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.

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.

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.

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.

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.

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.

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.

LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin.

Mice with disruption of Lrrk1 and patients with nonfunctional mutant Lrrk1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how Lrrk1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild-type mice and Lrrk1-deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L-plastin, an actin bundling protein. We found that phosphorylation of L-plastin at serine (Ser) residues 5 was present in wild-type osteoclasts but not in Lrrk1-deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L-plastin confirmed the reduced L-plastin Ser5 phosphorylation in Lrrk1 knockout (KO) osteoclasts. micro computed tomography (Micro-CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21-week-old L-plastin KO females as compared with the wild-type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P < 0.05), respectively, in L-plastin KO mice. Our data suggest that targeted disruption of L-plastin increases trabecular bone volume, and phosphorylation of Ser5 in L-plastin in the Lrrk1 signaling pathway may in part contribute to actin assembly in mature osteoclasts.

EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.

In this study, we examined the functional importance of EZH2 during skeletal development and homeostasis using the conditional deletion of Ezh2 (Ezh2fl/fl ) in early mesenchyme with the use of a Prrx-1-cre driver mouse (Ezh2+/+). Heterozygous (Ezh2+/-) newborn and 4-wk-old mice exhibited increased skeletal size, growth plate size, and weight when compared to the wild-type control (Ezh2+/+), whereas homozygous deletion of Ezh2 (Ezh2-/-) resulted in skeletal deformities and reduced skeletal size, growth plate size, and weight in newborn and 4-wk-old mice. Ezh2-/- mice exhibited enhanced trabecular patterning. Osteogenic cortical and trabecular bone formation was enhanced in Ezh2+/- and Ezh2-/- animals. Ezh2+/- and Ezh2-/- mice displayed thinner cortical bone and decreased mechanical strength compared to the wild-type control. Differences in cortical bone thickness were attributed to an increased number of osteoclasts, corresponding with elevated levels of the bone turnover markers cross-linked C-telopeptide-1 and tartrate-resistant acid phosphatase, detected within serum. Moreover, Ezh2+/- mice displayed increased osteoclastogenic potential coinciding with an upregulation of Rankl and M-csf expression by mesenchymal stem cells (MSCs). MSCs isolated from Ezh2+/- mice also exhibited increased trilineage potential compared with wild-type bone marrow stromal/stem cells (BMSCs). Gene expression studies confirmed the upregulation of known Ezh2 target genes in Ezh2-/- bone tissue, many of which are involved in Wnt/BMP signaling as promoters of osteogenesis and inhibitors of adipogenesis. In summary, EZH2 appears to be an important orchestrator of skeletal development, postnatal bone remodelling and BMSC fate determination in vitro and in vivo-Hemming, S., Cakouros, D., Codrington, J., Vandyke, K., Arthur, A., Zannettino, A., Gronthos, S. EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.

New bone formation and trabecular bone microarchitecture of highly porous tantalum compared to titanium implant threads: A pilot canine study.

AIM: This study evaluated new bone formation activities and trabecular bone microarchitecture within the highly porous region of Trabecular Metal™ Dental Implants (TM) and between the threads of Tapered Screw-Vent® Dental Implants (TSV) in fresh canine extraction sockets. MATERIALS AND METHODS: Eight partially edentulated dogs received four implants (4.1 mmD × 13 mmL) bilaterally in mandibular fresh extraction sockets (32 TM, 32 TSV implants), and allowed to heal for 2, 4, 8, and 12 weeks. Calcein was administered to label mineralizing bone at 11 and 4 days before euthanasia for dogs undergoing all four healing periods. Biopsies taken at each time interval were examined histologically. Histomorphometric assay was conducted for 64 unstained and 64 stained slides at the region of interest (ROI) (6 mm long × 0.35 mm deep) in the midsections of the implants. Topographical and chemical analyses were also performed. RESULTS: Histomorphometry revealed significantly more new bone in the TM than in the TSV implants at each healing time (p = .0014, .0084, .0218, and .0251). Calcein-labeled data showed more newly mineralized bone in the TM group than in the TSV group at 2, 8, and 12 weeks (p = .045, .028, .002, respectively) but not at 4 weeks (p = .081). Histologically TM implants exhibited more bone growth and dominant new immature woven bone at an earlier time point than TSV implants. The parameters representing trabecular bone microarchitecture corroborated faster new bone formation in the TM implants when compared to the TSV implants. TM exhibited an irregular faceted topography compared to a relatively uniform microtextured surface for TSV. Chemical analysis showed peaks associated with each implant's composition material, and TSV also showed peaks reflecting the elements of the calcium phosphate blasting media. CONCLUSIONS AND CLINICAL IMPLICATIONS: Results suggest that the healing pathway associated with the highly porous midsection of TM dental implant could enable faster and stronger secondary implant stability than conventional osseointegration alone; however, prospective clinical studies are needed to confirm these potential benefits in patients with low bone density, compromised healing, or prior implant failure.

Age-dependent alterations in osteoblast and osteoclast activity in human cancellous bone.

It is assumed that the activity of osteoblasts and osteoclasts is decreased in bone tissue of aged individuals. However, detailed investigation of the molecular signature of human bone from young compared to aged individuals confirming this assumption is lacking. In this study, quantitative expression analysis of genes related to osteogenesis and osteoclastogenesis of human cancellous bone derived from the distal radius of young and aged individuals was performed. Furthermore, we additionally performed immunohistochemical stainings. The young group included 24 individuals with an average age of 23.2 years, which was compared to cancellous bone derived from 11 body donators with an average age of 81.0 years. In cancellous bone of young individuals, the osteogenesis-related genes RUNX-2, OSTERIX, OSTEOPONTIN and OSTEOCALCIN were significantly up-regulated compared to aged individuals. In addition, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in cancellous bone of young individuals, as well as the WNT gene family member WNT5a and the matrix metalloproteinases MMP-9. However, quantitative RT-PCR analysis of BMP-2, ALP, FGF-2, CYCLIN-D1, MMP-13, RANK, OSTEOPROTEGERIN and TGFb1 revealed no significant difference. Furthermore, Tartrate-resistant acid phosphatase (TRAP) staining was performed which indicated an increased osteoclast activity in cancellous bone of young individuals. In addition, pentachrome stainings revealed significantly less mineralized bone matrix, more osteoid and an increased bone density in young individuals. In summary, markers related to osteogenesis as well as osteoclastogenesis were significantly decreased in the aged individuals. Thus, the present data extends the knowledge about reduced bone regeneration and healing capacity observed in aged individuals.

The development of inter-strain variation in cortical and trabecular traits during growth of the mouse lumbar vertebral body.

How cortical and trabecular bone co-develop to establish a mechanically functional structure is not well understood. Comparing early postnatal differences in morphology of lumbar vertebral bodies for three inbred mouse strains identified coordinated changes within and between cortical and trabecular traits. These early coordinate changes defined the phenotypic differences among the inbred mouse strains. INTRODUCTION: Age-related changes in cortical and trabecular traits have been well studied; however, very little is known about how these bone tissues co-develop from day 1 of postnatal growth to establish functional structures by adulthood. In this study, we aimed to establish how cortical and trabecular tissues within the lumbar vertebral body change during growth for three inbred mouse strains that express wide variation in adult bone structure and function. METHODS: Bone traits were quantified for lumbar vertebral bodies of female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse strains from 1 to 105 days of age (n = 6-10 mice/age/strain). RESULTS: Inter-strain differences in external bone size were observed as early as 1 day of age. Reciprocal and rapid changes in the trabecular bone volume fraction and alignment in the direction of axial compression were observed by 7 days of age. Importantly, the inter-strain difference in adult trabecular bone volume fraction was established by 7 days of age. Early variation in external bone size and trabecular architecture was followed by progressive increases in cortical area between 28 and 105 days of age, with the greatest increases in cortical area seen in the mouse strain with the lowest trabecular mass. CONCLUSION: Establishing the temporal changes in bone morphology for three inbred mouse strains revealed that genetic variation in adult trabecular traits were established early in postnatal development. Early variation in trabecular architecture preceded strain-specific increases in cortical area and changes in cortical thickness. This study established the sequence of how cortical and trabecular traits co-develop during growth, which is important for identifying critical early ages to further focus on intervention studies that optimize adult bone strength.

Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period.

OBJECTIVES: Parallel with body growth and development, bone structure in non-adults is reorganized to achieve the particular design observed in mature individuals. We traced the changes in three-dimensional trabecular microarchitectural design during the phases of locomotor maturation to clarify how human bone adapts to mechanical demands. MATERIALS AND METHODS: Micro-CT was performed on biomechanically-relevant subregions of the proximal femur (medial, intermediate and lateral neck regions, intertrochanteric region, metaphyseal region) from early postnatal period to late childhood. RESULTS: Developmental patterns of trabecular microarchitecture showed that gestationally overproduced bone present at birth underwent the most dramatic reduction during the first year, followed by a reversing trend in some of the quantitative parameters (e.g., bone volume fraction, trabecular anisotropy). Certain regional anisotropy already present at birth is further accentuated into the childhood suggesting an adaptation to differential loading environments. Trabecular eccentricity in the femoral neck was particularly accentuated during childhood, giving the medial neck-the site mostly loaded in walking-superior microarchitectural design (high bone volume fraction and anisotropy, the earliest appearance and predominance of plate- and honeycomb-shaped trabeculae). DISCUSSION: While providing quantitative data on how bone microarchitecture adapts to increasing mechanical demands occurring during the phases of locomotor maturation, the study reveals how regional anisotropy develops in the proximal femur to ensure a functional and competent bone structure. Decomposing the region-specific patterns of bone mass accrual is important in understanding skeletal adaptations to bipedalism, as well for understanding why fractures often occur location-dependent, both in pediatric and elderly individuals.

[Effects of 1.8 mT sinusoidal alternating electromagnetic fields of different frequencies on bone biomechanics of young rats].

Objective: To study the effects of 1.8 mT sinusoidal electromagnetic fields of different frequencies on bone mineral density (BMD) and biomechanical properties in young rats. Methods: A total of 32 female SD rats (6-week-old) were randomly divided into 4 groups (8 in each):control group, 10 Hz group, 25 Hz group and 40 Hz group. The experimental groups were given 1.8 mT sinusoidal electromagnetic field intervention 90 min per day. The whole body BMD of rats was detected with dual-energy X-ray absorptiometry after 4 and 8 weeks of intervention. After 8 weeks of intervention, all rats were sacrificed, and the BMD of femur and lumbar vertebra, the length and diameter of femur, the width between medial and lateral malleolus were measured. Electronic universal material testing machine was used to obtain biomechanical properties of femur and lumbar vertebra, and micro CT scan was performed to observe micro structures of tibial cancellous bone. Results: Compared with the control group, rats in 10 Hz and 40 Hz groups had higher whole body BMD, BMD of femur, maximum load and yield strength of femur, as well as maximum load and elastic modulus of lumbar vertebra (all P<0.05). But no significant differences in the length and diameter of femur, and the width between medial and lateral malleolus were observed between control group and experimental groups (all P>0.05). Micro CT scan showed that the trabecular number and separation degree, bone volume percentage were significantly increased in 10 Hz and 40 Hz groups (all P<0.01). Rats in 25 Hz group also had higher BMD and better in biomechanical properties than control group, but the differences were not statistically significant (all P>0.05). Conclusion: 10 and 40 Hz of 1.8 mT sinusoidal electromagnetic field can significantly improve the bone density, microstructure and biomechanical properties in young rats.

The ontogeny of human fetal trabecular bone architecture occurs in a limb-specific manner.

Gestational growth and development of bone is an understudied process compared to soft tissues and has implications for lifelong health. This study investigated growth and development of human fetal limb bone trabecular architecture using 3D digital histomorphometry of microcomputed tomography data from the femora and humeri of 35 skeletons (17 female and 18 male) with gestational ages between 4 and 9 months. Ontogenetic data revealed: (i) fetal trabecular architecture is similar between sexes; (ii) the proximal femoral metaphysis is physically larger, with thicker trabeculae and greater bone volume fraction relative to the humerus, but other aspects of trabecular architecture are similar between the bones; (iii) between 4 and 9 months gestation there is no apparent sexual or limb dimorphism in patterns of growth, but the size of the humerus and femur diverges early in development. Additionally, both bones exhibit significant increases in mean trabecular thickness (and for the femur alone, bone volume fraction) but minimal trabecular reorganisation (i.e., no significant changes in degree of anisotropy, connectivity density, or fractal dimension). Overall, these data suggest that in contrast to data from the axial skeleton, prenatal growth of long bones in the limbs is characterised by size increase, without major reorganizational changes in trabecular architecture.

Fibronectin isoforms promote postnatal skeletal development.

Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-ß1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth.

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.

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.