The periodontal ligament (PDL) contains heterogeneous mesenchymal stem/progenitor cells that contribute to periodontal homeostasis and regeneration. However, the developmental origin and behavior of α-smooth muscle actin (αSMA)-positive cells remain poorly understood. This study investigated the spatiotemporal distribution of αSMA-positive cells during tooth development and their response to altered mechanical loading following extraction of the opposing mandibular molars in mice. Before tooth eruption, αSMA-positive cells were widely distributed throughout the dental follicles but became progressively restricted to the apical PDL during root formation and eruption. In contrast, c-kit immunoreactivity gradually disappeared from the apical PDL, whereas Gli1/tdTomato-positive cells partially co-localized with αSMA-positive cells. Following extraction of the opposing molars, the αSMA-positive area and the number of PCNA-positive cells transiently increased, while endomucin-positive blood vessels became dilated in the apical PDL. These findings demonstrate that αSMA-positive cells undergo dynamic developmental redistribution and rapidly respond to altered mechanical loading. Furthermore, the partial co-localization of αSMA and Gli1 suggests that at least a subset of αSMA-positive cells may represent a resident mechanosensitive mesenchymal progenitor population involved in periodontal homeostasis and remodeling, providing new insight into the biological basis of periodontal remodeling during orthodontic tooth movement and periodontal regeneration.

Skeletal stem/progenitor cells (SSPCs) play a fundamental role in maintaining skeletal homeostasis throughout life. Leptin receptor (Lepr)-positive cells were initially identified as SSPCs in the long bone marrow and later in the craniofacial skeleton, where they localize within the periodontal ligament (PDL), particularly among fibroblastic cells (PDLCs). However, their hierarchical organization, maintenance of stemness, and relationships with other SSPC populations remain poorly understood. Through lineage tracing and single-cell RNA sequencing facilitated by Runx2-GFP expression, we identified a Lepr+ subpopulation characterized by Thy1 expression but lacking Runx2, which is hierarchically positioned at the apex of the Lepr+ PDLC lineage and gives rise to downstream osteogenic progeny. However, their SSPC capacity to contribute to osteocytes and cementocytes is quantitatively limited and declines with age, and this contribution may exhibit sexual dimorphism. At the singlecell transcriptome level, Lepr+ PDLCs shared an expression profile with long bone-derived Lepr+ SSPCs; however, the expression of Kit ligand (Kitl) was uniquely restricted to Lepr+ cells within the PDLCs. In contrast, they were not enriched for other PDLC-associated SSPC markers, such as Axin2, Gli1, and Acta2. Although Lepr+ PDLCs contributed to bone regeneration by differentiating into osteocytes after tooth extraction, their contribution was limited. Moreover, genetic ablation of Lepr+ PDLCs did not reduce the regenerated bone volume, suggesting compensatory contributions from other SSPC populations. Collectively, these findings reveal a previously unrecognized hierarchical and functional organization of Lepr+ PDLCs and demonstrate that their contribution to skeletal homeostasis progressively declines with age, with potential compensation by other SSPC populations.

Background Alveolar ridge preservation (RP) is performed to reduce post-extraction alveolar bone resorption and support bone regeneration. Octacalcium phosphate collagen complex (OCP/Col) has osteoconductive potential; however, its effects on early vascular and osteogenic responses during extraction socket healing remain incompletely understood. This study investigated early bone formation, CD31/endomucin double-positive vessel distribution, and Osterix expression following RP with OCP/Col in mouse extraction sockets. Methods RP was performed after extraction of the lower first molars in C57BL/6J mice using OCP/Col, β-tricalcium phosphate (β-TCP), or Bio-Oss. Extraction-only sockets served as controls. Samples were collected on Days 1, 4, 7, and 14. Micro-computed tomography, bone morphometric analysis, histological assessment, and fluorescence immunohistochemistry were used to evaluate bone formation, CD31/endomucin double-positive vascular signals, and Osterix-positive cells. Results RP reduced alveolar bone resorption compared with extraction alone. On Day 14, bone volume/tissue volume and trabecular thickness were significantly greater in the OCP/Col group than in the other groups. Residual granules persisted in the Bio-Oss and β-TCP groups, whereas OCP/Col was associated with extensive new bone formation. CD31/endomucin double-positive vascular signals were observed throughout the healing period and were significantly more abundant in the OCP/Col group. Osterix-positive cells were frequently observed adjacent to these vascular structures and newly formed bone. Conclusions RP with OCP/Col was associated with reduced alveolar bone resorption, enhanced early bone formation, increased CD31/endomucin double-positive vascular signals, and Osterix-positive cells in mouse extraction sockets. These findings suggest that OCP/Col may provide a favorable microenvironment for early bone regeneration during extraction socket healing.

BACKGROUND:Leptin receptor (LepR)+ stromal cells are widely recognized as skeletal stem/progenitor cells (SSPCs) in the bone marrow that generate osteolineage cells, and contribute to skeletal homeostasis under steady-state conditions and tissue repair. By expressing representative niche factors, including stem cell factor and C-X-C motif chemokine ligand 12, LepR+ SSPCs function as key microenvironmental components of the hematopoietic stem cell (HSC) niche. Over the past decade, genetic cell-labeling studies have identified LepR+ SSPCs in craniofacial tissues, including in the periodontal ligament and alveolar bone marrow, where they differentiate into osteoblasts and cementoblasts. HIGHLIGHT:Emerging evidence indicates that periodontal LepR + cells contribute to orthodontic remodeling, periodontal repair, and extraction socket healing. Independent SSPC populations may coexist with LepR+ SSPCs in oral tissues and contribute in parallel to tissue homeostasis. Periodontal LepR+ cells also express HSC niche-associated factors, suggesting their possible role in the regulation of local immune and hematopoietic environments. Because oral tissues are continuously exposed to mechanical forces and the oral microbiome, LepR+ SSPCs in the oral environment may function through unique regulatory mechanisms that influence skeletal homeostasis and hematopoiesis. CONCLUSION:In this review, current knowledge regarding LepR+ SSPCs in periodontal tissues is summarized, and their emerging roles in skeletal maintenance, tissue repair, and hematopoietic regulation is discussed. Further elucidation of the regulatory mechanisms governing these cells will advance understanding of oral skeletal and hematopoietic biology, and may provide new insights into oral health and regenerative strategies.

Reduced masticatory muscle function during the growth period is implicated in the development of craniofacial deformities; however, its effects on skeletal development remain unclear. This study investigated systemic and local skeletal responses to reduced masticatory muscle function using botulinum neurotoxin type A (BTX) in growing mice. Four-week-old male mice were divided into three groups: a saline-injected Sham group; a BTX-injected group (both injected into the right masseter muscle); and an untreated control group. Blood samples, body weight, and mandibular deviation were assessed 2, 4, and 8 weeks after injection or control treatment. Mandibular morphology was evaluated using micro-computed tomography and histology. Masseter muscle weight was measured, and bulk RNA sequencing of the right mandibular condyle was performed. Body weight and masseter muscle weight were significantly lower in the BTX group throughout the experimental period. BTX treatment induced mandibular deviation and transient changes in condylar cartilage thickness and bone morphology, which became comparable to those of the Sham group at week 8. No significant differences in circulating bone metabolism markers were observed between the BTX and Sham groups. Transcriptome analysis indicated that genes associated with the canonical Wnt signaling pathway (GO:0044338), which is involved in mesenchymal stem cell differentiation, exhibited dynamic expression changes during recovery. These findings demonstrate that unilateral reduction of masseter muscle function during the growth phase induces transient mandibular deformation accompanied by histological, morphological, and gene expression changes, and they suggest that specific gene regulatory pathways may contribute to the regulation and recovery of jaw deformities.

Purpose Alveolar bone resorption after tooth extraction leads to gingival recession and invagination, which may result in the stalling of tooth movement and difficulties in orthodontic tooth movement. Bone substitutes used for alveolar ridge preservation should exhibit bioaffinity, appropriate initial calcification, ease of handling, replacement by bone, long-term stability, and formation of an abundant vascular network. Therefore, this study focused on ridge preservation (RP) using an octacalcium phosphate collagen composite (OCP/Col). Type H vessels are known to appear at sites of active bone formation, and osteoblast markers are observed in their vicinity, creating a microenvironment conducive to osteogenesis. The objective of this study was to investigate the distribution of newly formed bone and Type H vessels in extraction sockets following ridge preservation with OCP/Col, as well as the expression of Osterix. Methods RP of the lower first molars in mice was performed using OCP/Col. Bone mass, bone quality, and angiogenesis were analyzed by micro-computed tomography, bone morphometry, and histological and fluorescence immunohistochemical staining. Results In RP with OCP/Col, the granularity of the bone substitute tended not to be preserved, and immature woven bone formation was observed, which prevented alveolar bone resorption. A microcapillary vascular network including Type H vessels was formed, and osteoblast markers were induced in the surrounding area, promoting new bone formation. Conclusions These findings suggest that RP with OCP/Col promotes early new bone formation associated with Type H vessels and may be beneficial for orthodontic treatment.

The aim of this study was to elucidate the localization of Glioma-associated oncogene homolog 1–expressing (Gli1⁺) cells during the healing process of pulp revascularization. In addition, we investigated their contribution to newly formed mineralized tissue using lineage-tracing analysis. Five-week-old Gli1-CreERT2-Tomato mice underwent pulp revascularization in maxillary right first molars and were sacrificed at 1 h and 3, 7, 14, and 21 days postoperatively. At 3 days, co-localization of Gli1⁺ cells with osterix, a cementoblast marker, was observed, whereas nestin-positive odontoblast-lineage cells were not detected in the treated root canal. Because proliferative activity is an important component of early healing, Ki-67–immunoreactive cells were also examined. Ki-67–immunoreactive cells were present in the periapical tissues and within the root canal at 3 days, with partial overlap with Gli1⁺ cells, but no Ki-67–positive cells were found at 14 days. Periostin, a marker of periodontal ligament fibroblasts, was not expressed in the root canal or in regions populated by Gli1⁺ cells. These findings suggest that Gli1⁺ cells migrate from periapical tissues into the root canal and contribute to the formation of cementum-like mineralized tissue during wound healing after pulp revascularization.

Senescent cell accumulation in the bone marrow promotes inflammaging, hematopoietic stem cell dysfunction, and myeloid-biased hematopoiesis, contributing to the onset of age-related diseases. However, the mechanisms underlying the emergence of senescent cells in the bone marrow remain poorly understood. Here, we identify endothelial RANK signaling as a key regulator of bone marrow senescence. Young Tnfrsf11b (osteoprotegerin, OPG)-deficient mice exhibited premature accumulation of senescent cells and myeloid-biased hematopoiesis, phenocopying middle-aged wild-type mice. Endothelial-specific deletion of Tnfrsf11a (RANK), blockade of RANKL, and in vivo tracing with fluorescent-labelled RANKL revealed that RANK signaling in endothelial cells drives senescent cell accumulation in the bone marrow. Bulk and single-cell RNA sequencing, and IL-1β neutralization demonstrate that RANK signaling induces IL-1β expression in endothelial cells, which in turn promotes bone marrow inflammaging. These findings identify endothelial RANK as a critical initiator of bone marrow senescence, thereby driving inflammaging, a potential therapeutic target for age-related diseases.

The parotid papilla is the epithelial outlet through which the parotid duct opens into the oral cavity. Although the papilla is thought to open postnatally, its precise timing and morphogenetic events during the initial opening have not been anatomically defined. Sox9 is essential for salivary gland development; however, its role in parotid papilla formation and opening remains unclear. Here, we (i) determined when and how the parotid papilla opens after birth and (ii) tested whether Sox9 is required for this process. We analyzed C57BL/6 J mice at postnatal days (P) 2-6 and Sox9CreER;Sox9fl/+ mice following tamoxifen administration at P2. In wild-type mice, the parotid papilla opened into the oral cavity at P4. Immediately prior to opening (P2-P3), Sox9-high epithelial cells transiently accumulated within the papillary epithelium and rapidly decreased after opening. In Sox9CreER;Sox9fl/+ mice, papilla opening was absent at P5 and the papilla itself displayed marked hypoplasia. These findings demonstrate that Sox9 haploinsufficiency disrupts parotid papilla morphogenesis and impairs the formation of a patent salivary duct outlet, thereby providing an anatomical basis for the postnatal opening of the parotid papilla.

Bone is a multifunctional organ that provides structural support and hosts the bone marrow, a key site for hematopoiesis and systemic homeostasis. These dual features have long attracted the attention of both bone biologists and hematologists. Each field has pursued the identification of stem-like cells responsible for hard tissue formation and the regulatory microenvironment/niche that supports hematopoietic stem cells (HSCs), which give rise to all blood cell lineages. Converging advances in bone and hematopoietic biology have led to the identification of skeletal stem/progenitor cells (SSPCs), a multifunctional population that gives rise to osteolineage cells and serves as a principal component of the HSC niche. This landmark discovery was largely enabled by Cre/loxP-based genetic mouse models. Among them, the leptin receptor (LepR)-Cre system has become one of the most widely used tools in skeletal stem cell research worldwide. In this review, we summarize the historical background and recent advances in SSPC research, specifically LepR+ SSPCs, highlighting their function and lineage plasticity during development, adolescence, aging, and fracture healing. Advanced genetic labeling-based studies and single-cell transcriptomics unveiled the fate, dynamics and indispensible roles of LepR⁺ SSPCs under both homeostatic and pathological conditions.

Alveolar bone resorption following tooth extraction often poses challenges to orthodontic tooth movement. Conventional bone grafting materials used for alveolar ridge preservation (RP) are typically designed to achieve high bone strength; however, such properties may limit the dynamic bone remodeling required for efficient orthodontic tooth movement. This study investigated orthodontic tooth movement into RP sites treated with octacalcium phosphate collagen (OCP/Col) in a rat model. OCP/Col was applied to the extraction socket of first molars (n = 5 per group), followed by orthodontic movement of the second molars into the treated region. Tooth movement, root resorption, bone morphometric parameters (BMD and BV/TV), and angiogenesis were evaluated using micro-CT and histological analyses. The rate of tooth movement on Days 21-22 was significantly greater in the OCP/Col group than in the CTL group (0.0475 mm/day vs. 0.00375 mm/day, P < 0.05). Root resorption showed no statistically significant differences among groups (P = 0.62). Alveolar bone resorption in both width and height was significantly reduced in the OCP/Col group (P < 0.05), with lower resorption rates at Day 28 (width: 3.1% vs. 12.3%; height: 11.9% vs. 28.8%). Histological analysis demonstrated increased bone turnover and vascularization, with abundant Type H vessels being qualitatively observed on the tension side. These findings indicate that OCP/Col-based ridge preservation was associated with enhanced orthodontic tooth movement and reduced alveolar bone resorption without increased root resorption in this rat model. Further mechanistic and clinical studies are required to determine its translational relevance.

Objectives Temporomandibular joint osteoarthritis (TMJ-OA) involves cartilage degeneration and bone changes; however, the mechanisms of onset and progression are unclear. Recently, it has been shown that asporin, an extracellular matrix protein, increases as knee osteoarthritis progresses and suppresses the TGF-β pathway. However, the asporin levels in later stages of TMJ-OA, when cartilage destruction occurs, are not known. In this study, we examined the relationship between asporin and the pathological progression of TMJ-OA. Methods We constructed TMJ-OA models in 12-week-old male C57BL/6J mice by partial discectomy and evaluated these at 3 days and 2, 4, 8, 12, and 16 weeks postoperatively. Cartilage degeneration was evaluated using a modified Mankin scoring system after safranin O staining. The mRNA and protein expression of asporin, Tgfb1, and Smad2/3 were compared between experimental and sham-operated control mice using reverse-transcription polymerase chain reaction and immunohistochemical staining, respectively. Results Safranin O staining intensity increased at 8 weeks postoperatively, and partial cartilage loss was observed at 16 weeks postoperatively. The modified Mankin score was higher in the experimental group than in the control group, and peaked at 16 weeks postoperatively. Asporin mRNA and protein expression was higher in the experimental group at all time points. Smad2/3 mRNA expression was higher, whereas p-SMAD2/3 levels were lower in the experimental group.TGF-β levels were higher in the experimental group. Conclusion Asporin mRNA and protein levels are high in the early and late stages of TMJ-OA. Asporin may be a potential biomarker of TMJ-OA and a potential therapeutic target.

Ectopic calcification is an abnormal regenerative response occurring in various tissues following injury, surgery, or genetic mutations. However, its underlying mechanisms remain unclear. By comparing three macrophage depletion methods using clodronate liposome, Csf1r neutralizing antibody, and macrophage-specific Csf1r gene deletion (Csf1r cKO), we found that F4/80(+)Csf1r(-) macrophages were specifically increased in calcified muscles in notexin-injected mice and BaCl2-injected Csf1r cKO mice. Mechanistically, bone morphogenetic protein (BMP) signaling was found to contribute to ectopic calcification. Reanalysis of public single-cell sequencing data and lineage-tracing analysis using Cdh5 creERT2 mice revealed that endothelial-to-mesenchymal transition (EndoMT) is also a key contributor to ectopic calcification, as evidenced by the high expression of EndoMT-related markers in mesenchymal progenitor cells. Notably, the administration of BMP inhibitors reduced calcification and promoted muscle regeneration. Thus, F4/80(+)Csf1r(-) macrophages and BMP signals represent promising therapeutic targets for preventing ectopic calcifications triggered by trauma, burns, infections, or surgical interventions.

Odontoblasts are terminally differentiated cells that exhibit mechanosensitivity and mineralization capacity. Mechanosensitive ion channels such as Piezo1 are present in odontoblasts and are associated with their physiological functions via Ca2+ signaling. Both Ca2+ signals via Ca2+ influx from mechanosensitive ion channels and Ca2+ release from Ca2+ stores function as secondary messenger systems for various biological phenomena. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store that mobilizes intracellular Ca2+. Changes in Ca2+ concentration inside the ER are among the factors that cause ER stress. Perivascular cells are located around odontoblasts in the dental pulp. Although such formation indicates that perivascular cells interact with odontoblasts, their detailed profiles under developmental and pathological conditions remain unclear. In this study, we revealed that pericyte marker, neural/glial antigen 2 (NG2)-positive cells, in cell-rich zones (CZs) can differentiate into Piezo1-positive odontoblasts following genetic odontoblast depletion in mice, and modeled as odontoblast death after severe dentin injury and as reparative dentin formation. NG2-positive pericytes differentiated into odontoblasts faster than glial cells. To determine how NG2-positive cells differentiate into Piezo1-positive odontoblasts, we focused on the ER-stress sensor protein, activating transcription factor 6a (ATF6a). After genetic odontoblast depletion, NG2-positive cells regenerated in the odontoblast layer and were capable of acting as functional odontoblasts. In the presence of extracellular Ca2+, the application of a sarco/ER Ca2+-ATPase (SERCA) inhibitor, thapsigargin, known as an ER-stress inducer, increased the intracellular Ca2+ concentration in the odontoblast lineage cells (OLCs). The increase was significantly inhibited by the application of a pharmacologic Piezo1 inhibitor, indicating that ER stress by SERCA inhibition augmented Piezo1-induced responses in odontoblast progenitor cells. However, the physiological activation of Gq-coupled receptors by adenosine diphosphate did not induce Piezo1 activation. Gene silencing of ATF6a and/or NG2 impaired the mineralization of OLCs. Overall, ATF6a orchestrates the differentiation of NG2-positive pericytes into functional odontoblasts that act as sensory receptor cells and dentin-forming cells.

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury.

Bone tissue provides structural support for our bodies, with the inner bone marrow (BM) acting as a hematopoietic organ. Within the BM tissue, two types of stem cells play crucial roles: mesenchymal stem cells (MSCs) (or skeletal stem cells) and hematopoietic stem cells (HSCs). These stem cells are intricately connected, where BM-MSCs give rise to bone-forming osteoblasts and serve as essential components in the BM microenvironment for sustaining HSCs. Despite the mid-20th century proposal of BM-MSCs, their in vivo identification remained elusive owing to a lack of tools for analyzing stemness, specifically self-renewal and multipotency. To address this challenge, Cre/loxP-based cell lineage tracing analyses are being employed. This technology facilitated the in vivo labeling of specific cells, enabling the tracking of their lineage, determining their stemness, and providing a deeper understanding of the in vivo dynamics governing stem cell populations responsible for maintaining hard tissues. This review delves into cell lineage tracing studies conducted using commonly employed genetically modified mice expressing Cre under the influence of LepR, Gli1, and Axin2 genes. These studies focus on research fields spanning long bones and oral/maxillofacial hard tissues, offering insights into the in vivo dynamics of stem cell populations crucial for hard tissue homeostasis.

OBJECTIVE:Leptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells.DESIGN:Bone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers.RESULTS:Bone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly.CONCLUSION:LRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

Objectives: Factors that induce bone formation during orthodontic tooth movement (OTM) remain unclear. Gli1 was recently identified as a stem cell marker in the periodontal ligament (PDL). Therefore, we evaluated the mechanism of differentiation of Cre/LoxP-mediated Gli1/Tomato+ cells into osteoblasts during OTM. Methods: After the final administration of tamoxifen to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato mice for 2 days, nickel-titanium closed coil springs were attached between the upper anterior alveolar bone and the first molar. Immunohistochemical localizations of 8-catenin, Smad4, and Runx2 were observed in the PDL on 2, 5, and 10 days after OTM initiation. Results: In the untreated tooth, few Gli1/Tomato+ cells were detected in the PDL. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased in the PDL on the tension side. On this side, 49.3 +/- 7.0% of 8-catenin+ and 48.7 +/- 5.7% of Smad4+ cells were found in the PDL, and Runx2 expression was detected in some Gli1/Tomato+ cells apart from the alveolar bone. The number of positive cells in the PDL reached a maximum on day 5. In contrast, on the compression side, 8-catenin and Smad4 exhibited less immunoreactivity. On day 10, Gli1/Tomato+ cells were aligned on the alveolar bone on the tension side, with some expressing Runx2. Conclusions: Gli1+ cells in the PDL differentiated into osteoblasts during OTM. Wnt and bone morphogenetic proteins signaling pathways may be involved in this differentiation.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.