarchitecture, biomechanical strength, and sclerostin expression in male mice. At 12 weeks of age, male C57BL/6J ERKO and WT animals were randomized into two groups: exercise treatment (EX) and sedentary (SED) controls, until 22 weeks of age. Cortical geometry and trabecular microarchitecture were measured via µCT; biomechanical strength was assessed via three-point bending; sclerostin expression was measured via immunohistochemistry. Two-way ANOVA was used to assess sclerostin expression and trabecular microarchitecture; two-way ANCOVA with body weight was used to assess cortical geometry and biomechanical strength. ERKO positively impacted trabecular microarchitecture, and exercise had little effect on these outcomes. ERKO significantly impaired cortical geometry, but exercise was able to partially reverse these negative alterations. EX increased cortical thickness regardless of genotype. There were no effects of genotype or exercise on sclerostin expression. In conclusion, male ERKO mice retain the ability to build bone in response to exercise, but altering sclerostin expression is not one of the mechanisms involved.

The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression.

Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3' untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones.

Mineralization of cortical bone during maturation and growth in rabbits.

INTRODUCTION: The composite nature of bone as a material governs its structure and mechanical behavior. How the collagenous matrix mineralizes, in terms of both mineral deposition and structure of the mineral crystals, is highly interesting when trying to elucidate the complex structural changes that occur during bone growth and maturation. We have previously looked at mineral deposition and structural evolution of the collagenous matrix, linking both to changes in mechanics. The purpose of this study was to provide specific information on changes in crystal size and organization as a function of growth and maturation. MATERIALS AND METHODS: Using micro-computed tomography (µCT) and micro-focused scanning small-angle X-ray scattering (SAXS) we investigated cortical bone in two orthogonal directions relative to the long axis of the humeri of New Zealand White rabbits spanning from new-born to 6-months of age. We also investigated the changes with tissue age by looking at radial profiles of osteonal structures in the 6-months old rabbits. The findings were compared to our previous compositional, structural and mechanical data on the same sample cohort. RESULTS: µCT showed a continuous mineral deposition up until 3-months of age, whilst the SAXS data showed an increase in both crystal thickness and degree of orientation up until 6-months of age. The osteonal profiles showed no statistically significant changes in crystal thickness. CONCLUSIONS: Comparison to previously collected mechanical data suggests that changes are not only explained by amount of mineral in the tissue but also by the crystal dimensions.

Compositional and mechanical properties of growing cortical bone tissue: a study of the human fibula.

Human cortical bone contains two types of tissue: osteonal and interstitial tissue. Growing bone is not well-known in terms of its intrinsic material properties. To date, distinctions between the mechanical properties of osteonal and interstitial regions have not been investigated in juvenile bone and compared to adult bone in a combined dataset. In this work, cortical bone samples obtained from fibulae of 13 juveniles patients (4 to 18 years old) during corrective surgery and from 17 adult donors (50 to 95 years old) were analyzed. Microindentation was used to assess the mechanical properties of the extracellular matrix, quantitative microradiography was used to measure the degree of bone mineralization (DMB), and Fourier transform infrared microspectroscopy was used to evaluate the physicochemical modifications of bone composition (organic versus mineral matrix). Juvenile and adult osteonal and interstitial regions were analyzed for DMB, crystallinity, mineral to organic matrix ratio, mineral maturity, collagen maturity, carbonation, indentation modulus, indicators of yield strain and tissue ductility using a mixed model. We found that the intrinsic properties of the juvenile bone were not all inferior to those of the adult bone. Mechanical properties were also differently explained in juvenile and adult groups. The study shows that different intrinsic properties should be used in case of juvenile bone investigation.

Woven bone overview: structural classification based on its integral role in developmental, repair and pathological bone formation throughout vertebrate groups.

Cortical bone development is characterised by initial formation of woven bone followed by deposition of lamellar bone on the woven scaffold. This occurs in normal bone formation as an integral obligate self-assembly pattern throughout all vertebrate groups, with specific temporal and spatial features. It also occurs in repair bone, modified by the biophysical/mechanical environment, and in pathological bone, modified by the specific disorder and its severity. Two spatially distinct osteoblast cell populations synthesise woven and lamellar bone: mesenchymal osteoblasts surround themselves circumferentially with collagen in a random array to form woven bone; surface osteoblasts align themselves in a linear array on the woven bone surface (or adjacent lamellar bone) to synthesise parallel-fibred lamellar bone. Four specific stages of woven bone formation are defined: stage I, early differentiation of pre-osteoblasts from undifferentiated mesenchymal cells; stage II, mesenchymal osteoblasts surrounding themselves in a 360° arc with randomly oriented matrix fibres; stage III, woven matrix acting as a scaffold on which surface osteoblasts begin to synthesise bone in parallel-fibred lamellar conformation; stage IV, progressive relative diminution of woven bone in the woven bone/lamellar bone complex. Stages II and IV are further subdivided (in a, b and c) by shifting cell area/matrix area and woven bone/lamellar bone relationships. The under-appreciated biological significance of woven bone is that it initiates formation de novo at sites of no previous bone. This information allows for targeted assessment of molecular-biophysical mechanisms underlying woven bone formation and their utilisation for initiating enhanced bone formation.

Sfrp4 repression of the Ror2/Jnk cascade in osteoclasts protects cortical bone from excessive endosteal resorption.

Loss-of-function mutations in the Wnt inhibitor secreted frizzled receptor protein 4 (SFRP4) cause Pyle's disease (OMIM 265900), a rare skeletal disorder characterized by wide metaphyses, significant thinning of cortical bone, and fragility fractures. In mice, we have shown that the cortical thinning seen in the absence of Sfrp4 is associated with decreased periosteal and endosteal bone formation and increased endocortical resorption. While the increase in Rankl/Opg in cortical bone of mice lacking Sfrp4 suggests an osteoblast-dependent effect on endocortical osteoclast (OC) activity, whether Sfrp4 can cell-autonomously affect OCs is not known. We found that Sfrp4 is expressed during bone marrow macrophage OC differentiation and that Sfrp4 significantly suppresses the ability of early and late OC precursors to respond to Rankl-induced OC differentiation. Sfrp4 deletion in OCs resulted in activation of canonical Wnt/ß-catenin and noncanonical Wnt/Ror2/Jnk signaling cascades. However, while inhibition of canonical Wnt/ß-catenin signaling did not alter the effect of Sfrp4 on OCgenesis, blocking the noncanonical Wnt/Ror2/Jnk cascade markedly suppressed its regulation of OC differentiation in vitro. Importantly, we report that deletion of Ror2 exclusively in OCs (CtskCreRor2fl/fl ) in Sfrp4 null mice significantly reversed the increased number of endosteal OCs seen in these mice and reduced their cortical thinning. Altogether, these data show autocrine and paracrine effects of Sfrp4 in regulating OCgenesis and demonstrate that the increase in endosteal OCs seen in Sfrp4-/- mice is a consequence of noncanonical Wnt/Ror2/Jnk signaling activation in OCs overriding the negative effect that activation of canonical Wnt/ß-catenin signaling has on OCgenesis.

Undernutrition impairs the quality of growth plate and trabecular and cortical bones in growing rats1.

PURPOSE: To investigate the effects of dietary restriction on the growth plate and long bone tissue in growing rats. METHODS: Sixty male Wistar rats were randomly assigned to two groups: Control (Con) and Diet-restricted (Res). After weaning, the Res rats were offered 50% of the chow ingested by the control (ad libitum food intake). The animals were subdivided into two subgroups with follow-ups up to 56 or 70 days. After euthanasia, the growth plate of tibias was analyzed by histomorphometry, micro-computed tomography, and mechanical test. The trabecular and compact bones were evaluated by histomorphometry, dual-energy X-ray absorptiometry, and micro-computed tomography (µCT). Real-time PCR was used to analyze gene expression. RESULTS: Although dietary restriction did not alter gene expression, several phenotypic changes were seen in the growth plate; i.e., decrease in volume, reduction in total area and height, decrease in the area ossified zones, mechanical weakening, reduction in mass of trabecular and cortical bone, lower bone density, deterioration of the trabecular and cortical microarchitecture, and trabeculae with lower collagen deposition. CONCLUSION: Dietary restriction had severe detrimental effects on the growth plate and trabecular and cortical bone.

Undernutrition impairs the quality of growth plate and trabecular and cortical bones in growing rats

Abstract Purpose: To investigate the effects of dietary restriction on the growth plate and long bone tissue in growing rats. Methods: Sixty male Wistar rats were randomly assigned to two groups: Control (Con) and Diet-restricted (Res). After weaning, the Res rats were offered 50% of the chow ingested by the control (ad libitum food intake). The animals were subdivided into two subgroups with follow-ups up to 56 or 70 days. After euthanasia, the growth plate of tibias was analyzed by histomorphometry, micro-computed tomography, and mechanical test. The trabecular and compact bones were evaluated by histomorphometry, dual-energy X-ray absorptiometry, and micro-computed tomography (μCT). Real-time PCR was used to analyze gene expression. Results: Although dietary restriction did not alter gene expression, several phenotypic changes were seen in the growth plate; i.e., decrease in volume, reduction in total area and height, decrease in the area ossified zones, mechanical weakening, reduction in mass of trabecular and cortical bone, lower bone density, deterioration of the trabecular and cortical microarchitecture, and trabeculae with lower collagen deposition. Conclusion: Dietary restriction had severe detrimental effects on the growth plate and trabecular and cortical bone.

Fluoride intake and cortical and trabecular bone characteristics in adolescents at age 17: A prospective cohort study.

OBJECTIVE: To investigate the associations between period-specific and cumulative fluoride (F) intakes from birth to age 17 years, and radial and tibial bone measures obtained using peripheral quantitative computed tomography (pQCT). METHODS: Participants (n = 380) were recruited from hospitals at birth and continued their participation in the ongoing Iowa Fluoride Study/Iowa Bone Development Study until age 17. Fluoride intakes from water, other beverages, selected foods, dietary fluoride supplements and dentifrice were determined every 1.5-6 months using detailed questionnaires. Associations between F intake and bone measures (cortical and trabecular bone mineral content [BMC], density and strength) were determined in bivariate and multivariable analyses adjusted for height, weight, maturity offset, physical activity, and daily calcium and protein intake using robust regression analysis. RESULTS: Fluoride intake ranged from 0.7 to 0.8 mg F/d for females and from 0.7 to 0.9 mg F/d for males. Spearman correlations between daily F intake and pQCT bone measures were weak. For females, Spearman correlations ranged from r = -.08 to .21, and for males, they ranged from r = -.03 to .30. In sex-specific, height-, weight- and maturity offset- partially adjusted regression analyses, associations between females' fluoride intake and bone characteristics were almost all negative; associations for males were mostly positive. In the fully adjusted models, which also included physical activity, and protein and calcium intakes, no significant associations were detected for females; significant positive associations were detected between F intake from 14 to 17 years and tibial cortical bone content (ß = 21.40, P < .01) and torsion strength (ß = 175.06, P < .01) for males. CONCLUSION: In this cohort of 17-year-old adolescents, mostly living in optimally fluoridated areas, lifelong F intake from combined sources was weakly associated with bone pQCT measures.

The effect of growth rate on the three-dimensional orientation of vascular canals in the cortical bone of broiler chickens.

Vascular canals in cortical bone during growth and development typically show an anisotropic pattern with canals falling into three main categories: circumferential, radial, and longitudinal. Two major hypotheses attempt to explain the preferred orientations in bone: that vascular canal orientation is optimized to resist a predominant strain direction from functional loading, or that it reflects growth requirements and velocity. We use a controlled growth experiment in broiler chickens to investigate the effect of growth rate on vascular canal orientation. Using feed restriction we set up a fast growing control group and a slow growing restricted group. We compared the microstructure in the humerus and the femur at 42 days of age using synchrotron micro-computed tomography (micro-CT), a three-dimensional (3D) method that visualizes the full canal network. We measured the 3D orientation of each canal in the whole cross-section of the bone cortex using a set of custom ImageJ scripts. Using these orientations we compute laminar, radial, and longitudinal indices that measure the proportion of circumferential, radial, and longitudinal canals, by unit of length, in the cortex. Following previous studies we hypothesized that vascular canal orientation is related to growth, with radial canals linked to a faster growth rate and related to functional loading through a high laminar index in flight bones which reflects torsional loading resulting from active flight. The control group had final body weights that were nearly twice the final weights of the restricted group and higher absolute growth rates. We found consistent patterns in the comparison between the humerus and the femur in both groups, with the humerus having higher laminar and longitudinal indices, and a lower radial index than the femur. The control group had higher radial indices and lower laminar and longitudinal indices in both the humerus and the femur than the restricted group. The higher radial indices in our control group point to a link between radial canals and faster growth, and between laminar canals and slower growth, while the higher laminar indices in the humerus point to a link between circumferential canals and torsional loading. Overall, our results indicate that the orientation of the cortical canal network in a bone is the consequence of a complex interaction between the growth rate of that bone and functional loading environment.

Asymmetry in the Cortical and Trabecular Bone of the Human Humerus During Development.

Many studies have noted that the bones of the human upper limb display bilateral asymmetry, commonly linking this asymmetry in external and internal morphology to handedness and lateralization. Few studies, however, have attempted to track asymmetry throughout ontogeny. This study assesses the ontogenetic development of cortical and trabecular bone asymmetry in the humerus. We predict that directional asymmetry in structural properties will emerge in concert with hand preference and increased activity levels during the juvenile period. Paired humeri from 57 individuals from the Norris Farms #36 archaeological skeletal collection ranging in age from neonate to adult were used in the current study. Cortical bone cross-sectional properties and three-dimensional trabecular bone structure were quantified from microcomputed tomography data. The results indicate significant absolute asymmetry in all measured cortical and trabecular bone variables across all ages. Trabecular bone displays significantly higher absolute asymmetry than cortical bone. Contrary to expectations, however, this study found very little evidence for significant directional asymmetry in humeral length and cortical or trabecular bone variables, except in adults. The presence of significant absolute asymmetry in all age groups, and the lack of significant directional asymmetry in almost all variables at all ages, suggests that structural differences due to higher levels of habitual loading in the dominant arm are overlain on a template of potentially significant existing asymmetry. Anat Rec, 301:1012-1025, 2018. © 2017 Wiley Periodicals, Inc.

Long-Term Intake of Green Tea Extract Causes Mal-Conformation of Trabecular Bone Microarchitecture in Growing Rats.

The purpose of this study was to examine the effects of green tea extract (GTE) intake on bone structural and physiological properties, such as bone mass, trabecular bone microarchitecture, cortical bone geometry, and bone mechanical strength, in growing rats. Four-week-old male Wistar rats were divided into the following four groups: standard diet feeding for 85 days (S-CON) or 170 days (L-CON), and GTE diet feeding for 85 days (S-GTE) or 170 days (L-GTE). At the end of the experiment, in addition to measurement of circulating bone formation/resorption markers, bone mass, trabecular bone microarchitecture, and cortical bone geometry were analyzed in the left femur, and bone mechanical strength of the right femur was measured. There was no difference in all bone parameters between the S-CON and S-GTE groups. On the other hand, the L-GTE group showed the decrease in some trabecular bone mass/microarchitecture parameters and no change in cortical bone mass/geometry parameters compared with the L-CON group, and consequently the reduction in bone weight corrected by body weight. There was no difference in bone formation/resorption markers and bone mechanical strength between the S-CON and S-GTE groups and also between the L-CON and L-GTE groups. However, serum leptin levels were significantly lower in the L-GTE group than in the L-CON group. Thus, the long-term GTE intake had negative effects on bone, especially trabecular bone loss and microarchitecture mal-conformation, in growing rats.

Milk Basic Protein Facilitates Increased Bone Mass in Growing Mice.

Milk basic protein (MBP) comprises a group of basic whey proteins and is effective in preventing bone loss by promoting bone deposition (bone formation) and suppressing withdrawn (bone resorption). We previously revealed the bone protective effects of MBP during life phases involving excessive bone resorption, such as in adults and postmenopausal women, and in animal models (ovariectomized rats and mice). However, it was unclear whether MBP increases bone mass during the growth stage, when there is more bone formation than resorption. We therefore investigated the effect of MBP supplementation on bone mass in 6-wk-old mice provided water supplemented with MBP [0.01%, 0.1%, 1.0% (w/w)] or deionized water (control) ad libitum for 10 wk. Analysis by micro-computerized tomography showed that MBP significantly increased tibia cortical bone mineral density and femur trabecular bone volume to tissue volume compared with mice provided deionized water. Next, the function of MBP in bone remodeling (bone formation and resorption) was evaluated using an in vitro system and the results demonstrated that MBP directly promoted osteoblast proliferation and inhibited osteoclastogenesis. Moreover, the plasma level of insulin-like growth factor-1 was increased by MBP supplementation, suggesting that MBP indirectly promoted osteoblast proliferation/differentiation. These effects enhance bone formation and/or inhibit bone resorption, resulting in increased bone mass in growing mice.

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales - nano-, micro-, meso-, and macroscopic - fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

Chronic psychosocial stress disturbs long-bone growth in adolescent mice.

Although a strong association between psychiatric and somatic disorders is generally accepted, little is known regarding the interrelationship between mental and skeletal health. Although depressive disorders have been shown to be strongly associated with osteoporosis and increased fracture risk, evidence from post-traumatic stress disorder (PTSD) patients is less consistent. Therefore, the present study investigated the influence of chronic psychosocial stress on bone using a well-established murine model for PTSD. C57BL/6N mice (7 weeks old) were subjected to chronic subordinate colony housing (CSC) for 19 days, whereas control mice were singly housed. Anxiety-related behavior was assessed in the open-field/novel-object test, after which the mice were euthanized to assess endocrine and bone parameters. CSC mice exhibited increased anxiety-related behavior in the open-field/novel-object test, increased adrenal and decreased thymus weights, and unaffected plasma morning corticosterone. Microcomputed tomography and histomorphometrical analyses revealed significantly reduced tibia and femur lengths, increased growth-plate thickness and reduced mineral deposition at the growth plate, suggesting disturbed endochondral ossification during long-bone growth. This was associated with reduced Runx2 expression in hypertrophic chondrocytes in the growth plate. Trabecular thicknesses and bone mineral density were significantly increased in CSC compared to singly housed mice. Tyrosine hydroxylase expression was increased in bone marrow cells located at the growth plates of CSC mice, implying that local adrenergic signaling might be involved in the effects of CSC on the skeletal phenotype. In conclusion, chronic psychosocial stress negatively impacts endochondral ossification in the growth plate, affecting both longitudinal and appositional bone growth in adolescent mice.

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.

Cortical and trabecular morphology is altered in the limb bones of mice artificially selected for faster skeletal growth.

Bone strength is influenced by mineral density and macro- and microstructure. Research into factors that contribute to bone morphology and strength has focused on genetic, environmental and morphological factors (e.g., body mass index), but little is known regarding the impact of rates of skeletal elongation on adult skeletal morphology and strength. Using micro-CT, we examined the impact of rates of skeletal elongation on bone cortical and trabecular morphology, and on rates of estrogen-dependent bone loss in the tibia in CD-1 mice, and in mice with accelerated skeletal growth (Longshanks). Groups of adult mice (n = 7/group) were subjected to ovariectomy or sham surgeries, scanned for 6 weeks, and indices of bone morphology were collected. Results show that Longshanks mice had significantly less trabecular bone at skeletal maturity, characterized by fewer, thinner trabeculae, and furthermore lost trabecular bone more slowly in response to ovariectomy. Artificial selection for rapid skeletal growth relative to somatic growth thus had a significant impact on trabecular bone morphology in Longshanks. Our data do not unequivocally demonstrate a causal relationship between rapid bone growth and reduced trabecular bone quality, but suggest that rapid linear bone growth may influence the risk of cancellous bone fragility.

Sost deficiency led to a greater cortical bone formation response to mechanical loading and altered gene expression.

Bone adaptation optimizes mass and structure, but the mechano-response is already reduced at maturation. Downregulation of sclerostin was believed to be a mandatory step in mechano-adaptation, but in young mice it was shown that load-induced formation can occur independent of sclerostin, a product of the Sost gene. We hypothesized that the bone formation and resorption response to loading is not affected by Sost deficiency, but is age-specific. Our findings indicate that the anabolic response to in vivo tibial loading was reduced at maturation in Sost Knockout (KO) and littermate control (LC) mice. Age affected all anabolic and catabolic parameters and altered Sost and Wnt target gene expression. While load-induced cortical resorption was similar between genotypes, loading-induced gains in mineralizing surface was enhanced in Sost KO compared to LC mice. Loading led to a downregulation in expression of the Wnt inhibitor Dkk1. Expression of Dkk1 was greater in both control and loaded limbs of Sost KO compared to LC mice suggesting a compensatory role in the absence of Sost. These data suggest physical activity could enhance bone mass concurrently with sclerostin-neutralizing antibodies, but treatment strategies should consider the influence of age on ultimate load-induced bone mass gains.