Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signaling.

Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.

Combining sclerostin neutralization with tissue engineering: An improved strategy for craniofacial bone repair.

Scaffolds associated with different types of mesenchymal stromal stem cells (MSC) are extensively studied for the development of novel therapies for large bone defects. Moreover, monoclonal antibodies have been recently introduced for the treatment of cancer-associated bone loss and other skeletal pathologies. In particular, antibodies against sclerostin, a key player in bone remodeling regulation, have demonstrated a real benefit for treating osteoporosis but their contribution to bone tissue-engineering remains uncharted. Here, we show that combining implantation of dense collagen hydrogels hosting wild-type (WT) murine dental pulp stem cells (mDPSC) with weekly systemic injections of a sclerostin antibody (Scl-Ab) leads to increased bone regeneration within critical size calvarial defects performed in WT mice. Furthermore, we show that bone formation is equivalent in calvarial defects in WT mice implanted with Sost knock-out (KO) mDPSC and in Sost KO mice, suggesting that the implantation of sclerostin-deficient MSC similarly promotes new bone formation than complete sclerostin deficiency. Altogether, our data demonstrate that an antibody-based therapy can potentialize tissue-engineering strategies for large craniofacial bone defects and urges the need to conduct research for antibody-enabled local inhibition of sclerostin. STATEMENT OF SIGNIFICANCE: The use of monoclonal antibodies is nowadays broadly spread for the treatment of several conditions including skeletal bone diseases. However, their use to potentialize tissue engineering constructs for bone repair remains unmet. Here, we demonstrate that the neutralization of sclerostin, through either a systemic inhibition by a monoclonal antibody or the implantation of sclerostin-deficient mesenchymal stromal stem cells (MSC) directly within the defect, improves the outcome of a tissue engineering approach, combining dense collagen hydrogels and MSC derived from the dental pulp, for the treatment of large craniofacial bone defects.

Microvascular maturation by mesenchymal stem cells in vitro improves blood perfusion in implanted tissue constructs.

Blood perfusion of grafted tissue constructs is a hindrance to the success of stem cell-based therapies by limiting cell survival and tissue regeneration. Implantation of a pre-vascularized network engineered in vitro has thus emerged as a promising strategy for promoting blood supply deep into the construct, relying on inosculation with the host vasculature. We aimed to fabricate in vitro tissue constructs with mature microvascular networks, displaying perivascular recruitment and basement membrane, taking advantage of the angiogenic properties of dental pulp stem cells and self-assembly of endothelial cells into capillaries. Using digital scanned light-sheet microscopy, we characterized the generation of dense microvascular networks in collagen hydrogels and established parameters for quantification of perivascular recruitment. We also performed original time-lapse analysis of stem cell recruitment. These experiments demonstrated that perivascular recruitment of dental pulp stem cells is driven by PDGF-BB. Recruited stem cells participated in deposition of vascular basement membrane and vessel maturation. Mature microvascular networks thus generated were then compared to those lacking perivascular coverage generated using stem cell conditioned medium. Implantation in athymic nude mice demonstrated that in vitro maturation of microvascular networks improved blood perfusion and cell survival within the construct. Taken together, these data demonstrate the strong potential of in vitro production of mature microvasculature for improving cell-based therapies.

Growth-dependent phenotype in FasL-deficient mandibular/alveolar bone.

FASL (CD178) is known for its role in triggering apoptosis, mostly in relation with immune cells but additional functions have been reported more recently, including those in bone development. Examination of postnatal FasL-deficient mice (gld) showed an increased bone deposition in adult mice when compared with wild types. However, a different phenotype was observed prenatally, when the gld bone was underdeveloped. The aim of the following investigation was to evaluate this indication for an growth-dependent bone phenotype of gld mice and to search for the 'switch point'. This study focused on the mandibular/alveolar bone as an important structure for tooth anchorage. In vivo micro-computed tomography (CT) analysis was performed at different stages during the first month (6, 12 and 24 days) of postnatal bone development. In 6-day-old gld mice, a decrease in bone volume/tissue volume (BV/TV), trabecular thickness and trabecular number was revealed. In contrast, the 12-day-old gld mice showed an increased BV/TV and trabecular thickness in the alveolar bone. The same observation applied for bone status in 24-day-old gld mice. Therefore, changes in the bone phenotype occurred between day 6 and 12 of the postnatal development. The switch point is likely related to the changing proportion of bone cells at these stages of development, when the number of osteocytes increases. Indeed, the immunohistochemical analysis of FASL localized this protein in osteoblasts, whereas osteocytes were mostly negative at examined stages. The impact of FASL particularly on osteoblasts would agree with an earlier in vivo observed effect of FASL deficiency on expression of Mmp2, typical for osteoblasts, in the gld mandibular/alveolar bone. Notably, an age-dependent bone phenotype was reported in Mmp2-deficient mice.

Priming Dental Pulp Stem Cells from Human Exfoliated Deciduous Teeth with Fibroblast Growth Factor-2 Enhances Mineralization Within Tissue-Engineered Constructs Implanted in Craniofacial Bone Defects.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue-engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor-2 (FGF-2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF-2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF-2 priming. After implantation in immunodeficient mice, the tissue-engineered constructs seeded with FGF-2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF-2 priming in tissue engineering for craniofacial bone repair. Stem Cells Translational Medicine 2019;8:844&857.

Mouse Wnt1-CRE-RosaTomato Dental Pulp Stem Cells Directly Contribute to the Calvarial Bone Regeneration Process.

Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE- RosaTomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. Stem Cells 2019;37:701-711.

Early angiogenesis detected by PET imaging with 64Cu-NODAGA-RGD is predictive of bone critical defect repair.

Therapies using stem cells may be applicable to all fields of regenerative medicine, including craniomaxillofacial surgery. Dental pulp stem cells (DPSCs) have demonstrated in vitro and in vivo osteogenic and proangiogenic properties. The aim of the study was to evaluate whether early angiogenesis investigated by nuclear imaging can predict bone formation within a mouse critical bone defect. Two symmetrical calvarial critical-sized defects were created. Defects were left empty or filled with i) DPSC-containing dense collagen scaffold, ii) 5% hypoxia-primed DPSC-containing dense collagen scaffold, iii) acellular dense collagen scaffold, or iv) left empty. Early angiogenesis assessed by PET using 64Cu-NODAGA-RGD as a tracer was found to be correlated with bone formation determined by micro-CT within the defects from day 30, and to be correlated to the late calcium apposition observed at day 90 using 18F-Na PET. These results suggest that nuclear imaging of angiogenesis, a technique applicable in clinical practice, is a promising approach for early prediction of bone grafting outcome, thus potentially allowing to anticipate alternative regenerative strategies. STATEMENT OF SIGNIFICANCE: Bone defects are a major concern in medicine. As life expectancy increases, the number of bone lesions grows, and occurring complications lead to a delay or even lack of consolidation. Therefore, to be able to predict healing or the absence of scarring at early times would be very interesting. This would not "waste time" for the patient. We report here that early nuclear imaging of angiogenesis, using 64Cu-NODAGA-RGD as a tracer, associated with nuclear imaging of mineralization, using 18F-Na as a tracer, is correlated to late bone healing objectivized by classical histology and microtomography. This nuclear imaging represents a promising approach for early prediction of bone grafting outcome in clinical practice, thus potentially allowing to anticipate alternative regenerative strategies.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 RosaTomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.

Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.

EMMPRIN/CD147 deficiency disturbs ameloblast-odontoblast cross-talk and delays enamel mineralization.

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10±5µm thick (mean±SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition.

MEPE-derived ASARM peptide inhibits odontogenic differentiation of dental pulp stem cells and impairs mineralization in tooth models of X-linked hypophosphatemia.

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide - a substrate for PHEX and a strong inhibitor of mineralization - derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C.

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT(2B)R) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT(2B)R and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT(2B)R and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT(2B)R intrinsic activity or shRNA-mediated 5-HT(2B)R knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT(2B)R couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT(2B)R controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT(2B)R-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT(2B)R-null mice exhibit defects in TNAP activity.

Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells.

Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A-4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4).

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

UNLABELLED: The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. INTRODUCTION: Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. MATERIALS AND METHODS: C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. RESULTS AND CONCLUSIONS: Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.