Zone Chondrocytes Research Articles

7598 A Humanized PTH Receptor Mouse Model Of Eiken Syndrome: Delayed Bone Mineralization Caused By A Receptor C-tail Truncation

Abstract Disclosure: J. Höppner: None. P. Hanna: None. M.N. Wein: None. H. Jueppner: None. T.J. Gardella: None. R. Civitelli: None. I.A. Portales-Castillo: None. Abstract The parathyroid hormone receptor-1 (PTH1R) plays a key role in bone development and acts in the growth plates in response to PTHrP ligand. Eiken syndrome is characterized by a delay in bone mineralization and is caused by homozygous PTH1R mutations. One such mutation, R485X, truncates the receptor's C-tail and removes serine phosphorylation sites involved in βarrestin binding. In HEK293 cells, R485X-hPTH1R exhibits deficient interaction with βarrestin and increased cAMP signaling both basally and in response to PTHrP (Portales-Castillo et al., Comms. Biology 2023). To understand how the R485X mutation causes delayed bone mineralization, we generated humanized R485X-hPTH1R knock-in mice. Homozygous hPTH1RR485X/R485X mice closely recapitulate the delayed bone mineralization seen in Eiken patients, as skeletal whole mount preparations from neonatal mutant mice showed reduced Alizarin red staining compared to WT controls, and metatarsal explants from the mutant mice showed a pronounced absence of mineralized bone. Tails of hPTH1RR485X/R485X mice were ∼50% shorter than those of WT littermates, and H&E-stained sections revealed only proliferative chondrocytes in the growth plates of mutant mice. This delay in growth plate chondrocyte maturation in hPTH1RR485X/R485X mice resolved with age, although older long bones and tails remained smaller in size than WT controls (Höppner et al. Presented at ASBMR 2023).Based on our in vitro findings and initial mouse characterization, we reasoned that the phenotype of Eiken mice may be explained by increased PTHrP/PTH1R signaling in growth plate chondrocyte. The class IIa histone deacetylase HDAC4 suppresses chondrocyte maturation downstream of PTH1R signaling. Therefore, we generated compound mutant mice bearing both Hdac4 deletion and the mutant R485X PTH1R allele. Indeed, Hdac4 deletion largely rescued the mineralization defect apparent in metatarsals of P1 hPTH1RR485X/R485X mice. We then assessed the contribution of endogenous PTHrP to the Eiken-like phenotype by generating hPTH1RR485X/R485X/PTHrPflox/+/Col2-Cre(tg) mice. Remarkably, these mice exhibit approximately the same tail lengths as hPTH1R-WT littermate controls, indicating that reduced PTHrP production can rescue the effects of homozygosity for R485X-PTH1R. In line, vertebral growth plates of the rescued mice exhibited normal zones of chondrocytes, including hypertrophic cells. The overall results support a disease mechanism for Eiken syndrome by which excess cAMP signaling by PTH1R-R485X, as induced in part by endogenous PTHrP, results in delayed chondrocyte differentiation and bone mineralization. Further studies employing βarr1/2-KO mice may help shed light on the specific roles of βarrestins in growth plate development by PTH1R in Eiken syndrome. Presentation: 6/3/2024

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.

Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants

BackgroundLimited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.MethodsFull-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, −AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.ResultsSuperficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.ConclusionsIn enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Longitudinal Imaging of the Skull Base Synchondroses Demonstrate Prevention of a Premature Ossification After Recifercept Treatment in Mouse Model of Achondroplasia.

ABSTRACTAchondroplasia is the most common form of short‐limb dwarfism. In this disorder, endochondral ossification is impaired due to gain‐of‐function mutation in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene. In addition to short limbs, cranial base bones are also affected leading to shortening of the skull base and to serious neurological complications associated with foramen magnum stenosis. These complications are thought to be due to the delay or premature arrest of skull base growth, caused by an accelerated ossification of the sphenooccipital (SOS) and the intraoccipital (IOS) synchondroses. Skull synchondroses consist of two opposite growth plates sharing a common reserve zone of chondrocytes. In this study, we first characterized the skull base synchondroses ossification in a mouse model of achondroplasia carrying the human G380R mutation (Fgfr3 ach/+ ). We then addressed whether Recifercept, a soluble FGFR3, could prevent premature closure of these synchondroses. Postnatal radiological observations revealed the presence of bony bridge structures in one or more synchondroses in Fgfr3 ach/+ mice as early as postnatal day 3 in the most severe cases. The presence of early ossification correlated with the severity of the disease as it was associated with an arrest of the cranial base bone growth. Histological analyses indicated changes in the synchondroses structure and matrix proteoglycan contents confirming a process of ossification. Treatment of Fgfr3 ach/+ mice with Recifercept compared with vehicle prevented premature synchondrosis ossification and the transition to bone, resulting in improved skull shape and cranium ratio. Given the impact of Recifercept on synchondrosis inactivation, it is possible that it could prevent one of the most severe complication of achondroplasia if used early enough during bone development. These data support the clinical development of Recifercept for achondroplasia, and suggests that early treatment may be required to best address impaired endochondral bone growth. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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.

Bone tissue morphology of rat offspring lactationally exposed to polychlorinated biphenyl 169 and 155

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent, organic pollutants also considered endocrine-disrupting chemicals. Our study examined the effects of lactational exposure to nondioxin-like PCB-155 and/or dioxin-like PCB-169 on longitudinal femur growth at the distal epiphyseal growth plate (EGP) in young rats at three different ages [postnatal days (PNDs) 9, 22, and 42]. After delivery, lactating rats were divided into four groups (PCB-169, PCB-155, PCB-155 + 169, and control) and administered PCBs intraperitoneally. The femurs of offspring were used to estimate growth rate (µm/day), and histomorphometric analysis on the distal femur included the thickness of the EGP and zones of proliferation and hypertrophy with calcification. Stereometry was used to determine trabecular bone volume density. In the PCB-169 and PCB-155 + 169 groups, PCB-169 affected longitudinal bone growth in the early postnatal period by interfering with chondrocytes in the EGP zone of proliferation and, to a lesser extent, the zone of hypertrophy. Morphometric alterations in EGP structure diminished until puberty. A slow growth rate persisted in the PCB-169 group until PND 42, while in the PCB-155 group, a fast growth rate between PNDs 9 to 22 was significantly reduced between PNDs 22 to 42. Sterometric assessment showed decreased trabecular bone volume in the PCB-155 + 169 group compared with that in the control on PND 9 and increased in the PCB-169 group compared with that in the PCB-155 group on PND 42. To summarize, studied PCB congeners exerted congener- and age-dependent effects on femur growth rate and its histomorphometric characteristics.

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.

Changes of microstructure in cartilage and subchondral bone of patients with knee osteoarthritis

Objective To investigate microstructure changes of cartilage and subchondral bone in the nonsclerotic and sclerotic zones of the tibial plateau in patients with knee osteoarthritis (OA), and explore the pathological features of different parts and stages of knee OA. Methods Tibial plateau tissues abandoned after total knee arthroplasty were collected in OA patients. After gross observation, the cartilage and subchondral bone were divided into nonsclerotic and sclerotic zones. The pathologic changes of tissue and cell microstructure characteristics were observed by scanning and transmission electron microscopies, and the microstructure of subchondral bone was evaluated by Micro-CT scanning system. Results As compared with the nonscleroic zone, scanning electron microscopy showed that the cartilage morphology of the scleroic zone was chaotic and thinner, and surface collagen fibers were disarranged. The cracks to the subchondral bone were observed. The subchondral bone plate and trabecular bone were thickened, and gap narrowed. The transmission electron microscopy showed that the nucleus of the chondrocytes in the scleroic zone was irregular, and the rough endoplasmic reticulum was dilated, the Golgi apparatus was underdeveloped, and mitochondria displayed edema. Bone matrix collagen arranged sparsely. Secondary lysosomes increased and bright microfilament reduced in osteoclasts. The osteoblast envelope was wrinkled with no small protuberances on the surface. The microvascular formation was seen in the scleroic zones. The micro-computed tomography showed bone mineral density (BMD, 98.040±20.600), BV/TV (40.060±7.110), Tb.N (1.540±0.181) and Tb.Th (0.250±0.039) were increased, and Tb.Sp (0.43±0.047) and SMI (0.83±0.124) decreased in the scleroic zone as compared with the non-sclerotic zone (t=7.931, 11.959, 6.212, 8.070, 8.566, 10.803, P<0.05). Conclusion The microstructure of cartilage and subchondral bone in OA sclerotic area is different from that in nonsclerotic area. The microstructure of cartilage and subchondral bone in the sclerotic area should be further studied. Key words: Osteoarthritis; Cartilage; Subchondral bone; Microstructure

Early morphological changes in the knee joint of rats after opioid exposure

The objective is to study early morphological changes of rats’ knee joint after experimental opioid exposure. Materials and methods. Research material is mature, non-breeding male rats 4.5 months old (n = 16) with body weight 80 g. Animals were divided into 2 groups: experimental and control. The experimental group of rats (n = 10) got the injection of nalbuphine hydrochloride intramuscularly, one time per day during 7 days; initial dose – 8 mg/kg. The condition of chronic opioid exposure has been modeled. The rats of control group (n = 6) got the injection of physiological solution intramuscularly during 7 days. The distal epiphysis of rat thigh and proximal epiphysis of rat tibia taking into account the preservation of topographical correlation of the knee joint structural components have been used for histological analysis. Following decalcination, the histologic specimens have been stained with hematoxylin, eosin, azure and azan by Heidenhain. The thickness of the cover synovial layer of articular cartilage has been measured. Results. The architectonics of articular cartilage distorts, the cover synovial layer of articular cartilage gets thinner due to experimental action of opiates. There is also tendency towards volume decrease of chondrocytes in proliferation zone and thickness loss of the cartilage. Conclusions. At the early stage of opioid exposure the structure abnormality of laboratory rats’ knee joint can be seen in the synovial layer and in the zone of chondrocytes proliferation. These research data can be a prerequisite for opioid chondropathy.

Endochondral ossification in hindlimbs during bufo gargarizans metamorphosis: A model of studying skeletal development in vertebrates

Background Endochondral ossification, the process by which most of the skeleton is formed, is accurately regulated by many specific groups of molecules and extracellular matrix components. The molecular mechanisms of endochondral ossification have been extensively investigated in mammals. However, there are few studies about it in amphibians. Results Ossification of femur and tibiofibula was observed at Gs 40, and tarsals and metatarsals were ossified at Gs 42. Most of the skeletons in hindlimbs were completely ossified except for articular cartilages at Gs 46. Thirty-two genes related to endochondral ossification were found in the differentially expressed genes (DEGs) library of hindlimbs, and nine of these genes were validated by qRT-PCR during metamorphosis. Sox9 was expressed in the columnar, prehypertrophic, and upper hypertrophic zones, and the expression of Ihh was observed in prehypertrophic chondrocyte zone in hindlimbs of B. gargarizans. Conclusions The ossification of hindlimbs increased gradually, and the ossification sequence was from proximal to distal in B. gargarizans during metamorphosis. Thirty-two genes found in the DEGs library were related to the regulation of endochondral ossification of hindlimbs in amphibians. The present study will provide a valuable genomic resource for the future study of endochondral ossification in amphibian. Developmental Dynamics 247:1121-1134, 2018. © 2018 Wiley Periodicals, Inc.