Zone Of Hypertrophic Chondrocytes Research Articles

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-β1 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.

FOXC1 and FOXC2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

Roles of Local Soluble Factors in Maintaining the Growth Plate: An Update

The growth plate is a cartilaginous tissue found at the ends of growing long bones, which contributes to the lengthening of bones during development. This unique structure contains at least three distinctive layers, including resting, proliferative, and hypertrophic chondrocyte zones, maintained by a complex regulatory network. Due to its soft tissue nature, the growth plate is the most susceptible tissue of the growing skeleton to injury in childhood. Although most growth plate damage in fractures can heal, some damage can result in growth arrest or disorder, impairing leg length and resulting in deformity. In this review, we re-visit previously established knowledge about the regulatory network that maintains the growth plate and integrate current research displaying the most recent progress. Next, we highlight local secretary factors, such as Wnt, Indian hedgehog (Ihh), and parathyroid hormone-related peptide (PTHrP), and dissect their roles and interactions in maintaining cell function and phenotype in different zones. Lastly, we discuss future research topics that can further our understanding of this unique tissue. Given the unmet need to engineer the growth plate, we also discuss the potential of creating particular patterns of soluble factors and generating them in vitro.

Roles of Sodium Hydrogen Exchanger (NHE1) and Anion Exchanger (AE2) across Chondrocytes Plasma Membrane during Longitudinal Bone Growth.

Mammalian long bone growth occurs through endochondral ossification, majorly regulated by the controlled enlargement of chondrocytes at the growth plate (GP). This study aimed to investigate the roles of Na+/H+ (sodium hydrogen exchanger (NHE1)) and HCO3− (anion exchanger [AE2]) during longitudinal bone growth in mammals. Bones from P10 SpragueDawley rat pups were cultured exvivo in the presence or absence of NHE1 and AE2 inhibitors to determine their effect on long bone growth. Gross morphometry, histomorphometry, and immunohistochemistry were used to assess the bone growth. The results revealed that the culture of the bones in the presence of NHE1 and AE2 inhibitors reduces bone growth significantly (p < 0.05) by approximately 11%. The inhibitor significantly (p < 0.05) reduces bone growth velocity and the length of the hypertrophic chondrocyte zone without any effect on the total GP length. The total GP chondrocyte density was significantly (p < 0.05) reduced, but hypertrophic chondrocyte densities remained constant. NHE1 fluorescence signaling across the GP length was higher than AE2, and their localization was significantly (p < 0.05) inhibited at the hypertrophic chondrocytes zone. The GP lengthening was majorly driven by an increase in the overall GP chondrocyte and hypertrophic chondrocyte densities apart from the regulatory volume phenomenon. This may suggest that NHE1 and AE2 could have a regulatory role in long bone growth.

Runx2 is required for hypertrophic chondrocyte mediated degradation of cartilage matrix during endochondral ossification

The RUNX2 transcription factor is a key regulator for the development of cartilage and bone. Global or resting chondrocyte-specific deletion of the Runx2 gene results in failure of chondrocyte hypertrophy, endochondral ossification, and perinatal lethality. The terminally mature hypertrophic chondrocyte regulates critical steps of endochondral ossification. Importantly, expression of the Runx2 gene starts in the resting chondrocyte and increases progressively, reaching the maximum level in hypertrophic chondrocytes. However, the RUNX2 role after chondrocyte hypertrophy remains unknown. To answer this question, we deleted the Runx2 gene specifically in hypertrophic chondrocytes using the Col10-Cre line. Mice lacking the Runx2 gene in hypertrophic chondrocytes (Runx2HC/HC) survive but exhibit limb dwarfism. Interestingly, the length of the hypertrophic chondrocyte zone is doubled in the growth plate of Runx2HC/HC mice. Expression of pro-apoptotic Bax decreased significantly while anti-apoptotic Bcl2 remains unchanged leading to a four-fold increase in the Bcl2/Bax ratio in mutant mice. In line with this, a significant reduction in apoptosis of Runx2HC/HC hypertrophic chondrocyte is noted. A large amount of cartilage matrix is present in the long bones that extend toward the diaphyseal region of Runx2HC/HC mice. This is not due to enhanced synthesis of the cartilage matrix as the expression of both collagen type 2 and aggrecan were comparable among Runx2HC/HC and WT littermates. Our qPCR analysis demonstrates the increased amount of cartilage matrix is due to impaired expression of cartilage degrading enzymes such as metalloproteinase and aggrecanase as well as tissue inhibitor of metalloproteinases. Moreover, a significant decrease of TRAP positive chondroclasts was noted along the cartilage islands in Runx2HC/HC mice. Consistently, qPCR data showed an 81% reduction in the Rankl/Opg ratio in Runx2HC/HC littermates, which is inhibitory for chondroclast differentiation. Finally, we assess if increase cartilage matrix in Runx2HC/HC mice serves as a template for bone and mineral deposition using micro-CT and Von Kossa. The mutant mice exhibit a significant increase in trabecular bone mass compared to littermates. In summary, our findings have uncovered a novel role of Runx2 in apoptosis of hypertrophic chondrocytes and degradation of cartilage matrix during endochondral ossification.

Characterisation of Growth Plate Dynamics in Murine Models of Osteoarthritis.

The purpose of this study was to investigate growth plate dynamics in surgical and loading murine models of osteoarthritis, to understand whether abnormalities in these dynamics are associated with osteoarthritis development. 8-week-old C57BL/6 male mice underwent destabilisation of medial meniscus (DMM) (n = 8) surgery in right knee joints. Contralateral left knee joints had no intervention (controls). In 16-week-old C57BL/6 male mice (n = 6), osteoarthritis was induced using non-invasive mechanical loading of right knee joints with peak force of 11N. Non-loaded left knee joints were internal controls. Chondrocyte transiency in tibial articular cartilage and growth plate was confirmed by histology and immunohistochemistry. Tibial subchondral bone parameters were measured using microCT and correlated to 3-dimensional (3D) growth plate bridging analysis. Higher expression of chondrocyte hypertrophy markers; Col10a1 and MMP13 were observed in tibial articular cartilage chondrocytes of DMM and loaded mice. In tibial growth plate, Col10a1 and MMP13 expressions were widely expressed in a significantly enlarged zone of proliferative and hypertrophic chondrocytes in DMM (p=0.002 and p<0.0001, respectively) and loaded (both p<0.0001) tibiae of mice compared to their controls. 3D quantification revealed enriched growth plate bridging and higher bridge densities in medial compared to lateral tibiae of DMM and loaded knee joints of the mice. Growth plate dynamics were associated with increased subchondral bone volume fraction (BV/TV; %) in medial tibiae of DMM and loaded knee joints and epiphyseal trabecular bone volume fraction in medial tibiae of loaded knee joints. The results confirm articular cartilage chondrocyte transiency in a surgical and loaded murine models of osteoarthritis. Herein, we reveal spatial variation of growth plate bridging in surgical and loaded osteoarthritis models and how these may contribute to anatomical variation in vulnerability of osteoarthritis development.

Liquid-phase ASEM imaging of cellular and structural details in cartilage\xa0and bone formed during endochondral ossification: Keap1-deficient osteomalacia

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1−/− femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Osteoclasts and Macrophages—Their Role in Bone Marrow Cavity Formation During Mouse Embryonic Development

The formation of the bone marrow cavity is a prerequisite for endochondral ossification. In reviews and textbooks, it is occasionally reported that osteoclasts are essential for bone marrow cavity formation removing hypertrophic chondrocytes. Mice lacking osteoclasts or having functionally defective osteoclasts have osteopetrotic bones, yet they still form a bone marrow cavity. Here, we investigated the role of osteoclasts and macrophages in bone marrow cavity formation during embryogenesis. Macrophages can assist osteoclasts in matrix removal by phagocytosing resorption byproducts. Rank-deficient mice, lacking osteoclasts, and Pu.1-deficient mice, lacking monocytes, macrophages, and osteoclasts, displayed a delay in bone marrow cavity formation and a lengthening of the zone of hypertrophic chondrocytes. F4/80-positive monocyte/macrophage numbers increased by about fourfold in the bone marrow cavity of E18.5 Rank-deficient mice. Based on lineage-tracing experiments, the majority of the excess F4/80 cells were derived from definitive hematopoietic precursors of the fetal liver. In long bones of both Rank-/- and Pu.1-/- specimens, Mmp9-positive cells were still present. In addition to monocytes, macrophages, and osteoclasts, Ctsb-positive septoclasts were lost in Pu.1-/- specimens. The mineralization pattern was altered in Rank-/- and Pu.1-/- specimens, revealing a significant rise in transverse-oriented mineralized structures. Taken together, our findings imply that early on during bone marrow cavity formation, osteoclasts facilitate the entry of blood vessels and later the turnover of hypertrophic chondrocytes, whereas macrophages appear to play no major role. Furthermore, the absence of septoclasts in Pu.1-/- specimens suggests that septoclasts are either derived from Pu.1-dependent precursors or require PU.1 activity for their differentiation. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Role of iRhoms 1 and 2 in Endochondral Ossification.

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell–cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2−/−) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2−/−iR1∆Ch mice had retarded bone growth compared to iR2−/− mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2−/−iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2−/−iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα−/− mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.

Histological evaluation of the possible role of Na+/ H+ entiporter and anion exchanger in endochondral ossification activities of secondary bone healing in rats

In secondary fracture healing, callus proliferate, undergo hypertrophy and the extracellular matrix becomes calcified. This step to some extent, recapitulates the embryological bone development with a combination of cellular proliferation and differentiation, increasing cellular volume and matrix deposition. The causes of the chondrocytes volume increase in secondary bone healing are poorly known, but cell membrane transporters perhaps could be implicated. We hypothesize that NHE-1 and AE-2 are among plasma membrane transporters that have a role in cellular differentiation and regulation of endochondral ossification for secondary bone fracture healing. Study of closed tibia fracture healing in 2 groups of 25 of 8-weeks-old Sprague-Dawley rats were undertaken and histological evaluation were made at 5 different time points at 1, 2, 3, 4, and 6 weeks after induction of the fracture. Histological evaluation of proliferative and hypertrophic chondrocyte zone area showed a significant difference in week 1 compared to other weeks. Immunohistochemistry study revealed a significant high level of labeling intensity of NHE-1 at the first four weeks. While labeling intensity of AE-2 showed moderate reaction at 1 and 2 weeks, that increased and reached the highest level at 3 and 4 weeks. These results suggested that NHE-1 and AE-2 had role in the endochondral ossification of secondary bone healing.

Fluoride Inhibits Longitudinal Bone Growth by Acting Directly at the Growth Plate in Cultured Neonatal Rat Metatarsal Bones.

Excessive intake of fluoride inhibits bone growth in both humans and animals. It is unknown whether fluoride acts directly on the growth plate to inhibit longitudinal bone growth, and its mechanism of action has not been elucidated. In this study, we used an organ culture system and SW1353 cells to evaluate the effects of fluoride on endochondral ossification. Neonatal rat metatarsal bones were dissected and cultured with or without fluoride for 7days. The total length and width of the metatarsal rudiments and the length of the calcification zone were measured. Chondrocyte proliferation, differentiation, and apoptosis were analyzed by immunohistochemistry and TUNEL assay in sectioned bones. The apoptosis was detected by flow cytometry, and the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 were detected by western blotting in SW1353 cells. Linear measurements demonstrated that fluoride induced a biphasic effect on longitudinal bone growth in organ culture, with a significant growth inhibition at a high concentration (10-4M) and a stimulatory action at low concentration (10-6M) of fluoride. Histomorphometrical analysis of growth plate from fluoride-exposed metatarsal rudiments showed a significant reduction in the height of the proliferative and hypertrophic chondrocyte zones. Analysis of the Col2α1 and Col10α1 expression by immunohistochemistry revealed fluoride-suppressed metatarsal growth plate chondrocyte proliferation and differentiation. In addition, fluoride increased the number of apoptotic chondrocytes in the metatarsal growth plate. Western blotting showed an up-regulated expression of Caspase-3 and Bax and down-regulated expression of anti-apoptotic protein Bcl-2 after treatment with 5 × 10-4M fluoride in SW1353 cells. Our findings indicated that fluoride inhibited longitudinal bone growth by acting directly at the growth plate in cultured neonatal rat metatarsal bones. Such growth inhibition was mediated by suppressing proliferation and differentiation, increasing apoptosis of resting chondrocytes and causing premature cell senescence in the growth plate.

Mineralization pathways in the active murine epiphyseal growth plate

Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature.

Altered chondrocyte differentiation, matrix mineralization and MEK-Erk1/2 signaling in an INPPL1 catalytic knock-out mouse model of opsismodysplasia.

Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.

C-type natriuretic peptide improves growth retardation in a mouse model of cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome, a genetic disorder caused by germline mutations in BRAF, KRAS, MAP2K1 and MAP2K2, is characterized by growth retardation, heart defects, dysmorphic facial appearance and dermatologic abnormalities. We have previously reported that knock-in mice expressing the CFC syndrome-associated mutation, Braf Q241R, showed growth retardation because of gastrointestinal dysfunction. However, other factors associated with growth retardation, including chondrogenesis and endocrinological profile, have not been examined. Here, we show that 3- and 4-week-old BrafQ241R/+ mice have decreased body weight and length, as well as reduced growth plate width in the proximal tibiae. Furthermore, proliferative and hypertrophic chondrocyte zones of the growth plate were reduced in BrafQ241R/+ mice compared with Braf+/+ mice. Immunohistological analysis revealed that extracellular signal-regulated kinase (ERK) activation was enhanced in hypertrophic chondrocytes in BrafQ241R/+ mice. In accordance with growth retardation and reduced growth plate width, decreased serum levels of insulin-like growth factor 1 (IGF-1) and IGF binding protein 3 (IGFBP-3) were observed in BrafQ241R/+ mice at 3 and 4 weeks of age. Treatment with C-type natriuretic peptide (CNP), a stimulator of endochondral bone growth and a potent inhibitor of the FGFR3-RAF1-MEK/ERK signaling, increased body and tail lengths in Braf+/+ and BrafQ241R/+ mice. In conclusion, ERK activation in chondrocytes and low serum IGF-1/IGFBP-3 levels could be associated with the growth retardation observed in BrafQ241R/+ mice. Our data also suggest that CNP is a potential therapeutic target in CFC syndrome.

Cardiac, bone and growth plate manifestations in hypocalcemic infants: revealing the hidden body of the vitamin D deficiency iceberg

BackgroundWhilst hypocalcemic complications from vitamin D deficiency are considered rare in high-income countries, they are highly prevalent among Black, Asian and Minority Ethnic (BAME) group with darker skin. To date, the extent of osteomalacia in such infants and their family members is unknown. Our aim was to investigate clinical, cardiac and bone histomorphometric characteristics, bone matrix mineralization in affected infants and to test family members for biochemical evidence of osteomalacia.Case presentationThree infants of BAME origin (aged 5–6 months) presented acutely in early-spring with cardiac arrest, respiratory arrest following seizure or severe respiratory distress, with profound hypocalcemia (serum calcium 1.22–1.96 mmol/L). All infants had dark skin and vitamin D supplementation had not been addressed during child surveillance visits. All three had severely dilated left ventricles (z-scores + 4.6 to + 6.5) with reduced ejection fraction (25–30%; normal 55–70), fractional shortening (7 to 15%; normal 29–40) and global hypokinesia, confirming hypocalcemic dilated cardiomyopathy. They all had low serum levels of 25 hydroxyvitamin D (25OHD < 15 nmol/L), and elevated parathyroid hormone (PTH; 219–482 ng/L) and alkaline phosphatase (ALP; 802–1123 IU/L), with undiagnosed rickets on radiographs.One infant died from cardiac arrest. At post-mortem examination, his growth plate showed a widened, irregular zone of hypertrophic chondrocytes. Histomorphometry and backscattered electron microscopy of a trans-iliac bone biopsy sample revealed increased osteoid thickness (+ 262% of normal) and osteoid volume/bone volume (+ 1573%), and extremely low bone mineralization density. Five of the nine tested family members had vitamin D deficiency (25OHD < 30 nmol/L), three had insufficiency (< 50 nmol/L) and 6/9 members had elevated PTH and ALP levels.ConclusionsThe severe, hidden, cardiac and bone pathology described here exposes a failure of public health prevention programs, as complications from vitamin D deficiency are entirely preventable by routine supplementation. The family investigations demonstrate widespread deficiency and undiagnosed osteomalacia in ethnic risk groups and call for protective legislation.

Role of Angiopoietin-like 4 on Bone Vascularization in Chickens Exposed to High-altitude Hypoxia

The aim of this study was to investigate the role and expression of a novel angiogenic factor (angiopoietin-like 4, ANGPTL4) in tibial growth plates of broiler chickens exposed to high-altitude hypoxia. One-day-old healthy broiler chickens (n=120) were transported from lowland to a high-altitude hypoxic region (nearly 3,000m above sea level) and were reared under hypoxic- (natural lower oxygen content) and normoxic conditions (nearly 21% oxygen content) for 14 days. The effect of hypoxia on angiogenesis in the tibial growth platesand hypoxia-inducible factor (HIF)-1α and ANGPTL4 expressions were determined by histological examination, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), western blot and enzyme-linked immunosorbent assay (ELISA) techniques. The increase in vascular distribution to the hypertrophic chondrocyte zone of tibial growth plates contributed to promoting growth and development of the tibia under hypoxic conditions, which was highly correlated with the upregulation of ANGPTL4 at both the mRNA and protein levels together with activation of HIF-1α under hypoxic conditions. These findings demonstrate that angiogenic factor ANGPTL4 upregulation is involved in tibial growth plate angiogenesis to promote the development of the tibia in broiler chickens under hypoxic conditions. They also suggest that ANGPTL4 may serve as a new molecular therapeutic target for ameliorating tibial dyschondroplasia chicken bone vascularization.

Ablation of Dnmt3b in chondrocytes suppresses cell maturation during embryonic development.

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization was delayed in Col2Cre+ ;Dnmt3bf/f (Dnmt3bCol2 ) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3bCol2 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3bCol2 growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3bCol2 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3bCol2 chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.

Role and regulation of growth plate vascularization during coupling with osteogenesis in tibial dyschondroplasia of chickens

Tibial dyschondroplasia (TD) is the most-prevalent leg disorder in fast-growing chickens; it is intractable and characterized by abnormal endochondral bone formation of proximal tibial growth-plates (TGPs). Previous studies have shown that bone is a highly vascularized tissue dependent on the coordinated coupling between angiogenesis and osteogenesis, but the underlying mechanisms of bone formation and bone remodeling are poorly defined in TD chickens. Here, we observed that inhibition of vasculogenesis and angiogenesis remarkably impaired vascular invasion in the hypertrophic chondrocyte zone of the TGPs, resulting in the massive death of chondrocytes due to a shortage of blood vessels and nutrients. Moreover, the balance of the OPG (osteoprotegerin)/RANKL (receptor activator of nuclear factor-kB ligand) system is also severely disrupted during the osteogenesis process while coupling with angiogenesis, both of which eventually lead to abnormal endochondral bone formation in TD chickens. Thus, the process of vascular formation in endochondral bone appears to initiate the pathological changes in TD, and improvement of this process during coupling with osteogenesis may be a potential therapeutic approach to treat this intractable disease.

Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsαR201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsαR201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsαR201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology.

The vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation

Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase) V0 subunit d2 (ATP6V0D2), was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium). In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.