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.

Trabecular Bone Parameters, TIMP-2, MMP-8, MMP-13, VEGF Expression and Immunolocalization in Bone and Cartilage in Newborn Offspring Prenatally Exposed to Fumonisins.

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group (n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

Podoplanin is dispensable for mineralized tissue formation and maintenance in the Swiss outbred mouse background.

Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

Functional interaction between Wnt and Bmp signaling in periosteal bone growth.

Wnt and Bmp proteins are well known to regulate bone development and homeostasis. Although both signals are extensively studied, their potential interaction in vivo is less well understood. Previous studies have shown that deletion of Bmpr1a, a type I receptor for Bmp signaling, results in excessive trabecular bone formation while diminishing periosteal bone growth. Moreover, forced-expression of the Wnt antagonist Sost suppresses the overgrowth of trabecular bone caused by Bmpr1a deletion, thus implicating hyperactive Wnt signaling in the excessive trabecular bone formation. However, it remains uncertain whether Wnt and Bmp signaling interacts in regulating the periosteal bone growth. Here we show that multiple Wnt genes are markedly suppressed in the cortical bone without Bmpr1a. Importantly, overexpression of Wnt7b fully rescues periosteal bone growth in the Bmpr1a-deficient mice. Thus, pharmacological activation of Wnt signaling can restore normal bone size without intact Bmp signaling.

Micro-CT data of early physiological cancellous bone formation in the lumbar spine of female C57BL/6 mice.

Micro-CT provides critical data for musculoskeletal research, yielding three-dimensional datasets containing distributions of mineral density. Using high-resolution scans, we quantified changes in the fine architecture of bone in the spine of young mice. This data is made available as a reference to physiological cancellous bone growth. The scans (n = 19) depict the extensive structural changes typical for female C57BL/6 mice pups, aged 1-, 3-, 7-, 10- and 14-days post-partum, as they attain the mature geometry. We reveal the micro-morphology down to individual trabeculae in the spine that follow phases of mineral-tissue rearrangement in the growing lumbar vertebra on a micrometer length scale. Phantom data is provided to facilitate mineral density calibration. Conventional histomorphometry matched with our micro-CT data on selected samples confirms the validity and accuracy of our 3D scans. The data may thus serve as a reference for modeling normal bone growth and can be used to benchmark other experiments assessing the effects of biomaterials, tissue growth, healing, and regeneration.

Early ontogeny of humeral trabecular bone in Neandertals and recent modern humans.

Trabecular bone ontogeny is well known in modern humans and unknown in Neandertals. Yet the bone developmental pattern is useful for interpreting fossils from evolutionary and functional perspectives. Interestingly, microstructure in early ontogeny is supposedly not influenced by high and specific mechanical loading related to the lifestyle of a human group and consequently does not directly depend on the activities of hunter-gatherers. Here, we specifically explored the early growth trajectories of the trabecular bone structure of the humerus and emphasized in particular how bone fraction (bone volume/total volume [BV/TV]) was built up in Neandertals, given the specific modern human bone loss after birth and the use of BV/TV in functional studies. Six Neandertals and 26 recent modern humans ranging from perinates to adolescents were included in this study. Six trabecular parameters were measured within a cubic region of interest extracted from the proximal metaphysis of the humerus. We found that the microstructural changes in Neandertals during early ontogeny (<1 year) fit with modern human growth trajectories for each parameter. The specific bone loss occurring immediately after birth in modern humans also occurred in Neandertals (but not in chimpanzees). However, the early childhood fossil Ferrassie 6 presented unexpectedly high BV/TV, whereas the high BV/TV in the Crouzade I adolescent was predictable. These results suggest that Neandertals and modern humans shared predetermined early growth trajectories and developmental mechanisms. We assume that the close relationship between skeletal characteristics in early ontogeny and adults in modern humans also existed in Neandertals. However, it was difficult to ensure that the high BV/TV in Neandertal early childhood, represented by only one individual, was at the origin of the high BV/TV observed in adults. Consequently, our study does not challenge the mechanical hypothesis that explains the trabecular gracilization of the humerus during the Holocene.

ADAMTS5 is required for normal trabeculated bone development in the mandibular condyle.

OBJECTIVE: Determine the role of the extracellular matrix protease ADAMTS5 in development of the trabeculated bone of the mandibular condyle. METHODS: The mandibular condyles of wild type and mice deficient in the protease ADAMTS5 were examined for histopathology with Safranin O staining. Microcomputed tomography was performed to analyze the developing bone of the mandibular condyle. RNAscope and immunohistochemistry were utilized to investigate cell type and extracellular matrix expression. RESULTS: Mice deficient in Adamts5, (Adamts5tm1Dgen/J) exhibit an increase in trabecular separation (n = 37 wild type; n = 27: P < 0.0001) and reduction of trabecular thickness P = 0.0116 and bone volume fraction P = 0.0869 in the mandibular condylar head compared to wild type littermates. The altered bone parameters were more pronounced in male Adamts5-/- mice compared to female Adamts5-/- mice (TbSp; P = 0.03). Adamts5 was co-expressed with versican and Gli1 in mesenchymal, stem-like cells in the transition zone where the trabeculated bone is adjacent to mature hypertrophic chondrocytes. Loss of Adamts5 caused a reduction of Bglap expressing osteoblasts throughout mandibular condylar development and in young adult mice. The protease Mmp13, that is involved in mineralization and is expressed by hypertrophic chondrocytes and osteoblasts, was reduced in the mandibular condyle of Adamts5 deficient mice. CONCLUSION: This is the first report of a novel and critical role for Adamts5 in bone formation within the mandibular condyle of the temporomandibular joint. These data indicate Adamts5 may be required in the transdifferentiation of hypertrophic chondrocytes to osteoblasts during trabecular bone formation in development of the mandibular condyle.

NLRP3 is involved in long bone edification and the maturation of osteogenic cells.

Overexpression of the nucleotide-binding leucine-rich repeat protein 3 (NLRP3) inflammasome in chronic auto-immune diseases leads to skeletal anomalies, with severe osteopenia due to the activation of osteoclasts. Reproducing this phenotype in Nlrp3 knock-in mice has provided insights into the role of NLRP3 in bone metabolism. We studied the role of NLRP3 in physiological bone development using a complete Nlrp3 knock-out mouse model. We found impaired skeletal development in Nlrp3-/- mice, resulting in a shorter stature than that of Nlrp3+/+  mice. These growth defects were associated with altered femur bone growth, characterized by a deficient growth plate and an osteopenic profile of the trabeculae. No differences in osteoclast recruitment or activity were observed. Instead, Nlrp3-/- femurs showed a less mineralized matrix in the trabeculae than those of Nlrp3+/+  mice, as well as less bone sialoprotein (BSP) expressing hypertrophic chondrocytes. In vitro, primary osteoblasts lacking NLRP3 expression showed defective mineralization, together with the downregulation of BSP expression. Finally, follow-up by micro-CT highlighted the role of NLPR3 in bone growth, occurring early in living mice, as the osteopenic phenotype diminishes over time. Overall, our data suggest that NLRP3 is involved in bone edification via the regulation of hypertrophic chondrocyte maturation and osteoblast activity. Furthermore, the defect appeared to be transitory, as the skeleton recovered with aging.

Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

Collagen VIα2 chain deficiency causes trabecular bone loss by potentially promoting osteoclast differentiation through enhanced TNFα signaling.

Type VI collagen is well known for its role in muscular disorders, however its function in bone is still not well understood. To examine its role in bone we analyzed femoral and vertebral bone mass by micro-computed tomography analysis, which showed lower bone volume/total volume and trabecular number in Col6α2-KO mice compared with WT. Dynamic histomorphometry showed no differences in trabecular bone formation between WT and Col6α2-KO mice based on the mineral appositional rate, bone formation rate, and mineralizing perimeter. Femoral sections were assessed for the abundance of Tartrate Resistant Acid Phosphatase-positive osteoclasts, which revealed that mutant mice had more osteoclasts compared with WT mice, indicating that the primary effect of Col6a2 deficiency is on osteoclastogenesis. When bone marrow stromal cells (BMSCs) from WT and Col6α2-KO mice were treated with rmTNFα protein, the Col6α2-KO cells expressed higher levels of TNFα mRNA compared with WT cells. This was accompanied by higher levels of p-p65, a down-stream target of TNFα, suggesting that BMSCs from Col6α2-KO mice are highly sensitive to TNFα signaling. Taken together, our data imply that Col6a2 deficiency causes trabecular bone loss by enhancing osteoclast differentiation through enhanced TNFα signaling.

Effects of chemotherapy agents used to treat pediatric acute lymphoblastic leukemia patients on bone parameters and longitudinal growth of juvenile mice.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Therapies for pediatric ALL have improved such that more than 80% of patients survive to 5 years post-therapy, and most survive to adulthood. These ALL patients experience long-term side effects that permanently affect their quality of life, with bone loss and reduced longitudinal growth being the most common skeletal complications. To determine the effects of the chemotherapeutic agents used in ALL induction therapy on bone density and longitudinal growth in mice, we treated juvenile mice with doxorubicin, dexamethasone, vincristine, l-asparaginase, or combination therapy. At adulthood, mice were culled and bones collected and scanned by micro-computed tomography (micro-CT). Mice that received doxorubicin and combination therapy exhibited reduced longitudinal growth and significant reductions in trabecular bone volume, trabecular thickness, and trabecular number, with increased trabecular separation. Mean cortical thickness, cortical area, marrow area, endocortical perimeter, and polar moment of inertia were significantly reduced by doxorubicin and combination therapy. Vincristine treatment significantly decreased trabecular bone volume, trabecular number, and increased trabecular separation but had no effects on cortical bone. Dexamethasone treatment increased trabecular bone separation, cortical marrow area, and cortical bone periosteal perimeter. Mice treated with l-asparaginase did not have any bone phenotypes. In conclusion, these data indicate that the majority of the chemotherapy agents used in induction therapy for pediatric ALL have long-term effects on bone in mice. A single dose of doxorubicin in juvenile mice was sufficient to cause the majority of the bone phenotypes, with combination therapy intensifying these effects.

Age- and Strain-Related Differences in Bone Microstructure and Body Composition During Development in Inbred Male Mouse Strains.

We explored age- and strain-related differences in bone microstructure and body composition in male C57BL/6J, DBA/2JRj and C3H/J mice. Bone microstructure of the femur, tibia and L4 was assessed by µCT at the age of 8, 16 and 24 weeks. The weight of several muscles and fat depots were measured at the same time points. At all timepoints, C3H/J mice had the thickest cortices followed by DBA/2JRj and C57BL/6J mice. Nevertheless, C57BL/6J mice had higher Tb.BV/TV and Tb.N, and lower Tb.Sp than DBA/2JRj and C3H/J mice at least at 24 weeks of age. Skeletal development patterns differed among strains. C57BL/6J and DBA/2JRj mice, but not C3H/J mice, experienced significant increases in the sum of the masses of 6 individual muscles by 24 weeks of age. In C57BL/6J and DBA/2JRj mice, the mass of selected fat depots reached highest values at 24 weeks, whist, in C3H/J mice, the highest values of fat depots masses were achieved at 16 weeks. Early strain differences in muscle and fat masses were largely diminished by 24 weeks of age. C3H/J and C57BL/6J mice displayed the most favorable cortical and trabecular bone parameters, respectively. Strain differences in body composition were less overt than strain specificity in bone microstructure, however, they possibly influenced aspects of skeletal development.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

BACKGROUND: Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. METHODS: Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n = 13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22 h/d, 7 d/wk, for a total of 12 months. Control animals (n = 9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3 days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. RESULTS: Monkeys in the EtOH group consumed an average of 2.8 ± 0.2 (mean ± SE) g/kg/d of EtOH (30 ± 2% of total calories), resulting in an average blood EtOH concentration of 88.3 ± 8.8 mg/dl 7 hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). CONCLUSIONS: Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Experimental Evaluation of the Properties of 3D Porous Bone Substitute Based on Calcium Phosphate on the Model of Monocortical Diaphysial Femur Defect in Rats.

A new alloplastic high-permeable material based on tricalcium phosphate with Kelvin architectonics created by stereolithographic 3D-printing was studied in vivo. A monocortical defect of the femur was modeled in rats and the material was implanted into the defect area. In 24 weeks, the animals were euthanized and histological examination of the defect area was performed. One femur fracture with fixator migration was recorded after implantation of the studied material and the reference chronOS synthetic material. The studied material demonstrated better osteoconductive properties then traditional osteoplastic material, which was seen from greater number of bone trabeculae and their area in the defect area.

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.

Ninjurin1 positively regulates osteoclast development by enhancing the survival of prefusion osteoclasts.

Osteoclasts (OCs) are bone-resorbing cells that originate from hematopoietic stem cells and develop through the fusion of mononuclear myeloid precursors. Dysregulation of OC development causes bone disorders such as osteopetrosis, osteoporosis, and rheumatoid arthritis. Although the molecular mechanisms underlying osteoclastogenesis have been well established, the means by which OCs maintain their survival during OC development remain unknown. We found that Ninjurin1 (Ninj1) expression is dynamically regulated during osteoclastogenesis and that Ninj1-/- mice exhibit increased trabecular bone volume owing to impaired OC development. Ninj1 deficiency did not alter OC differentiation, transmigration, fusion, or actin ring formation but increased Caspase-9-dependent intrinsic apoptosis in prefusion OCs (preOCs). Overexpression of Ninj1 enhanced the survival of mouse macrophage/preOC RAW264.7 cells in osteoclastogenic culture, suggesting that Ninj1 is important for the survival of preOCs. Finally, analysis of publicly available microarray data sets revealed a potent correlation between high NINJ1 expression and destructive bone disorders in humans. Our data indicate that Ninj1 plays an important role in bone homeostasis by enhancing the survival of preOCs.

Establishment and effects of allograft and synthetic bone graft substitute treatment of a critical size metaphyseal bone defect model in the sheep femur.

Assessment of bone graft material efficacy is difficult in humans, since invasive methods like staged CT scans or biopsies are ethically unjustifiable. Therefore, we developed a novel large animal model for the verification of a potential transformation of synthetic bone graft substitutes into vital bone. The model combines multiple imaging methods with corresponding histology in standardized critical sized cancellous bone defect. Cylindrical bone voids (10 ml) were created in the medial femoral condyles of both hind legs (first surgery at right hind leg, second surgery 3 months later at left hind leg) in three merino-wool sheep and either (i) left empty, filled with (ii) cancellous allograft bone or (iii) a synthetic, gentamicin eluting bone graft substitute. All samples were analysed with radiographs, MRI, µCT, DEXA and histology after sacrifice at 6 months. Unfilled defects only showed ingrowth of fibrous tissue, whereas good integration of the cancellous graft was seen in the allograft group. The bone graft substitute showed centripetal biodegradation and new trabecular bone formation in the periphery of the void as early as 3 months. µCT gave excellent insight into the structural changes within the defects, particularly progressive allograft incorporation and the bone graft substitute biodegradation process. MRI completed the picture by clearly visualizing soft tissue ingrowth into unfilled bone voids and presence of fluid collections. Histology was essential for verification of trabecular bone and osteoid formation. Conventional radiographs and DEXA could not differentiate details of the ongoing transformation process. This model appears well suited for detailed in vivo and ex vivo evaluation of bone graft substitute behaviour within large bone defects.

Normal trabecular vertebral bone is formed via rapid transformation of mineralized spicules: A high-resolution 3D ex-vivo murine study.

At birth, mouse vertebrae have a reticular fine spongy morphology, yet in the adult animal they exhibit elaborate trabecular architectures. Here, we characterize the physiological microstructural transformations in growing young female mice of the widely used C57BL/6 strain. Extensive architectural changes lead to the establishment of mature cancellous bone in the spine. Vertebrae were mapped in 3D by high resolution microcomputed tomography (µCT), backed by conventional histology. Three different phases are observed in the natural bony biomaterial: In a prenatal templating phase, early vertebrae are composed of foamy, loosely-packed mineralized spicules. During a consolidation phase in the first 7 days after birth, bone material condenses into struts and forms primitive trabeculae accompanied by a significant (>50%) reduction in bone volume/tissue volume ratio (BV/TV). After day 7, the trabeculae expand, reorient and increase in mineral density. Swift growth ensues such that by day 14 the young lumbar spine exhibits all morphological features observed in the mature animal. The greatly varied micro-morphologies of normal trabecular bone observed in 3D within a short timespan are typical for rodent and presumably for other mammalian forming spines. This suggests that fully structured cancellous bone emerges through rapid post-natal restructuring of a foamy mineralized scaffold. STATEMENT OF SIGNIFICANCE: Cancellous bone develops in stages that are not well documented. Using a mouse model, we provide an observer-independent quantification of normal bone formation in the spine. We find that within 14 days, the cancellous bone transforms in 3 phases from a scaffold of spicules into well organized, fully mineralized trabeculae in a functional spine. Detailed knowledge of the physiological restructuring of mineralized material may help to better understand bone formation and may serve as a blueprint for studies of pharmaceuticals effects, tissue healing and regeneration.

Differences in third metacarpal trabecular microarchitecture between the parasagittal groove and condyle at birth and in adult racehorses.

BACKGROUND: The aetiology of equine metacarpal condylar fractures is not completely understood and a developmental cause has been postulated. OBJECTIVES: To investigate the subchondral bone trabecular microarchitecture of the lateral parasagittal groove and condyle in equine neonates and its adaptation with maturation and athletic activity. STUDY DESIGN: Ex vivo observational study. METHODS: Distal metacarpi of neonates, yearlings and adult racehorses (n = 24) were harvested. Dorsal and palmar frontal histological sections, containing the lateral parasagittal groove and condyle, were studied. The sections were digitalised and subchondral trabecular bone quantity and quality parameters and trabecular orientation in the frontal plane were measured. RESULTS: Trabecular spacing and length were greater (P = 0.004 and P = 0.0005 respectively) whereas bone fraction, trabecular number and connectivity were all lower (P = 0.0004, P = 0.0001 and P = 0.001 respectively) in the lateral parasagittal groove compared with the condyle in neonatal foals. Trabecular thickness and bone fraction increased with age in racehorses and trabecular spacing decreased. The predominant trabecular orientation had a consistent pattern in neonates and it changed with maturity and the cumulative effect of racing at all the ROIs except for the palmar lateral parasagittal groove that retained a more 'immature' pattern. MAIN LIMITATIONS: Samples were investigated in 2D. 3D processing could have provided more information. CONCLUSIONS: Already at birth there are striking differences in the subchondral bone trabecular microarchitecture between the lateral parasagittal groove and condyle in foals. Adaptation of trabeculae is confirmed with maturity in racehorses, with the greatest adaptation measured in bone quantity parameters. The trabecular orientation had a unique and more immature orientation pattern in the lateral palmar parasagittal grooves in adult racehorses and may reflect a weaker structure at this site.

Release of CXCL12 From Apoptotic Skeletal Cells Contributes to Bone Growth Defects Following Dexamethasone Therapy in Rats.

Dexamethasone (Dex) is known to cause significant bone growth impairment in childhood. Although previous studies have suggested roles of osteocyte apoptosis in the enhanced osteoclastic recruitment and local bone loss, whether it is so in the growing bone following Dex treatment requires to be established. The current study addressed the potential roles of chemokine CXCL12 in chondroclast/osteoclast recruitment and bone defects following Dex treatment. Significant apoptosis was observed in cultured mature ATDC5 chondrocytes and IDG-SW3 osteocytes after 48 hours of 10-6 M Dex treatment, and CXCL12 was identified to exhibit the most prominent induction in Dex-treated cells. Conditioned medium from the treated chondrocytes/osteocytes enhanced migration of RAW264.7 osteoclast precursor cells, which was significantly inhibited by the presence of the anti-CXCL12 neutralizing antibody. To investigate the roles of the induced CXCL12 in bone defects caused by Dex treatment, young rats were orally gavaged daily with saline or Dex at 1 mg/kg/day for 2 weeks, and received an intraperitoneal injection of anti-CXCL12 antibody or control IgG (1 mg/kg, three times per week). Aside from oxidative stress induction systemically, Dex treatment caused reductions in growth plate thickness, primary spongiosa height, and metaphysis trabecular bone volume, which are associated with induced chondrocyte/osteocyte apoptosis and enhanced chondroclast/osteoclast recruitment and osteoclastogenic differentiation potential. CXCL12 was induced in apoptotic growth plate chondrocytes and metaphyseal bone osteocytes. Anti-CXCL12 antibody supplementation considerably attenuated Dex-induced chondroclast/osteoclast recruitment and loss of growth plate cartilage and trabecular bone. CXCL12 neutralization did not affect bone marrow osteogenic potential, adiposity, and microvasculature. Thus, CXCL12 was identified as a potential molecular linker between Dex-induced skeletal cell apoptosis and chondroclastic/osteoclastic recruitment, as well as growth plate cartilage/bone loss, revealing a therapeutic potential of CXCL12 functional blockade in preventing bone growth defects during/after Dex treatment. © 2018 American Society for Bone and Mineral Research.