A Narrative Review of the Roles of Chondromodulin-I (Cnmd) in Adult Cartilage Tissue.

Articular cartilage is crucial for joint function but its avascularity limits intrinsic repair, leading to conditions like osteoarthritis (OA). Chondromodulin-I (Cnmd) has emerged as a key molecule in cartilage biology, with potential implications for OA therapy. Cnmd is primarily expressed in cartilage and plays an important role in chondrocyte proliferation, cartilage homeostasis, and the blocking of angiogenesis. In vivo and in vitro studies on Cnmd, also suggest an involvement in bone repair and in delaying OA progression. Its downregulation correlates with OA severity, indicating its potential as a therapeutic target. Further research is needed to fully understand the mode of action of Cnmd and its beneficial implications for managing OA. This comprehensive review aims to elucidate the molecular characteristics of Cnmd, from its expression pattern, role in cartilage maintenance, callus formation during bone repair and association with OA.

Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.

Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.

A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis.

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10(-13); odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS.

Scleraxis and osterix antagonistically regulate tensile force-responsive remodeling of the periodontal ligament and alveolar bone.

The periodontal ligament (PDL) is a mechanosensitive noncalcified fibrous tissue connecting the cementum of the tooth and the alveolar bone. Here, we report that scleraxis (Scx) and osterix (Osx) antagonistically regulate tensile force-responsive PDL fibrogenesis and osteogenesis. In the developing PDL, Scx was induced during tooth eruption and co-expressed with Osx. Scx was highly expressed in elongated fibroblastic cells aligned along collagen fibers, whereas Osx was highly expressed in the perialveolar/apical osteogenic cells. In an experimental model of tooth movement, Scx and Osx expression was significantly upregulated in parallel with the activation of bone morphogenetic protein (BMP) signaling on the tension side, in which bone formation compensates for the widened PDL space away from the bone under tensile force by tooth movement. Scx was strongly expressed in Scx(+)/Osx(+) and Scx(+)/Osx(-) fibroblastic cells of the PDL that does not calcify; however, Scx(-)/Osx(+) osteogenic cells were dominant in the perialveolar osteogenic region. Upon BMP6-driven osteoinduction, osteocalcin, a marker for bone formation was downregulated and upregulated by Scx overexpression and knockdown of endogenous Scx in PDL cells, respectively. In addition, mineralization by osteoinduction was significantly inhibited by Scx overexpression in PDL cells without affecting Osx upregulation, suggesting that Scx counteracts the osteogenic activity regulated by Osx in the PDL. Thus, Scx(+)/Osx(-), Scx(+)/Osx(+) and Scx(-)/Osx(+) cell populations participate in the regulation of tensile force-induced remodeling of periodontal tissues in a position-specific manner.

THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.

[Homeostasis and Disorder of Musculoskeletal System.Enthesis formation and repair:Current understanding and perspectives for the future regenerative therapy.]

Tendons and ligaments are dense fibrous connective tissues mainly composed of type I collagen, aligned in highly ordered arrays along the axis of the tendon and ligament. The enthesis is defined as the attachment site of a tendon, ligament, joint capsule, or fascia to bone. During morphogenesis, the cell population co-expressing Scleraxis(Scx)and the SRY-box containing gene 9(Sox9)contributes to the formation of fibrocartilaginous entheses. Scx regulates tendon and ligament maturation, while Sox9 is a key regulatory factor for cartilage formation. The considerable mechanical forces transmitted through the enthesis and avascular properties of the tissue make it more prone to injuries and degenerative changes. Thus, integration of tendons or ligaments with bone following surgical repair remains a clinical challenge. In this review, we summarize the current knowledge regarding the formation, maintenance, damage, and repair of fibrocartilaginous entheses, focusing on the rotator cuff tendon-to-bone attachment sites.

Scx+/Sox9+ progenitors contribute to the establishment of the junction between cartilage and tendon/ligament.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx(+)/Sox9(+) progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx(+) domain revealed that Scx(+) progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. Tenocytes are derived from Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx(+)/Sox9(+) progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx(+)/Sox9(+) progenitors. Conditional inactivation of Sox9 in Scx(+)/Sox9(+) cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx(+)/Sox9(+) progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.

Pax1 acts as a negative regulator of chondrocyte maturation.

Paired box gene 1 (Pax1) indirectly promotes the early stages of chondrogenic differentiation through induction and transactivation of Nk3 homeobox 2 (Nkx3.2), a transcriptional repressor. Later in chondrogenic differentiation, Nkx3.2 blocks chondrocyte hypertrophy by repressing Runt-related transcription factor 2 (Runx2). Here we report the inhibitory action of Pax1 on chondrocyte maturation, independently of Nkx3.2. Upon cartilage formation, Pax1 expression in the ventral sclerotome was gradually decreased except for the perichondrial region of the vertebral bodies and the intervertebral region, both of which express SRY-box containing gene 9 (Sox9). Forced expression of Pax1 in the chick forelimb resulted in the formation of shortened skeletal elements with a significant reduction of proteoglycans (PGs) accumulation in cartilage as well as a lack of the cortical bone formation and vascular invasion into the primary ossification center. Pax1-misexpressing chondrocytes exhibited aberrant cell morphology with a marked downregulation of Aggrecan (Agc1). Pax1-misexpressing cultured chondrocytes failed to accumulate cartilaginous PGs and became fibroblastic, in association with downregulation of the expression of Sox9, Nkx3.2, Indian hedgehog (Ihh), type II collagen (Col2a1), Chondromodulin-1 (Chm1), and Agc1. Accumulation of cartilaginous PGs in chondrocytes was also reduced by forced expression of Pax1 and Sox9. Thus, chondrocyte maturation driven by Sox9 is antagonized by Pax1 that is downregulated during chondrogenic differentiation.

Generation and characterization of ScxCre transgenic mice.

Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is a marker for the tendon/ligament cell lineage. The ∼11 kb genomic region from the mouse Scx gene locus faithfully recapitulates the endogenous Scx expression pattern in ScxGFP transgenic (Tg) mice. We have established two Tg mouse lines expressing Cre-recombinase (Cre) using this regulatory region (ScxCre-L and ScxCre-H). The specificity and efficiency of Cre recombination in these Tg lines are evaluated by crossing with Rosa-CAG-LSL-tdTomato (Ai14) or ROSA26R (R26R) reporter mice. The recombination in ScxCre-H;Ai14 mice is efficiently achieved in the endogenous Scx expression domains including the branchial arches, the syndetome, and the lateral plate mesoderm. Further analysis of ScxCre-H;Ai14;ScxGFP embryos reveal that expression of the ScxGFP transgene largely overlaps with Cre activity detected by tdTomato at embryonic day 12.5 (E12.5). In ScxCre-L;R26R or ScxCre-H;R26R neonates, Cre activity is detected in tendons, ligaments, intervertebral discs, joints, and cartilage around the chondro-tendinous/ligamentous junction, the prospective enthesis. The present results suggest that ScxCre Tg lines are useful for targeting the gene specifically in the Scx-expressing domains.

Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis.

The avascularity of cardiac valves is abrogated in several valvular heart diseases (VHDs). This study investigated the molecular mechanisms underlying valvular avascularity and its correlation with VHD. Chondromodulin-I, an antiangiogenic factor isolated from cartilage, is abundantly expressed in cardiac valves. Gene targeting of chondromodulin-I resulted in enhanced Vegf-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography showed aortic valve thickening, calcification and turbulent flow, indicative of early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation and mobilization of endothelial cells and induced their apoptosis; these effects were partially inhibited by chondromodulin-I small interfering RNA. In human VHD, including cases associated with infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in areas of chondromodulin-I downregulation. These findings provide evidence that chondromodulin-I has a pivotal role in maintaining valvular normal function by preventing angiogenesis that may lead to VHD.

Direct conversion of tenocytes into chondrocytes by Sox9.

Sox9 is a high-mobility group box-containing transcription factor that functions as a key regulator of chondrogenesis. We here report that Sox9 mediates the direct conversion of tenocytes to chondrocytes through an intermediate state in which both differentiation programs are active. Sox9 is abundantly expressed in cartilage but is undetectable in limb tendons that express Scleraxis (Scx) and Tenomodulin (Tnmd), tendon-specific early and late molecular markers, respectively. Upon forced expression of Sox9 in the chick forelimb, ectopic cartilage formation is preferentially observed in fibrous tissues including the tendons, ligaments, perichondrium/periosteum, dermis, and muscle connective tissues. Tnmd expression in tenocytes isolated from leg tendons was markedly upregulated by forced expression of basic helix-loop-helix (b-HLH) activators including Scx, Paraxis, Twist1 and Twist2. In contrast, the overexpression of Sox9 in monolayer tenocytes resulted in the downregulation of Tnmd and Scx expressions during passaging in culture, and the induction of cartilage molecular markers such as type II collagen (Col2a1) and Chondromodulin-I (ChM-I). This Sox9-driven switching from a tenocytic to a chondrocytic gene expression profile was associated with a dramatic change from a spindle to a polygonal cellular morphology. The extracellular accumulation of cartilage-characteristic proteoglycans was also observed. These data suggest that tenocytes have a strong potential for conversion into chondrocytes through the activities of Sox9 both in vitro and in vivo.

Chondromodulin is necessary for cartilage callus distraction in mice.

Chondromodulin (Cnmd) is a glycoprotein known to stimulate chondrocyte growth. We examined in this study the expression and functional role of Cnmd during distraction osteogenesis that is modulated by mechanical forces. The right tibiae of the mice were separated by osteotomy and subjected to slow progressive distraction using an external fixator. In situ hybridization and immunohistochemical analyses of the lengthened segment revealed that Cnmd mRNA and its protein in wild-type mice were localized in the cartilage callus, which was initially generated in the lag phase and was lengthened gradually during the distraction phase. In Cnmd null (Cnmd-/-) mice, less cartilage callus was observed, and the distraction gap was filled by fibrous tissues. Additionally, radiological and histological investigations demonstrated delayed bone consolidation and remodeling of the lengthened segment in Cnmd-/- mice. Eventually, Cnmd deficiency caused a one-week delay in the peak expression of VEGF, MMP2, and MMP9 genes and the subsequent angiogenesis and osteoclastogenesis. We conclude that Cnmd is necessary for cartilage callus distraction.

Synthetic disulfide-bridged cyclic peptides mimic the anti-angiogenic actions of chondromodulin-I.

Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein in cartilage matrix that inhibits angiogenesis. It contains two distinctive structural domains: the N-terminal third of the molecule is a hydrophilic domain that contains O-linked and N-linked oligosaccharide chains, and the C-terminal two-thirds is a hydrophobic domain that contains all of the cysteine residues. In the present study, we have attempted to further uncover the structural requirements for ChM-I to exert anti-angiogenic activity by monitoring its inhibition of the vascular endothelial growth factor (VEGF)-A-induced migration of HUVEC in vitro. Site-directed mutagenesis experiments revealed that the cyclic structure formed by the disulfide bridge between Cys(83) and Cys(99) in human ChM-I is indispensable for its anti-angiogenic function. Moreover, the C-terminal hydrophobic tail (from Trp(111) to Val(120) ) was found to play an important role in ensuring the effectiveness of ChM-I activity on HUVEC. A synthetic cyclic peptide corresponding to the ChM-I region between Ile(82) to Arg(100) also inhibited the migration of HUVEC, while replacing the Cys(83) and Cys(99) residues in this peptide with Ser completely negated this inhibitory activity. An additional synthetic cyclic peptide harboring the hydrophobic C-terminal tail of ChM-I clearly mimicked the inhibitory action of this protein on the migration of HUVEC and successfully inhibited tumor angiogenesis and growth in a xenograft mouse model of human chondrosarcoma.

Factors contributing to changes in viscosity and flow rate of a dedicated gel for gel immersion endoscopy.

Background and study aims Gel immersion endoscopy is a novel technique for securing the visual field during endoscopy. Clinical application of a dedicated gel for this technique with an appropriate viscosity to prevent mixing with blood and its efficacy was reported. The aim of this study was to evaluate changes in gel viscosity and flow rate under different conditions. Methods The viscosity of the gel after injection and flow rate were measured under various conditions changing the injection route and method. Gel viscosity was measured at 25, 10, and 4 °C. Results A decrease in gel viscosity was found when the gel was injected via the water jet channel compared to the accessory channel. The flow rate and decrease in viscosity of the gel injected via the water jet channel were 220 mL/min and 63.2 %, while when injected via the accessory channel with a clip device inserted were 560 mL/min and 35.8 %. When the gel was kept at a low temperature, the viscosity increased. Conclusions Gel injection via the accessory channel should be the first choice for efficient use considering the viscosity and flow rate.

Localization of chondromodulin-I at the feto-maternal interface and its inhibitory actions on trophoblast invasion in vitro.

BACKGROUND: Chondromodulin-I (ChM-I) is an anti-angiogenic glycoprotein that is specifically localized at the extracellular matrix of the avascular mesenchyme including cartilage and cardiac valves. In this study, we characterized the expression pattern of ChM-I during early pregnancy in mice in vivo and its effect on invasion of trophoblastic cells into Matrigel in vitro. RESULTS: Northern blot analysis clearly indicated that ChM-I transcripts were expressed in the pregnant mouse uterus at 6.5-9.5 days post coitum. In situ hybridization and immunohistochemistry revealed that ChM-I was localized to the mature decidua surrounding the matrix metalloproteinase-9 (MMP-9)-expressing trophoblasts. Consistent with this observation, the expression of ChM-I mRNA was induced in decidualizing endometrial stromal cells in vitro, in response to estradiol and progesterone. Recombinant human ChM-I (rhChM-I) markedly inhibited the invasion through Matrigel as well as the chemotactic migration of rat Rcho-1 trophoblast cells in a manner independent of MMP activation. CONCLUSIONS: This study demonstrates the inhibitory action of ChM-I on trophoblast migration and invasion, implying the potential role of the ChM-I expression in decidual cells for the regulated tissue remodeling and angiogenesis at feto-maternal interface.

A functional role of the glycosylated N-terminal domain of chondromodulin-I.

Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein that specifically localizes in the extracellular matrix of cartilage and negatively regulates angiogenesis. ChM-I comprises two domains: an N-terminal hydrophilic domain (domain 1) containing an N-linked glycosylation site and a C-terminal hydrophobic domain (domain 2) with all four disulfide bonds that are present in this protein. We generated a nonglycosylated recombinant human ChM-I (NG-hChM-I) and compared its bioactivity with that of the glycosylated form of human ChM-I (G-hChM-I) expressed in Chinese hamster ovary cells in vitro. NG-hChM-I exhibited the growth factor/inhibitor activity in the cultures of chondrocytes and vascular endothelial cells but required markedly higher doses. Although domain 1 is predicted to be hydrophilic per se on the basis of its amino acid sequence, NG-hChM-I remains insoluble in aqueous solution as much as ΔN-hChM-I that lacks the N-terminal 37 amino acids containing an N-glycosylation site. Circular dichroism measurements revealed that the content of α-helix was calculated to be 34% in G-hChM-I, whereas the content of the characteristic secondary structures in NG-hChM-I was distinctly lower than those in G-hChM-I. These results indicate that glycosylation in domain 1 is critical for the structural integrity for biological functions of ChM-I in vitro.

Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by chondromodulin-I, a cartilage-derived angiogenesis inhibitor.

Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.

Development of a gel dedicated to gel immersion endoscopy.

Backgrounds and study aims Gel immersion endoscopy is a novel technique to secure the visual field during endoscopy. The aim of this study was to develop a dedicated gel for this technique. Methods To identify appropriate viscoelasticity and electrical conductivity, various gels were examined. Based on these results, the dedicated gel "OPF-203" was developed. Efficacy and safety of OPF-203 were evaluated in a porcine model. Results In vitro experiments showed that a viscosity of 230 to 1900 mPa·s, loss tangent (tanδ) ≤ 0.6, and hardness of 240 to 540 N/cm 2 were suitable. Ex vivo experiments showed electrical conductivity ≤ 220 µS/cm is appropriate. In vivo experiments using gastrointestinal bleeding showed that OPF-203 provided clear visualization compared to water. After electrocoagulation of gastric mucosa in OPF-203, severe coagulative necrosis was not observed in the muscularis but limited to the mucosa. Conclusions OPF-203 is useful for gel immersion endoscopy.

Scleraxis upregulated by transforming growth factor-ß1 signaling inhibits tension-induced osteoblast differentiation of priodontal ligament cells via ephrin A2.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-ß1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-ß receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-ß1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.