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PubMed2002

Transgenic mouse models for studying the role of cartilage macromolecules in osteoarthritis.

The development of transgenic technology has made possible the generation of targeted gene-mutated mouse lines suitable for use in experimental osteoarthritis (OA) research. Transgenic mice harbouring mutations in cartilage collagen types II and IX develop early-onset OA and are therefore promising models of age-related OA, even though the mice often show signs of chondrodysplasia. Also, mouse lines harbouring other engineered mutations of the extracellular molecules have given rise to early OA. The molecular background of a few spontaneous mutations in mice has also been clarified and the characterization of the OA phenotype is now in progress. These mutations cause severe chondrodysplasia and death in homozygous mice, but the heterozygous offspring develop the early-onset OA phenotype.

Animals↗
PubMed1996

Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells.

Osteogenic protein-1 (OP-1, BMP-7), a bone morphogenetic protein in the transforming growth factor-beta superfamily, induces endochondral bone formation in vivo, but the mechanism of action of OP-1 in osteogenesis is not yet established. Three murine clonal cell lines in different stages of differentiation exhibit graded responses to recombinant human OP-1: the mouse embryonal carcinoma ATDC5 cell, with potential for chondroblastic differentiation; the osteoblast-like MC3T3-E1 cell derived from mouse calvaria; and the multipotent fibroblastic C3H10T1/2 cell derived from mouse embryo connective tissue. We show that OP-1 acts on early stage mesenchymal progenitor cells (ATDC5, C3H10T1/2) to induce chondroblastic differentiation, while OP-1 strongly enhances the osteoblastic phenotype of committed osteoblasts (MC3T3-E1), possibly explaining its induction of the endochondral ossification cascade in vivo. Markers of osteoblastic, chondroblastic, and adipocytic differentiation are compared. OP-1 is strongly mitogenic for ATDC5, showing dose-dependent (2.5-80 ng/ml) induction of Alcian blue staining, alkaline phosphatase activity, and mRNA expression for collagen types II and IX, and matrix Gla protein. MC3T3-E1 cells do not proliferate or stain with Alcian blue in response to OP-1, but express elevated levels of alkaline phosphatase and osteocalcin. While low-dose OP-1 treatment of C3H10T1/2 induces only adipocyte-like cells filled with lipid droplets, a high dose (500 ng/ml) causes the same cells to also exhibit chondrocytic properties. Thus, OP-1 can induce differentiation along elements of the endochondral ossification pathway according to the stage and potential of the target cell.

Adipocytes↗
PubMed2006

Cartducin, a paralog of Acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes.

We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor-beta1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it "cartducin". Cartducin shares a similar modular organization to adipocyte-derived hormone, adiponectin. In this study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole-mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein-2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo-oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS-2/8 cells in a dose-dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosaminoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes.

Adipokines↗
PubMed1990

An immunohistochemical study of the distribution of matrical proteins in the mandibular condyle of neonatal mice. II. Non-collagenous proteins.

This study has illustrated the localisation of several non-collagenous proteins in the extracellular matrix of neonatal condylar cartilage (chondrocalcin, proteoglycans and a 100 kDa protein). Also, it has been shown that chondrocytes synthesise laminin and anchorin CII. The bone trabeculae in the primary spongiosa reacted positively for chondrocalcin, osteopontin and BMP. The cartilaginous wealth of collagens (Type I, II, VI, IX, X and XI), together with the various non-collagenous proteins, are considered to possess an important role in the maintenance of chondrogenic expression as well as in the regulation of cartilage differentiation by means of an interaction with the undifferentiated precursor cells.

Animals↗
PubMed2001

Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/alginate amalgam.

Bone marrow-derived cells are considered as candidate cells for cartilage tissue engineering by virtue of their ability to undergo chondrogenesis in vitro when cultured in high density or when embedded within a three-dimensional matrix in the presence of growth factors. This study evaluated the potential of human bone marrow-derived cells for cartilage tissue engineering by examining their chondrogenic properties within a three-dimensional amalgam scaffold consisting of the biodegradable polymer, poly-L-lactic acid (PLA) alone, and with the polysaccharide gel, alginate. Cells were suspended either in alginate or medium and loaded into porous PLA blocks. Alginate was used to improve cell loading and retention within the construct, whereas the PLA polymeric scaffold provided appropriate mechanical support and stability to the composite culture. Cells seeded in the PLA/alginate amalgams and the plain PLA constructs were treated with different concentrations of recombinant human transforming growth factor-beta1 (TGF-beta 1) either continuously (10 ng/mL) or only for the initial 3 days of culture (50 ng/mL). Chondrogenesis was assessed at weekly intervals with cultures maintained for up to 3 weeks. Histological and immunohistochemical analysis of the TGF-beta 1-treated PLA/alginate amalgam and PLA constructs showed development of a cartilaginous phenotype from day 7 to day 21 as demonstrated by colocalization of Alcian blue staining with collagen type II and cartilage proteoglycan link protein. Expression of cartilage specific genes, including collagen types II and IX, and aggrecan, was detected in TGF-beta 1-treated cultures by reverse transcription-polymerase chain reaction analysis. The initiation and progression of chondrogenic differentiation within the polymeric macrostructure occurred with both continuous and the initial 3-day TGF-beta 1 treatment regimens, suggesting that key regulatory events of chondrogenesis take place during the early period of cell growth and proliferation. Scanning electron microscopy revealed abundant cells with a rounded morphology in the PLA/alginate amalgam. These findings suggest that the three-dimensional PLA/alginate amalgam is a potential candidate bioactive scaffold for cartilage tissue engineering applications.

Alginates↗
PubMed1987

A major, six-armed glycoprotein from embryonic cartilage.

Here we describe the isolation and identification of a major cartilage glycoprotein which is co-extracted along with typical hyaline cartilage components such as collagen types II and IX from chicken embryo sternum. In polyacrylamide gel electrophoresis it migrates as a high molecular mass protein (greater than 10(6) daltons) which on reduction gives rise to a prominent doublet at 205/195 kd and minor bands at 220 and 170 kd. The intact molecule sediments as a 13S component in rate zonal centrifugation, indicative of a highly extended conformation in solution. In these properties it closely resembles myotendinous antigen, a glycoprotein recently detected in a number of embryonic tissues, including cartilage. This identity was confirmed by immunoblotting using a monoclonal antibody (M1) specific for myotendinous antigen. Electron micrographs of the rotary shadowed molecule revealed an unusual six-armed structure, indistinguishable in form and dimensions from hexabrachion, a recently discovered contaminant of cellular fibronectin preparations. These structures could be decorated with the M1-antibody, demonstrating that hexabrachion is myotendinous antigen. This extended, potentially multivalent molecule could provide an ideal substrate to connect widely spaced components in a highly hydrated tissue such as cartilage.

Animals↗
PubMed2002

Combined autoimmune disease in a patient with AIDS.

Immune dysregulation in HIV-infected patients, along with the new medications for treatment of AIDS that possess immunomodulating potential, may lead to an increased incidence of autoimmune diseases in this patient population. However, the presence of combined autoimmune diseases in an AIDS patient is rare. Relapsing polychondritis (RP) is an uncommon inflammatory disease manifested by recurrent attacks of auricular chondritis. The presence of type II and IX collagen antibodies, and their association with HLA-DR4 and other autoimmune diseases, suggests that antiself reactions may be present. Sarcoidosis is a granulomatous disease manifested by inflammation of the lungs, eyes and joints. In the peripheral blood there is depressed cellular immunity and enhanced humoral immunity. We here describe a case of coexisting RP and sarcoidosis in an AIDS patient.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel.

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1-3 microns in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4-6 weeks in culture and progressively developed a broad territorial region up to 12 microns wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly.

Agar↗
PubMed2006

The functional (-1171 5A-->6A) polymorphisms of matrix metalloproteinase 3 gene as a risk factor for oral submucous fibrosis among male areca users.

BACKGROUND: Insertion/deletion (-1171 5A-->6A) polymorphisms in the promoter region of matrix metalloproteinase 3 (MMP3) gene result in different transcriptional activities. MMP3 is able to degrade collagens types II, V, IX, and X, and other extracellular matrix. The functional promoter polymorphism of MMP3 has been related to the susceptibility in some inflammatory diseases and metastasis of cancers. METHODS: Oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC) are prevalent among Asian areca users. In this study, genomic DNA obtained from the blood of OSCC (n = 150), OSF (n = 71), and control non-diseased areca user (n = 98) in male were subjected to polymerase chain reaction (PCR)-based genotyping of MMP3. RESULTS: The 5A genotype in MMP3 promoter was observed more frequently in OSF group than in control group (P = 0.01). No significant difference was noted between OSCC and control groups on the 5A genotype frequency (P = 0.18). No association was found between 5A genotype in MMP3 promoter and site or lymph node metastasis and stage of OSCC. CONCLUSION: The results indicated that the 5A genotype of MMP3 promoter was associated with the risk of OSF but not OSCC.

Adenine↗
PubMed2005

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.

BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARgamma, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.

Journal Article↗
PubMed2006

The pericellular matrix as a transducer of biomechanical and biochemical signals in articular cartilage.

The pericellular matrix (PCM) is a narrow tissue region surrounding chondrocytes in articular cartilage, which together with the enclosed cell(s) has been termed the "chondron." While the function of this region is not fully understood, it is hypothesized to have important biological and biomechanical functions. In this article, we review a number of studies that have investigated the structure, composition, mechanical properties, and biomechanical role of the chondrocyte PCM. This region has been shown to be rich in proteoglycans (e.g., aggrecan, hyaluronan, and decorin), collagen (types II, VI, and IX), and fibronectin, but is defined primarily by the presence of type VI collagen as compared to the extracellular matrix (ECM). Direct measures of PCM properties via micropipette aspiration of isolated chondrons have shown that the PCM has distinct mechanical properties as compared to the cell or ECM. A number of theoretical and experimental studies suggest that the PCM plays an important role in regulating the microenvironment of the chondrocyte. Parametric studies of cell-matrix interactions suggest that the presence of the PCM significantly affects the micromechanical environment of the chondrocyte in a zone-dependent manner. These findings provide support for a potential biomechanical function of the chondrocyte PCM, and furthermore, suggest that changes in the PCM and ECM properties that occur with osteoarthritis may significantly alter the stress-strain and fluid environments of the chondrocytes. An improved understanding of the structure and function of the PCM may provide new insights into the mechanisms that regulate chondrocyte physiology in health and disease.

Animals↗
PubMed2006

Regulation of osteoblast differentiation: a novel function for fibroblast growth factor 8.

Several members of the fibroblast growth factor (FGF) family have an important role in the development of skeletal tissues. FGF-8 is widely expressed in the developing skeleton, but its function there has remained unknown. We asked in this study whether FGF-8 could have a role in the differentiation of mesenchymal stem cells to an osteoblastic lineage. Addition of FGF-8 to mouse bone marrow cultures effectively increased initial cell proliferation as well as subsequent osteoblast-specific alkaline phosphatase production, bone nodule formation, and calcium accumulation if it was added to the cultures at an early stage of osteoblastic differentiation. Exogenous FGF-8 also stimulated the proliferation of MG63 osteosarcoma cells, which was blocked by a neutralizing antibody to FGF-8b. In addition, the heparin-binding growth factor fraction of Shionogi 115 (S115) mouse breast cancer cells, which express and secrete FGF-8 at a very high level, had an effect in bone marrow cultures similar to that of exogenous FGF-8. Interestingly, experimental nude mouse tumors of S115 cells present ectopic bone and cartilage formation as demonstrated by typical histology and expression of markers specific for cartilage (type II and IX collagen) and bone (osteocalcin). These results demonstrate that FGF-8 effectively predetermines bone marrow cells to differentiate to osteoblasts and increases bone formation in vitro. It is possible that FGF-8 also stimulates bone formation in vivo. The results suggest that FGF-8, which is expressed by a great proportion of malignant breast and prostate tumors, may, among other factors, also be involved in the formation of osteosclerotic bone metastases.

Alkaline Phosphatase↗
PubMedDate not supplied

Cartilage oligomeric matrix protein (COMP) is modified by intra-articular liposomal clodronate in an experimental model of arthritis.

OBJECTIVE: High-dose liposomal bisphosphonates exert apoptotic effects. This work studies the chondroprotective and anti-inflammatory properties of intra-articularly administered low-dose, non-cytotoxic liposomal clodronate. METHODS: Antigen induced arthritis in rabbits was treated with intra-articular injections of liposomal clodronate. Drug effects on cartilage oligomeric matrix protein COMP was assessed using immunohistochemistry and morphometry of synovial membrane and hyaline articular cartilage. RESULTS: COMP remained close to normal in liposomal clodronate treated superficial articular cartilage compared to a significant loss of COMP in arthritis controls treated with empty liposomes. The middle and deep layers of the hyaline articular cartilage were characterized by highly increased COMP expression in liposomal clodronate treated AIA joints compared to controls. In contrast to cartilage, synovial COMP expression was slightly decreased as a result of liposomal clodronate treatment. CONCLUSION: Low-dose, non-cytotoxic liposomal clodronate exerts a dichotomous effect on synovial membrane and articular cartilage COMP in the AIA model. COMP is a useful inflammation marker in the synovial tissue, but it also contributes to the structural integrity of the hyaline articular cartilage forming bridges between type II and IX collagens. Enhancement of COMP in clodronate treated AIA cartilage suggests a chondroprotective and anti-inflammatory effect in the inflammatorily damaged and mechanically strained cartilage.

Animals↗
PubMed1989

[Erysipelas as a disease factor].

The infection with Erysipelas rhusiopathiae demonstrates that it is possible to characterize the significance of predisposing factors. The virulence of the agent is most important among the exogenous factors; it determines decisively the course of the disease: 1. paracute course as septicemia; 2. chronic course as polyarthritis; 3. subclinical course nearly without any symptoms. The immune status and the genotype of the host are predominant out of endogenous factors. The importance of immunity is known since a long time. A series of field observations supported the potential genetical influence in the pig. Within the hereditability an association to the MHC (in special genetic lines of rats to the RT 1 system of the MHC) was most recently determined in inbred laboratory animals. Additionally several environmental conditions, which can be summarized as stress, and as endogenous factor the age of the animals are relevant for the pathogenesis of the erysipelas infection. A non variable but most important disposition for special tissues are the so-called "borderline tissues", where accumulation, sedimentation and persistence of bacterial antigens are wellknown as described earlier. This phenomenon is determined by hemodynamic manifestation and quantifiably regulated by cytokines especially interleukin (IL 1) as well as by the tumor necrosis factor (TNF alpha) and prostaglandin PgE2. Additionally the cross reactivity of antibodies of Erysipelas rhusiopathiae against most specific collagen of type II, IX and XI in the pig and in laboratory animals was elaborated. This autoimmune phenomenon called "immunologic mimicry" supports besides the special physiologic conditions as niche of defense a very successful evolutionary adaptation of the agent.

Age Factors↗
PubMed1987

The role of humoral immunity in Lactobacillus casei cell wall induced arthritis.

Although both cellular and humoral immunity have been shown to play important roles in the development of collagen induced arthritis, their roles in the pathogenesis of bacterial cell wall induced arthritis remain unclear. Previous studies of humoral immunity in bacterial cell wall induced arthritis were carried out in outbred animals and measured the humoral response to only the eliciting bacteria. We compared the humoral immune responses of arthritis susceptible Lewis and arthritis resistant Fisher rats after the injection of arthritogenic group B Lactobacillus casei cell wall fragments. Both strains developed significant IgG and IgM responses to the Lactobacillus casei cell wall fragments. Neither strain developed a significant immune response to types I, II, IX or 1a2a3a collagens. These data suggest that humoral immune responses to the Lactobacillus casei cell wall fragments and types I, II, IX, or 1a2a3a collagens do not play a significant role in determining the differing arthritis susceptibilities of Lewis and Fisher rats.

Animals↗
PubMed2000

Mutations in cartilage oligomeric matrix protein causing pseudoachondroplasia and multiple epiphyseal dysplasia affect binding of calcium and collagen I, II, and IX.

Mutations in type 3 repeats of cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We expressed recombinant wild-type COMP that showed structural and functional properties identical to COMP isolated from cartilage. A fragment encompassing the eight type 3 repeats binds 14 calcium ions with moderate affinity and high cooperativity and presumably forms one large disulfide-bonded folding unit. A recombinant PSACH mutant COMP in which Asp-469 was deleted (D469 Delta) and a MED mutant COMP in which Asp-361 was substituted by Tyr (D361Y) were both secreted into the cell culture medium of human cells. Circular dichroism spectroscopy revealed only small changes in the secondary structures of D469 Delta and D361Y, demonstrating that the mutations do not dramatically affect the folding and stability of COMP. However, the local conformations of the type 3 repeats were disturbed, and the number of bound calcium ions was reduced to 10 and 8, respectively. In addition to collagen I and II, collagen IX also binds to COMP with high affinity. The PSACH and MED mutations reduce the binding to collagens I, II, and IX and result in an altered zinc dependence. These interactions may contribute to the development of the patient phenotypes and may explain why MED can also be caused by mutations in collagen IX genes.

Achondroplasia↗
PubMed1991

Collagens of the bovine vitreous.

The predominant structural components of the vitreous are collagenous fibrils. Prior biochemical analyses have been limited by incomplete solubilization of the constituent collagen types. The techniques described enable an effective separation and nearly complete solubilization of calf vitreous fibrils. Semiquantitative analysis has led to the detection of types II, V, and IX collagen in a ratio of 69:24:7, respectively. Qualitatively similar ratios were found in vitreous from other mammals, including rabbits, monkeys, and humans. Electron microscopic evidence suggests that type VI collagen may be distributed selectively near the vitreous base. These observations are pertinent to vitreous gel stabilization, aging, and disease.

Aging↗
PubMed1990

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen.

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.

Aged↗