Chondrocyte Hypertrophic Differentiation Research Articles

A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

Ddx5 participates in regulation of Col10a1 expression and chondrocyte hypertrophic differentiation in vitro.

The type X collagen gene (Col10a1), is a specific molecular marker of hypertrophic chondrocytes during endochondral ossification. Col10a1 expression is known to be influenced by many regulators. In this study, we aim to investigate how DEAD-box helicase 5 (DDX5), a potential binding factor for Col10a1 enhancer, may play a role in Col10a1 expression and chondrocyte hypertrophic differentiation in vitro. The potential binding factors of the 150-bp Col10a1 cis-enhancer were identified with the hTFtarget database. The expression of DDX5 and COL10A1 was detected by quantitative real-time PCR (qRT-PCR) and Western blot in chondrogenic ATDC5 and MCT cell models with or without Ddx5 knockdown or overexpression. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were performed to determine the interaction between DDX5 and the Col10a1 enhancer. The effect and mechanism of DDX5 on chondrocyte differentiation and maturation was evaluated by alcian blue, alkaline phosphatase (ALP), and alizarin red staining in ATDC5 cell lines with stable knockdown of Ddx5. DDX5 was identified as a potential binding factor for the Col10a1 enhancer. The expression of DDX5 in hypertrophic chondrocytes was higher than that in proliferative chondrocytes. Knockdown of Ddx5 decreased, while overexpression of Ddx5 slightly increased COL10A1 expression. DDX5 promotes the enhancer activity of Col10a1 as demonstrated by dual-luciferase reporter assay, and the ChIP experiment suggests a direct interaction between DDX5 and the Col10a1 enhancer. Compared to the control (NC) group, we observed weaker alcian blue and ALP staining intensity in the Ddx5 knockdown group of ATDC5 cells cultured both for 7 and 14 days. Whereas weaker alizarin red staining intensity was only found in the Ddx5 knockdown group of cells cultured for 7 days. Meanwhile, knockdown of Ddx5 significantly reduced the level of runt-related transcription factor 2 (RUNX2) in related ATDC5 cells examined. Our results suggest that DDX5 acts as a positive regulator for Col10a1 expression and may cooperate with RUNX2 together to control Col10a1 expression and promote the proliferation and maturation of chondrocytes.

Collagen type X expression and chondrocyte hypertrophic differentiation during OA and OS development.

Chondrocyte hypertrophy and the expression of its specific marker, the collagen type X gene (COL10A1), constitute key terminal differentiation stages during endochondral ossification in long bone development. Mutations in the COL10A1 gene are known to cause schmid type metaphyseal chondrodysplasia (SMCD) and spondyloepiphyseal dyschondrodysplasia (SMD). Moreover, abnormal COL10A1 expression and aberrant chondrocyte hypertrophy are strongly correlated with skeletal diseases, notably osteoarthritis (OA) and osteosarcoma (OS). Throughout the progression of OA, articular chondrocytes undergo substantial changes in gene expression and phenotype, including a transition to a hypertrophic-like state characterized by the expression of collagen type X, matrix metalloproteinase-13, and alkaline phosphatase. This state is similar to the process of endochondral ossification during cartilage development. OS, the most common pediatric bone cancer, exhibits characteristics of abnormal bone formation alongside the presence of tumor tissue containing cartilaginous components. This observation suggests a potential role for chondrogenesis in the development of OS. A deeper understanding of the shifts in collagen X expression and chondrocyte hypertrophy phenotypes in OA or OS may offer novel insights into their pathogenesis, thereby paving the way for potential therapeutic interventions. This review systematically summarizes the findings from multiple OA models (e.g., transgenic, surgically-induced, mechanically-loaded, and chemically-induced OA models), with a particular focus on their chondrogenic and/or hypertrophic phenotypes and possible signaling pathways. The OS phenotypes and pathogenesis in relation to chondrogenesis, collagen X expression, chondrocyte (hypertrophic) differentiation, and their regulatory mechanisms were also discussed. Together, this review provides novel insights into OA and OS therapeutics, possibly by intervening the process of abnormal endochondral-like pathway with altered collagen type X expression.

Preparation of Novel Sea Cucumber Intestinal Peptides to Promote Tibial Fracture Healing in Mice by Inducing Differentiation of Hypertrophic Chondrocytes to the Osteoblast Lineage.

Hypertrophic chondrocytes have a decisive regulatory role in the process of fracture healing, and the fate of hypertrophic chondrocytes is not only apoptosis. However, the mechanism of sea cucumber (Stichopus japonicus) intestinal peptide (SCIP) on fracture promotion is still unclear. This study aims to investigate the effect of sea cucumber intestinal peptide on the differentiation fate of hypertrophic chondrocytes in a mouse tibial fracture model. Mice are subjected to open fractures of the right tibia to establish a tibial fracture model. The results exhibit that the SCIP intervention significantly promotes the mineralization of cartilage callus, decreases the expression of the hypertrophic chondrocyte marker Col X, and increases the expression of the osteoblast marker Col I. Mechanically, SCIP promotes tibial fracture healing by promoting histone acetylation and inhibiting histone methylation, thereby upregulating pluripotent transcription factors induced the differentiation of hypertrophic chondrocytes to the osteoblast lineage in a manner distinct from classical endochondral ossification. This study is the first to report that SCIP can promote tibial fracture healing in mice by inducing the differentiation of hypertrophic chondrocytes to the osteoblast lineage. SCIP may be considered raw material for developing nutraceuticals to promote fracture healing.

Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1.

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing low-intensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higher-intensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.

Squid cartilage type II collagen accelerates osteoporotic fractures healing through regulating homocysteine mediated EGF and FBN1 signals

Osteoporosis fracture (OPF) is a serious social public health concern in humans. Type II collagen from squid cartilage (SCII) has been proven to promote the healing process in normal fracture mice, however, the effect and mechanism of SCII to improve osteoporosis fracture remain unknown. In this study, type II collagen was obtained from squid laryngeal cartilage, and the effects of SCII on OPF were investigated in mice that experienced bilateral ovariectomy and open tibial fracture surgery after oral administration. The results showed that SCII supplementation significantly decreased the serum TNF-α, increased aggrecan levels on the 5 d, and elevated the serum TGF-β and Col10α levels on 13 d after fracture. Notably, H&E staining exhibited an obvious chondrocyte proliferation on 5 d, and a specific promotion of chondrocyte hypertrophic differentiation and mineralization in the SCII group on 13 d. These findings suggested that SCII promoted osteoporosis fracture healing by enhancing the proliferation and hypertrophy of chondrocyte. Mechanically, SCII supplementation significantly reduced homocysteine levels through down-regulating TMAO production and bile acid levels and activated the EGF/AKT/CylinD1 and TGFβ/Smad1/5/8 pathways, thereby recovering the delay of chondrocyte proliferation and hypertrophy in OPF mice. This study investigated the effects of SCII on osteoporosis fracture healing for the first time and provided a new theoretical basis and options for the high-value application of squid cartilage.

GPx1 promotes hypertrophic differentiation of chondrocytes through modulation of akt signaling in a non-monotonic manner

GPx1 promotes hypertrophic differentiation of chondrocytes through modulation of akt signaling in a non-monotonic manner

Pth1r Signal in Gli1+ Cells Maintains Postnatal Cranial Base Synchondrosis.

Cranial base synchondroses are the endochondral ossification centers for cranial base growth and thus indispensable for proper skull, brain, and midfacial development. The synchondroses are composed of mirror-image growth plates that are continuously maintained from the embryonic to postnatal stage through chondrocyte differentiation. Several factors, including Pth1r signaling, are known to control fetal synchondrosis development. However, there are currently no reports regarding any role for Pth1r signaling in postnatal cranial base and synchondrosis development. Also, the mesenchymal cells that source Pth1r signaling for synchondroses are not known. Here, we employed an inducible mouse model, a hedgehog-responsive Gli1-CreERT2 driver, focusing on the postnatal study. We performed 2 inducible protocols using Gli1-CreERT2;Tomatofl/+ mice that uncovered distinct patterning of Gli1-positive and Gli1-negative chondrocytes in the synchondrosis cartilage. Moreover, we generated Gli1-CreERT2;Pth1rfl/fl;Tomatofl/+ mice to assess their functions in postnatal synchondrosis and found that the mutants had survived postnatally. The mutant skulls morphologically presented unambiguous phenotypes where we noticed the shortened cranial base and premature synchondrosis closure. Histologically, gradual disorganization in mutant synchondroses caused an uncommon remaining central zone between hypertrophic zones on both sides while the successive differentiation of round, flat, and hypertrophic chondrocytes was observed in control sections. These mutant synchondroses disappeared and were finally replaced by bone. Of note, the mutant fusing synchondroses lost their characteristic patterning of Gli1-positive and Gli1-negative chondrocytes, suggesting that loss of Pth1r signaling alters the distribution of hedgehog-responsive chondrocytes. Moreover, we performed laser microdissection and RNA sequencing to characterize the flat proliferative and round resting chondrocytes where we found flat chondrocytes have a characteristic feature of both chondrocyte proliferation and maturation. Taken together, these data demonstrate that Pth1r signaling in Gli1-positive cells is essential for postnatal development and maintenance in cranial base synchondroses. Our findings will elucidate previously unknown aspects of Pth1r functions in cranial biology and development.

Adseverin, an actin-binding protein, modulates hypertrophic chondrocyte differentiation and osteoarthritis progression.

In osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin-/-) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin-/- mice who underwent surgical induction of OA. Adseverin-/- chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.

A Bioglass-Poly(lactic-co-glycolic Acid) Scaffold@Fibrin Hydrogel Construct to Support Endochondral Bone Formation.

Bone tissue engineering using stem cells to build bone directly on a scaffold matrix often fails due to lack of oxygen at the injury site. This may be avoided by following the endochondral ossification route; herein, a cartilage template is promoted first, which can survive hypoxic environments, followed by its hypertrophy and ossification. However, hypertrophy is so far only achieved using biological factors. This work introduces a Bioglass-Poly(lactic-co-glycolic acid@fibrin (Bg-PLGA@fibrin) construct where a fibrin hydrogel infiltrates and encapsulates a porous Bg-PLGA. The hypothesis is that mesenchymal stem cells (MSCs) loaded in the fibrin gel and induced into chondrogenesis degrade the gel and become hypertrophic upon reaching the stiffer, bioactive Bg-PLGA core, without external induction factors. Results show that Bg-PLGA@fibrin induces hypertrophy, as well as matrix mineralization and osteogenesis; it also promotes a change in morphology of the MSCs at the gel/scaffold interface, possibly a sign of osteoblast-like differentiation of hypertrophic chondrocytes. Thus, the Bg-PLGA@fibrin construct can sequentially support the different phases of endochondral ossification purely based on material cues. This may facilitate clinical translation by decreasing in-vitro cell culture time pre-implantation and the complexity associated with the use of external induction factors.

Activation of Nrf2 by sulfuretin stimulates chondrocyte differentiation and increases bone lengths in zebrafish

Elongation of most bones occur at the growth plate through endochondral ossification in postnatal mammals. The maturation of chondrocyte is a crucial factor in longitudinal bone growth, which is regulated by a complex network of paracrine and endocrine signaling pathways. Here, we show that a phytochemical sulfuretin can stimulate hypertrophic chondrocyte differentiation in vitro and in vivo. We found that sulfuretin stabilized nuclear factor (erythroid-derived 2)-like 2 (Nrf2), stimulated its transcriptional activity, and induced expression of its target genes. Sulfuretin treatment resulted in an increase in body length of zebrafish larvae and induced the expression of chondrocyte markers. Consistently, a clinically available Nrf2 activator, dimethyl fumarate (DMF), induced the expression of hypertrophic chondrocyte markers and increased the body length of zebrafish. Importantly, we found that chondrocyte gene expression in cell culture and skeletal growth in zebrafish stimulated by sulfuretin were significantly abrogated by Nrf2 depletion, suggesting that such stimulatory effects of sulfuretin were dependent on Nrf2, at least in part. Taken together, these data show that sulfuretin has a potential use as supporting ingredients for enhancing bone growth.

Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC-Derived Chondrocytes.

Collagen X is a non-fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss of Col10a1 gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous (COL10A1 +/-) or homozygous (COL10A1 -/-) deletions of COL10A1 gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet-derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger in COL10A1 -/- tissues. Prehypertrophic pellet-derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental- and mutant-derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating-phase genes and a higher expression of calcification-phase genes in COL10A1 -/- pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC-derived chondrocytes, though it may facilitate the differentiation process. Thus, COL10A1 -/- iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

Neuropeptide Y Promotes mTORC1 to Regulate Chondrocyte Proliferation and Hypertrophy.

Peripheral neuropeptide Y (NPY) has been reported to regulate bone metabolism and homeostasis; however, its potential roles in growth plate chondrogenesis remain unclear. Here, we found that NPY expression decreased during chondrocyte differentiation in vitro and in vivo. NPY was required for chondrocyte proliferation; in contrast, knockdown of NPY facilitated chondrocyte hypertrophic differentiation. Administration of recombinant NPY in rat chondrocytes and metatarsal bones uncoupled normal proliferation and hypertrophic differentiation during chondrogenesis and thereby inhibited growth plate chondrogenesis and longitudinal bone growth. Remarkably, NPY activated the mTORC1 pathway in chondrocytes, whereas attenuation of mTORC1 activity by administration of rapamycin in vitro partially abrogated NPY-mediated effects on chondrocyte proliferation and hypertrophic differentiation. In addition, a combination of Y2R antagonist but not Y1R antagonist with NPY abolished NPY-mediated inhibition of metatarsal growth and growth plate chondrogenesis. Mechanistically, NPY activated Erk1/2 by NPY2R, then phosphorylated ERK1/2 activated mTORC1 to initiate PTHrP expression, which in turn promoted chondrocyte proliferation and inhibited chondrocyte hypertrophic differentiation. In conclusion, our data identified NPY as a crucial regulator of chondrogenesis and may provide a promising therapeutic strategy for skeletal diseases.

P21 deficiency exhibits delayed endochondral ossification during fracture healing.

Endochondral ossification is a complex biological phenomenon involving a variety of factors and cells. Cyclin-dependent kinase inhibitor 1 (p21) inhibits cell cycle progression and is affected by external stress. We recently reported that embryonic endochondral ossification is unaffected by endogenous p21 deficiency. In this study, we evaluated whether p21 expression affects endochondral ossification during fracture healing. Tibial fractures were introduced into p21 knockout (p21-/-) (n=24) and wild-type C57BL/6 (p21+/+) (n=24) mice at age 10weeks. Fracture healing was evaluated using radiological, histological, and immunohistochemical (IHC) analyses. The effect of p21 small interfering RNA (siRNA) on ATDC5 cells was assessed in vitro. The Allen score for fracture healing was lower in p21-/- mice than in p21+/+ mice. In addition, p21-/- mice exhibited larger calluses and lower bone mineral density. IHC analyses showed that p21-/- mice exhibited delayed endochondral ossification via the Ihh-Runx2-Osterix pathway in vivo. Down-regulation of p21 expression in ATDC5 cells delayed endochondral ossification in vitro. p21 deficiency leads to delayed endochondral ossification by attenuating the Ihh-Runx2-Osterix pathway in vivo, and p21 deficiency in hypertrophic chondrocytes causes delayed differentiation of hypertrophic chondrocytes in vitro. p21 plays a role in endochondral ossification during fracture healing.

Mettl3 regulates hypertrophic differentiation of chondrocytes through modulating Dmp1 mRNA via Ythdf1-mediated m6A modification.

As the main cells in endochondral osteogenesis, chondrocytes have limited self-repair ability due to weak proliferation activity, low density, and dedifferentiation tendency. Here, a thorough inquiry about the effect and underlying mechanisms of methyltransferase like-3 (Mettl3) on chondrocytes was made. Functionally, it was indicated that Mettl3 promoted the proliferation and hypertrophic differentiation of chondrocytes. Mechanically, Dmp1 (dentin matrix protein 1) was proved to be the downstream direct target of Mettl3 for m6A modification to regulate the differentiation of chondrocytes through bioinformatics analysis and correlated experiments. The Reader protein Ythdf1 mediated Dmp1 mRNA catalyzed by Mettl3. In vivo, the formation of subcutaneous ectopic cartilage-like tissue further supported the in vitro results. In conclusion, the gene regulation of Mettl3/m6A/Ythdf1/Dmp1 axis in hypertrophic differentiation of chondrocytes for the development of endochondral osteogenesis may supply a promising treatment strategy for the repair and regeneration of bone defects.

Downregulation of miR-30b-5p Facilitates Chondrocyte Hypertrophy and Apoptosis via Targeting Runx2 in Steroid-Induced Osteonecrosis of the Femoral Head.

As a widely used steroid hormone medicine, glucocorticoids have the potential to cause steroid-induced osteonecrosis of the femoral head (SONFH) due to mass or long-term use. The non-coding RNA hypothesis posits that they may contribute to the destruction and dysfunction of cartilages as a possible etiology of SONFH. MiR-30b-5p was identified as a regulatory factor in cartilage degeneration caused by methylprednisolone (MPS) exposure in our study through cell transfection. The luciferase reporter assay confirmed that miR-30b-5p was downregulated and runt-related transcription factor 2 (Runx2) was mediated by miR-30b-5p. The nobly increased expression of matrix metallopeptidase 13 (MMP13) and type X collagen (Col10a1) as Runx2 downstream genes contributed to the hypertrophic differentiation of chondrocytes, and the efficiently upregulated level of matrix metallopeptidase 9 (MMP9) may trigger chondrocyte apoptosis with MPS treatments. The cell transfection experiment revealed that miR-30b-5p inhibited chondrocyte hypertrophy and suppressed MPS-induced apoptosis. As a result, our findings showed that miR-30b-5p modulated Runx2, MMP9, MMP13, and Col10a1 expression, thereby mediating chondrocyte hypertrophic differentiation and apoptosis during the SONFH process. These findings revealed the mechanistic relationship between non-coding RNA and SONFH, providing a comprehensive understanding of SONFH and other bone diseases.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

SUMMARYThe transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Expressions of parathyroid hormone-related protein (PTHrP) and parathyroid hormone receptor-1 (PTH1R) in the condylar cartilage of temporomandibular joint modulated by occlusal elevation

Parathyroid hormone-related protein (PTHrP) is an important regulatory factor in the growth, development and remodeling of bone or cartilage, and acts through its sole receptor, parathyroid hormone receptor-1 (PTH1R). The present study aimed to research the expression changes of PTHrP, PTH1R and other relevant factors in condylar cartilage during the progress of temporomandibular joint osteoarthritis (TMJOA). The animal model of TMJOA was constructed by the "resin-modified method", and Sprague Dawley (SD) rats were euthanized at 2 weeks, 4 weeks, 6 weeks and 8 weeks after occlusal elevation. The histological changes of condylar cartilage were observed by X-ray, hematoxylin-eosin (HE) and safranine O-fast green (SO-FG) staining. The expressions of PTHrP, PTH1R, Ki67, Collagen II (Col II), Collagen X (Col X) and Caspase 3 in each group were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). TMJOA progression was time-dependent. In the experimental group, PTHrP expression was unimodal with a peak at 4 weeks, but PTH1R expression showed a decreasing trend. From 2 weeks to 8 weeks in the experimental group, Col X expression rather than Caspase 3 expression was negatively related to PTHrP's, which has no positive relation to Ki67 or Col II. These results demonstrated abnormal occlusal load may be an important pathogenic factor of TMJOA. It may be one of the reasons of TMJOA progression that PTHrP can't play an effective role due to the low expression of PTH1R. PTHrP may be a direct factor regulating the hypertrophic differentiation of chondrocytes, but it does not directly regulate the proliferation and apoptosis of chondrocytes, and the realization of both regulatory effects may depend on the inhibition of hypertrophic differentiation.

Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification.

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provokedthe osteocyte differentiation of osteoblast precursor cells under a three-dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self-organized scaffold-free spheroid model reconstructed by pre-chondrocyte cells. Within 2-day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long-term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre-chondrocyte cells.