Chondrocyte Differentiation in Human Osteoarthritis: Expression of Osteocalcin in Normal and Osteoarthritic Cartilage and Bone

Osteocalcin (OC), which is a marker of the mature osteoblasts, can also be found in posthypertrophic chondrocytes of the epiphyseal growth plate, but not in chondrocytes of the resting zone or in adult cartilage. In human osteoarthritis (OA), chondrocytes can differentiate to a hypertrophic phenotype characterized by type X collagen. The protein- and mRNA-expression pattern of OC was systematically analyzed in decalcified cartilage and bone sections and nondecalcified cartilage sections of human osteoarthritic knee joints with different stages of OA to investigate the differentiation of chondrocytes in OA. In severe OA, we found an enhanced expression of the OC mRNA in the subchondral bone plate, demonstrating an increased osteoblast activity. Interestingly, the OC protein and OC mRNA were also detected in osteoarthritic chondrocytes, whereas in chondrocytes of normal adult cartilage, both the protein staining and the specific mRNA signal were negative. The OC mRNA signal increased with the severity of OA and chondrocytes from the deep cartilage layer, and proliferating chondrocytes from clusters showed the strongest signal for OC mRNA. In this late stage of OA, chondrocytes also stained for alkaline phosphatase and type X collagen. Our results clearly show that the expression of OC in chondrocytes correlates with chondrocyte hypertrophy in OA. Although the factors including this phenotypic shift in OA are still unknown, it can be assumed that the altered microenvironment around osteoarthritic chondrocytes and systemic mediators could be potential inducers of this differentiation. Received: 20 May 1999 / Accepted: 10 February 2000     

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.     

ODDD‐Linked Cx43 Mutants Reduce Endogenous Cx43 Expression and Function in Osteoblasts and Inhibit Late Stage Differentiation

Introduction: Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD. Materials and Methods: We expressed human and mouse ODDD‐linked Cx43 mutants in MC3T3‐E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin. Results: We showed that ODDD‐linked Cx43 mutants are loss‐of‐function and dominant‐negative to co‐expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation. Conclusions: Expression of human and mouse ODDD‐linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43‐based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.     

Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) mutations have been identified as responsible for two arthritic disorders, multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). However, the function of COMP in chondrogenic differentiation is largely unknown. Our investigation focuses on analyzing the function of normal COMP protein in cartilage biology. METHODS AND RESULTS: To explore the function of COMP we make use of an in vitro model system for chondrogenesis, consisting of murine C3H10T1/2 mesenchymal cells maintained as a high-density micromass culture and stimulated with bone morphogenetic protein 2 (BMP-2). Under these culture conditions, C3H10T1/2 cells undergo active chondrogenesis in a manner analogous to that of embryonic limb mesenchymal cells, and have been shown to serve as a valid model system to investigate the mechanisms regulating mesenchymal chondrogenesis. Our results indicate that ectopic COMP expression enhances several early aspects of chondrogenesis induced by BMP-2 in this system, indicating that COMP functions in part to positively regulate chondrogenesis. Additionally, COMP has inhibitory effects on proliferation of cells in monolayer. However, at later times in micromass culture, ectopic COMP expression in the presence of BMP-2 causes an increase in apoptosis, with an accompanying reduction in cell numbers in the micromass culture. However, the remaining cells retain their chondrogenic phenotype. CONCLUSIONS: These data suggest that COMP and BMP-2 signaling converge to regulate the fate of these cells in vitro by affecting both early and late stages of chondrogenesis.     

Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotype.

Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies.     

Cartilage oligomeric matrix protein: Isolation and characterization from human articular cartilage

Cartilage oligomeric matrix protein was purified in a native form from normal adult human articular cartilage. The key steps in the purification scheme were selective extraction with buffer containing EDTA, wheat germ agglutinin affinity chromatography, and removal of the related protein thrombospondin by heparin affinity chromatography. Particles of cartilage oligomeric matrix protein viewed by electron microscopy after rotary shadowing revealed structures similar to the prototype molecule purified from Swarm rat chondrosarcoma. The protein demonstrated a bouquet-like five-armed structure, with peripheral globular domains connected by thin flexible strands to a central assembly domain. Immunohistochemistry revealed age-dependent differences in the protein's distribution in cartilage. In normal human adult articular cartilage, there was a relatively uniform distribution throughout the interterritorial extracellular matrix, whereas in fetal articular cartilage, immunostaining was localized to the extracellular matrix directly adjacent to the chondrocytes. The isolation and characterization of human cartilage oligomeric matrix protein will facilitate its study in pathological conditions of human cartilage.     

Localization of Pigment Epithelium-Derived Factor in Growing Mouse Bone

Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor found in a wide range of fetal and adult tissues, where it is thought to play a role in the regulation of angiogenesis during development. The temporal expression of PEDF during endochondral bone formation has not previously been reported. In this study, we analysed the expression pattern of PEDF in growing mouse hindlimbs from newborn day one through to maturation at week 9, using immunohistochemistry and in situ hybridization. PEDF expression was demonstrated in chondrocytes within the resting, proliferative and upper hypertrophic zones of the epiphyseal growth plate. The pattern of expression was consistent throughout the developmental stages of the mouse. In addition, PEDF was expressed by osteoblasts lining the bone spicules in the ossification zone of metaphyseal bone, as well as by osteoblasts lining cortical periosteum. These novel results demonstrate that PEDF is developmentally expressed in both cartilage and bone cells during endochondral bone formation, and strongly suggest that it may play a regulatory role in the processes of chondrocyte and osteoblast differentiation, endochondral ossification, and bone remodelling during growth and development of long bones.     

Localization and expression of cartilage oligomeric matrix protein by human rheumatoid and osteoarthritic synovium and cartilage.

Synovium and cartilage from patients with osteoarthritis or rheumatoid arthritis were analyzed for expression of cartilage oligomeric matrix protein. Immunostaining of synovium with antiserum to cartilage oligomeric matrix protein demonstrated positive staining in both diseases. In osteoarthritis, there was positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. In rheumatoid arthritis, there was less intense staining within the synovial cells and marked intense staining in the deeper connective tissue. In situ hybridization performed with an antisense digoxigenin-labeled riboprobe to human cartilage oligomeric matrix protein confirmed the presence of cartilage oligomeric matrix protein mRNA in the cells of the synovial lining in both types of synovium. Quantitative polymerase chain reaction with a cartilage oligomeric matrix protein MIMIC demonstrated increased cartilage oligomeric matrix protein mRNA in rheumatoid cartilage and synovium as compared with osteoarthritic cartilage and synovium, respectively; mRNA levels in rheumatoid synovium were similar to those from osteoarthritic chondrocytes. As a result of the high expression of cartilage oligomeric matrix protein from rheumatoid synovium, inflammatory synovium should be considered as a potential tissue source of cartilage oligomeric matrix protein in any investigation of biological markers of cartilage metabolism. The upregulated expression of cartilage oligomeric matrix protein in inflammatory tissues suggests its in vivo regulation by cytokines.     

Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.     

Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis

Levels of HtrA1 protein in cartilage have been reported to elevate in joints of human osteoarthritis patients. To understand roles of HtrA1 in normal osteogenesis as well as in pathogenesis of arthritis, we examine HtrA1 expression pattern during bone and cartilage development and in articular cartilage affected by experimental arthritis. HtrA1 is not expressed in mesenchymal or cartilage condensations before initiation of ossification. When ossification begins in the condensations, the expression of HtrA1 starts in chondrocytes undergoing hypertrophic differentiation near the ossification center. Hypertrophic chondrocytes found in adult articular cartilage and epiphyseal growth plates also express HtrA1. When arthritis is induced by injection of anti-collagen antibodies and lipopolysaccharide, resting chondrocytes proceed to terminal hypertrophic differentiation and start expressing HtrA1. These data suggest that hypertrophic change induces HtrA1 expression in chondrocytes both in normal and pathological conditions. HtrA1 has been reported to inhibit TGF-beta signaling. We show that HtrA1 digests major components of cartilage, such as aggrecan, decorin, fibromodulin, and soluble type II collagen. HtrA1 may, therefore, promote degeneration of cartilage by inducing terminal hypertrophic chondrocyte differentiation and by digesting cartilage matrix though its TGF-beta inhibitory activity and protease activity, respectively. In bone, active cuboidal osteoblasts barely express HtrA1, but osteoblasts which flatten and adhere to the bone matrix and osteocytes embedded in bone are strongly positive for HtrA1 production. The bone matrix shows a high level of HtrA1 protein deposition akin to that of TGF-beta, suggesting a close functional interaction between TGF-beta and HtrA1.     

Human cartilage glycoprotein 39 (HC gp-39) mRNA expression in adult and fetal chondrocytes, osteoblasts and osteocytes by in-situ hybridization

OBJECTIVE: To examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in human cartilage and bone. DESIGN: In-situ hybridization analysis was used to examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in adult human osteoarthritic articular cartilage from various stages of disease, as well as in human osteophytic tissue and in human fetal bone. RESULTS: In cartilage from patients with mild osteoarthritic cartilage degeneration, HC gp-39 was expressed at moderate to high levels only in chondrocytes of the superficial zone. In advanced OA cartilage, cloning chondrocytes of the superficial zone expressed high levels of HC gp-39 and chondrocytes of the mid- and deep zones were also positive. HC gp-39 was undetectable in the chondrocytes of normal articular cartilage. In osteophytic tissue, the expression of HC gp-39 mRNA was intense in flattened, end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation. Proliferating osteoblasts expressed low to moderate levels. Notably, mature osteocytes were negative for HC gp-39 expression. Chondrocytes in the secondary ossification center of developing fetal cartilage demonstrated high expression while growth plate and mineralized cartilage chondrocytes had lower expression. Osteoblasts at sites of endochondral and intramembranous bone formation were positive for expression of HC gp-39. CONCLUSIONS: The stage-specific expression of HC gp-39 in fetal development and adult remodelling bone and cartilage provides evidence for a specific functional or structural role for HC gp-39 in bone and cartilage tissue. HC gp-39 is expressed in diseased human osteoarthritic cartilage and osteophyte, but not in non-diseased tissue, and its distribution within the tissue changes as disease progresses. OA is characterized not only by cartilage degeneration, but by increased subchondral bone formation and osteophytosis. The results from this study indicate that the increased HC gp-39 expression in OA serum and synovial fluid may reflect not only cartilage degeneration but increased osteogenesis.     

甲状旁腺激素和甲状旁腺激素相关蛋白在正常及骨性关节炎软骨中作用机制的研究进展

目的综述在正常和骨性关节炎(osteoarthritis,OA)的关节软骨及软骨下骨中,甲状旁腺激素(parathyroid hormone,PTH)和甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)的作用机制研究进展。方法广泛查阅近年来有关PTH和PTHrP对正常和OA关节软骨作用机制的文献,并进行总结与分析。结果 PTH和PTHrP可抑制OA软骨细胞的肥大分化及凋亡,促进其增殖,从而对OA软骨细胞起到保护作用;OA软骨下骨成骨细胞对PTH的反应下降。结论 PTH、PTHrP可能通过多种信号通路参与软骨降解和软骨下骨重塑,并对OA进展起到延缓和保护作用。     

A monoclonal antibody against dentin phosphophoryn recognizes a bone protein(s) appearing at the beginning of ossification

Summary Decalcified and nondecalcified sections of fetal bovine tibia were stained immunohistochemically with a monoclonal antibody against dentin phosphophoryn. In the epiphyseal portion of the long bone, osteoblasts, osteocytes and the bone matrix were stained, but chondrocytes and the cartilage matrix were not. Similar staining was observed in the epiphyseal and diaphyseal portions of bones. These findings suggest that a protein(s) with the same epitope as phosphophoryn may be synthesized and secreted by osteoblasts at the beginning of ossification and may be involved in mineralization of bone tissue. On Western blots of proteins extracted from fetal bovine bone, the antibody reacted with two bands of molecular weights of about 71,000 and 63,000. These proteins and antibody(s) to the proteins may be useful for detection of the phenotype of osteogenesis     

Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts

OBJECTIVE: To determine the effects of osteoarthritic (OA) subchondral osteoblasts on the metabolism of human OA chondrocytes in alginate beads. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 days in the absence or in the presence of osteoblasts isolated from non-sclerotic (N) or sclerotic (SC) zones of human OA subchondral bone in monolayer (co-culture system). Before co-culture, osteoblasts were incubated for 72 h with or without 1.7ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M (OSM). Aggrecan (AGG) and matrix metalloproteases (MMP)-3 and -13 mRNA levels in chondrocytes were quantified by real-time polymerase chain reaction. AGG production was assayed by a specific enzyme amplified sensitivity immunoassay. RESULTS: SC, but not N, osteoblasts induced a significant inhibition of AGG production and AGG gene expression by human OA chondrocytes in alginate beads, and significantly increased MMP-3 and MMP-13 gene expression by chondrocytes. When they were pre-incubated with IL-1beta, IL-6 or OSM, N osteoblasts inhibited AGG synthesis and increased MMP-3 and -13 gene expression by chondrocytes in alginate beads in a same order of magnitude as SC osteoblasts. CONCLUSIONS: These results demonstrate that SC OA subchondral osteoblasts could contribute to cartilage degradation by stimulating chondrocytes to produce more MMP and also by inhibiting AGG synthesis.     

Tissue distribution and measurement of cartilage oligomeric matrix protein in patients with magnetic resonance imaging-detected bone bruises after acute anterior cruciate ligament tears.

Histologic and immunostaining analyses were performed on articular cartilage/subchondral bone biopsy specimens overlying MRI-detected bone bruises in 12 patients with anterior cruciate ligament (ACL) tears. Staining with toluidine blue for proteoglycan revealed loss of staining from the superficial portion of the articular cartilage. Immunostaining for cartilage oligomeric matrix protein (COMP) showed an increased staining in the superficial matrix of the articular cartilage. Using polyclonal antisera against COMP, the authors performed a competitive enzyme-linked immunosorbent assay (ELISA) on the synovial fluid from the injured and uninjured knees. There was an approximately 10-fold higher synovial fluid COMP levels in injured knees. The COMP levels were greater in those patients who had synovial fluid samples harvested closer to the date of initial injury. Western blot analysis of the synovial fluid showed an increased presence of COMP degradation fragments from injured knees. These results are indicative of a significant injury to the articular cartilage, and may represent preclinical posttraumatic osteoarthritic lesions.