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Osteophytes in osteoarthritis (OA) joints contribute to restriction of joint movement, joint pain, and OA progression, but little is known about osteophyte regulators. Examination of gene expression related to cartilage extracellular matrix, endochondral ossification, and growth factor signaling in articular cartilage and osteophytes obtained from OA knee joints showed that several genes such as COL1A1, VCAN, BGLAP, BMP8B, RUNX2, and SOST were overexpressed in osteophytes compared with articular cartilage. Ratios of mesenchymal stem/progenitor cells, which were characterized by co-expression of CD105 and CD166, were significantly higher in osteophytic cells than articular cells. A three-dimensional culture method for cartilage and osteophyte cells was developed by modification of cultures of self-assembled spheroid cell organoids (spheroids). These spheroids cultured in the media for mesenchymal stem cells containing transforming growth factor-β3 showed characteristic morphologies and gene expression profiles of articular cartilage and osteophytes, respectively. The effects of IL-1β, tumor necrosis factor-α, and IL-6 on the spheroids of articular and osteophytic cells were studied. To the best of our knowledge, they provide the first evidence that IL-6 suppresses the spheroid size of osteophytic cells by inducing apoptosis and reducing extracellular matrix molecules. These data show that IL-6 is the suppressor of osteophyte growth and suggest that IL-6 expression and/or activity are implicated in the regulation of osteophyte formation in pathologic joints., Competing Interests: Disclosure Statement K.T. is an employee of Suntory Global Innovation Center, Ltd. Y.O. is an employee of Suntory Wellness, Ltd., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Histone hypermethylation represses gene transcription, which affects cartilage homeostasis or joint remodeling. Trimethylation of lysine 27 of histone 3 (H3K27me3) changes epigenome signatures, regulating tissue metabolism. This study aimed to investigate whether loss of H3K27me3 demethylase Kdm6a function affected osteoarthritis development. We revealed that chondrocyte-specific Kdm6a knockout mice developed relatively long femurs and tibiae as compared to wild-type mice. Kdm6a deletion mitigated osteoarthritis symptoms, including articular cartilage loss, osteophyte formation, subchondral trabecular bone loss, and irregular walking patterns of destabilized medial meniscus-injured knees. In vitro, loss of Kdm6a function compromised the loss in expression of key chondrocyte markers Sox9, collagen II, and aggrecan and improved glycosaminoglycan production in inflamed chondrocytes. RNA sequencing showed that Kdm6a loss changed transcriptomic profiles, which contributed to histone signaling, NADPH oxidase, Wnt signaling, extracellular matrix, and cartilage development in articular cartilage. Chromatin immunoprecipitation sequencing uncovered that Kdm6a knockout affected H3K27me3 binding epigenome, repressing Wnt10a and Fzd10 transcription. Wnt10a was, among others, functional molecules regulated by Kdm6a. Forced Wnt10a expression attenuated Kdm6a deletion-induced glycosaminoglycan overproduction. Intra-articular administration with Kdm6a inhibitor GSK-J4 attenuated articular cartilage erosion, synovitis, and osteophyte formation, improving gait profiles of injured joints. In conclusion, Kdm6a loss promoted transcriptomic landscapes contributing to extracellular matrix synthesis and compromised epigenetic H3K27me3-mediated promotion of Wnt10a signaling, preserving chondrocytic activity to attenuate osteoarthritic degeneration. We highlighted the chondroprotective effects of Kdm6a inhibitor for mitigating the development of osteoarthritic disorders., Competing Interests: Conflict of interest The authors have no competing financial interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The literature implies that large enthesophytes are exclusive to genetically predisposed individuals and to Spondyloarthropathies sufferers. Accordingly, the aim of this investigation and report was to assess the involvement of genetic predisposition, inflammatory and/or mechanical influences in the development of large enthesophytes in a sample population of teenagers presenting with large enthesophytes emanating from the external occipital protuberance., Methods: Analysis was based on four teenage males (13-16 year-old) possessing 14.5-30.5 mm enthesophytes projecting from the external occipital protuberance. This study included assessment of radiographs, MRI scans, blood-work, history, the SF-36 health survey, and the comparison of these data with the relevant literature to describe the interrelationships between the presence of enlarged external occipital protuberance, forward head protraction, active inflammation and/or genetic factors., Findings: Known genetic markers (e.g. HLA-B27) were not detected by allele-specific primers and both ESR and CRP tests were negative. Additionally, MRI analyses failed to detect active localised inflammation at the external occipital protuberance and surrounding structures. The health survey yielded normal parameters for all participants. All participants displayed significantly large Forward Head Protraction values (>40 mm), and interviews with participants and their parents indicated that concerns related to posture were prevalent since early childhood., Interpretation: This report suggests that mechanical load has an important role in enthesophyte development, irrespective the involvement of inflammatory or genetic factors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Osteophyte is one of the inevitable consequences of progressive osteoarthritis with the main characteristics of cartilage degeneration and endochondral ossification. The pathogenesis of osteophyte formation is not fully understood to date. In this work, metabolomic approaches were employed to explore potential mechanisms of osteophyte formation by detecting metabolic variations between extracts of osteophyte cartilage tissues (n = 32) and uninvolved control cartilage tissues (n = 34), based on the platform of ultraperformance liquid chromatography tandem quadrupole time-of-flight mass spectrometry, as well as the use of multivariate statistic analysis and univariate statistic analysis. The osteophyte group was significantly separated from the control group by the orthogonal partial least-squares discriminant analysis models, indicating that metabolic state of osteophyte cartilage had been changed. In total, 28 metabolic variations further validated by mass spectrum (MS) match, tandom mass spectrum (MS/MS) match, and standards match mainly included amino acids, sulfonic acids, glycerophospholipids, and fatty acyls. These metabolites were related to some specific physiological or pathological processes (collagen dissolution, boundary layers destroyed, self-restoration triggered, etc.) which might be associated with the procedure of osteophyte formation. Pathway analysis showed phenylalanine metabolism (PI = 0.168, p = 0.004) was highly correlative to this degenerative process. Our findings provided a direction for targeted metabolomic study and an insight into further reveal the molecular mechanisms of ostophyte formation.

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG-2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene-knockout model., Methods: Maturational changes were evaluated histologically in the knees of 3-, 6-, and 12-week-old wild-type (WT) mice and Hspg2(Δ3-/Δ3-) mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2(Δ3-/Δ3-) mice. Changes in cartilage expression of FGF-2, FGF-18, HSPG-2, FGF receptor 1 (FGFR-1), and FGFR-3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin-1α (IL-1α), FGF-2, and FGF-18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified., Results: No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2(Δ3-/Δ3-) mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA-induced loss of chondrocyte expression of FGF-2, FGF-18, and HSPG-2 occurred in both genotypes. Expression of FGFR-1 after OA induction was maintained in WT mice, while FGFR-3 loss after OA induction was significantly reduced in Hspg2(Δ3-/Δ3-) mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL-1α-stimulated cartilage. However, IL-1α-induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2(Δ3-/Δ3-) mice. Cartilage GAG release in either the presence or absence of IL-1α was unaltered by FGF-2 in both genotypes. In cartilage cultures with FGF-18, IL-1α-stimulated GAG loss was significantly reduced only in Hspg2(Δ3-/Δ3-) mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA., Conclusion: Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR-3 and increased FGF signaling., (© 2016, American College of Rheumatology.)

Objective: To identify the molecular differences between the transient and permanent chondrocyte phenotype in osteophytic and articular cartilage., Methods: Total RNA was isolated from the cartilaginous layer of osteophytes and from intact articular cartilage from knee joints of 15 adult human donors and subjected to cDNA microarray analysis. The differential expression of relevant genes between these two cartilaginous tissues was additionally validated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry., Results: Among 47,000 screened transcripts, 600 transcripts were differentially expressed between osteophytic and articular chondrocytes. Osteophytic chondrocytes were characterized by increased expression of genes involved in the endochondral ossification process [bone gamma-carboxyglutamate protein/osteocalcin (BGLAP), bone morphogenetic protein-8B (BMP8B), collagen type I, alpha 2 (COL1A2), sclerostin (SOST), growth arrest and DNA damage-induced gene 45ß (GADD45ß), runt-related transcription factor 2 (RUNX2)], and genes encoding tissue remodeling enzymes [matrix metallopeptidase (MMP)9, 13, hyaluronan synthase 1 (HAS1)]. Articular chondrocytes expressed increased transcript levels of antagonists and inhibitors of the BMP- and Wnt-signaling pathways [Gremlin-1 (GREM1), frizzled-related protein (FRZB), WNT1 inducible signaling pathway protein-3 (WISP3)], as well as factors that inhibit terminal chondrocyte differentiation and endochondral bone formation [parathyroid hormone-like hormone (PTHLH), sex-determining region Y-box 9 (SOX9), stanniocalcin-2 (STC2), S100 calcium binding protein A1 (S100A1), S100 calcium binding protein B (S100B)]. Immunohistochemistry of tissue sections for GREM1 and BGLAP, the two most prominent differentially expressed genes, confirmed selective detection of GREM1 in articular chondrocytes and that of BGLAP in osteophytic chondrocytes and bone., Conclusions: Osteophytic and articular chondrocytes significantly differ in their gene expression pattern. In articular cartilage, a prominent expression of antagonists inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, osteophytic chondrocytes express genes with roles in the endochondral ossification process, which may account for their transient phenotype., (Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism., Methods: Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR., Results: Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage., Conclusions: The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Ventrodorsal extended hip radiographs were analyzed from Foxhounds, Irish setters, Greyhounds, and Labrador retrievers radiographed four to seven times between 8 and 110 weeks of age. Occurrence in these 91 dogs of a puppy line, an ill-defined zone of proximal femoral metaphyseal sclerosis, a femoral neck linear sclerosis, or circumferential linear femoral head osteophytosis at 15-17 weeks of age were compared with hip joint laxity, as measured by distraction index, and to later findings of caudal curvilinear femoral neck osteophytes, circumferential femoral head osteophytes, hip incongruity consistent with hip dysplasia and degenerative joint disease by 52 weeks of age. A puppy line and/or femoral metaphyseal sclerosis was common at 15-17 weeks of age for dogs at mimimal risk (Greyhounds) and high risk (Foxhounds) of developing early degenerative joint disease associated with canine hip dysplasia. Though 44% of Greyhound hips had puppy lines and 28% had femoral metaphyseal sclerosis at 15-17 weeks of age, no Greyhound had a caudolateral curvilinear osteophyte or circumferential femoral head osteophyte at 24-27 or 52 weeks of age. No significant relationship was found between occurrence of a puppy line, a circumferential femoral head osteophyte or femoral metaphyseal sclerosis at 15-17 weeks and canine hip dysplasia or degenerative joint disease incidence at 42-52 weeks. Presence of a caudolateral curvilinear osteophyte in at least one hip at 24-27 weeks was significantly related to the diagnosis of canine hip dysplasia by 42-52 weeks. When both a caudolateral curvilinear osteophyte and a circumferential femoral head osteophyte were present in a hip at 24-27 weeks, degenerative joint disease was evident in all such hips by 42-52 weeks of age.

Osteophytes are a distinct feature of osteoarthritis (OA). Their formation may be related to pluripotential cells in the periosteum responding to stimulus during OA. This study aimed to isolate stem cells from osteophyte tissues and to characterize their phenotype, proliferation, and differentiation potential, as well as their immunomodulatory properties. Osteophyte-derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterized by the expression of cell-surface antigens, differentiation potential into mesenchymal lineages, growth kinetics, and modulation of alloimmune responses. Multipotential stem cells were identified from all osteophyte samples, namely osteophyte-derived mesenchymal stem cells (oMSCs). The surface antigen expression of oMSCs was consistent with that of MSCs; they lacked the hematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSCs in culture was superior to that of bone marrow-derived MSCs (bMSCs), and these cells readily differentiated into tissues of the mesenchymal lineages. oMSCs also demonstrated the ability to suppress allogeneic T cell proliferation, which was associated with the expression of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). Our results showed that osteophyte-derived cells had similar properties to MSCs in the expression of antigen phenotype, differential potential, and suppression of alloimmune response. Furthermore, when compared to bMSCs, oMSCs maintained a higher proliferative capacity, which may offer new insights of the tissue formation and potentially an alternative source for therapeutic stem cell-based tissue regeneration.

Objective: The aims of the present study were (1) to evaluate whether development of enthesophytes is an age- and/or sex-associated phenomenon; (2) to clarify whether enthesophyte development is controlled by genetics; (3) to evaluate the correlations between the enthesophytes and osteophytes of the hand joints., Design: The studied cohort comprised 359 Chuvashian (Russian Federation) pedigrees (424 nuclear families) and included 786 males and 723 females aged 18-90 years. The enthesophyte score (ES) was constructed as the overall number of enthesophytes at the midshaft of the phalanges of the second to the fifth fingers of both hands. The osteophyte score (OS) was constructed similarly. We used variance component (VC) analysis to examine the age-related patterns and compare the contribution of the genetic and common environmental factors to ES and OS variations., Results and Conclusions: After age 25, ES increases with age (on average linearly). Age explains 45% of the ES variation in males but only 25% of the variation in females, in contrast to about 75% of the variation of OS in both sexes. At any age, males showed higher ES than females and the difference between sexes increased with age. Genetic components explained 20% of enthesophyte development variation. We did not find common additive genetic factors for ES and OS. The correlation coefficients between ES and OS were r=0.62 (P=0.0001) in males and r=0.50 (P=0.0001) in females. After age adjustment, the correlation decreased to r=0.087 (P=0.014) and r=0.14 (P=0.001) correspondingly. Most probably, enthesophytes and osteophytes are manifestations of different etiological processes.

Objective: To investigate the gene expression and potential function of insulinlike-Iin osteophyte development., Methods: Specimens were obtained from 25 individuals undergoing total knee arthroplasty due to severe primary osteoarthritis and from 3 healthy adults. The tissue samples were embedded in paraffin wax and made into sections to undergo HE and toluidine blue staining. Immunohistochemistry was used to detect the expression of collagen I, IIa, IIb, and X. The osteophytic tissues were grouped into different types based on the results by histomorphology and expression of different collagens and glycosaminoglycans. Immunohistochemistry and in situ hybridization were used to detect the expression of insulinlike growth factor-I in the osteophyte. The images obtained by imunohistochemistry and in situ hybridization were put into the image analysis system to be analyzed semi-quantitatively., Results: Based on the characteristics of histomorphology and expression of different collagens and glycosaminoglycans, the osteophytic tissues were grouped into five different types. The type I tissue mainly consists of mesenchymal fibroblast-like cells producing collagen I. The type II tissue was characterized by the existence of collagen IIA expressed in prechondrocyte and glycosaminoglycans in the extracellular mesenchyma. he type III tissue was marked by the co-expression of collagen IIB, glycosaminoglycans, collagen IIA, and collagen I. The type IV tissue showed a clear zonal organization of chondrocytes, abundant expression of collagen IIB and glycosaminoglycans, and hypertrophic deep chondrocytes expressing Collagen X. The type V tissue had a composition similar to that of the normal articular hyaline cartilage with a predominance of collagen IIB and abundant glycosaminoglycans. These five types seemed to indicate the different stages of osteophyte development: mesenchymal cell, prechondrocyte, chondrocyte, early osteophyte and mature osteophyte. Immunohistochemistry showed that the expression level of insulinlike growth factor-I in the type I tissue was 14.6 +/- 3.8, significantly higher than those in the types IV and V tissues (7.7 +/- 3.5 and 6.0 +/- 1.9 respectively, both P = 0.000); and the expression level of insulinlike growth factor-I in the type II tissue was 13.6 +/- 4.7, significantly higher than those in the types IV and V tissues (both P = 0.000). The results of in situ hybridization were similar to those by imunohistochemistry., Conclusion: Insulinlike growth factor-I mRNA is mainly expressed in the early stage osteophyte, and it may play an important role in initiation of the chondrogenic differentiation of mesenchymal progenitor cells in the early stage of osteophyte development.