Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25.

We have previously identified megakaryocyte stimulating factor (MSF) gene expression by synovial fibroblasts as the origin of lubricin in the synovial cavity. Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage. MSF has a significant homology to vitronectin and is composed of 12 exons. RNA was purified from human synovial fibroblasts and articular chondrocytes grown in vitro from tissue explants obtained from subjects without degenerative joint disease. RT-PCR was used with multiple complimentary primer pairs spanning the central mucin expressing exon 6 of the MSF gene and individual exons on both the N- and C-terminal sides of exon 6. Exons 2, 4 and 5 appear to be variably expressed by synovial fibroblasts and articular chondrocytes. Lubricating mucin, in the form of MSF, is expressed by both chondrocytes and synovial fibroblasts in vitro. Both lubricin and superficial zone protein (SZP), a related proteoglycan, share a similar primary structure but could differ in post-translational modifications with O-linked oligosaccharides which are predominant in lubricin and with limited amounts chondroitin and keratan sulfate found in SZP. Since most of the MSF exons are involved in the expression of lubricating mucin, a strong homology to vitronectin persists. It is therefore appropriate to consider that both SZP and lubricin occupy a new class of biomolecules termed tribonectins. Screening of a human genome bacterial artificial chromsome (BAC) library with a cDNA primer pair complimentary for exon 6 identified two clones. Both clones were complimentary for chromosome 1q25 by in situ hybridization. This same locus was previously implicated in camptodactyl-arthropathy-pericarditis syndrome (CAP) by genetic mapping. It is hypothesized that CAP, a large joint arthropathy, may be associated with ineffective boundary lubrication provided by synovial fluid.

Weight-bearing alters the expression of collagen types I and II, BMP 2/4 and osteocalcin in the early stages of distraction osteogenesis.

This study was performed to investigate the effect of loading on the biology of newly forming bone during limb lengthening. Unilateral 2.0 mm femoral lengthenings were performed in 20 male Sprague Dawley rats. Half (n = 10) of the animals were allowed to bear weight freely, while the other half were prevented from weight-bearing via an ipsilateral through-knee amputation. The animals in each group were sacrificed after one (n = 5) or four (n = 5) days of consolidation (post-operative days seven and 10, respectively). In situ hybridization for osteocalcin and collagen I, and antibody staining for collagen II and BMP 2/4 were used to evaluate the molecular influence of loading. There was more new bone in the distraction gap of the weight-bearing animals than there was in the non-weight-bearing animals. BMP 2/4 expression, and the messages for collagen I and osteocalcin, were more abundant in tissue from the weight-bearing animals; collagen II was higher in the non-weight-bearing animals. This suggests that early regenerate tissue is capable of responding to loading, and that weight-bearing appears to stimulate intramembranous ossification. These findings support the concept of early weight-bearing after limb lengthening.

Articular collagen degradation in the Hulth-Telhag model of osteoarthritis.

OBJECTIVE: This study employed immunohistochemistry to investigate the pattern of type II collagen (CII) degradation in an acute injury model of osteoarthritis. It was hypothesized, based on previous studies of primary osteoarthritis (OA), that the worst areas of CII degradation would be located in the superficial and upper middle zones of the articular cartilage, with staining extending into the deep zone as the OA became more severe. DESIGN: In this model of secondary OA, rabbits were made osteoarthritic by transecting the anterior and posterior cruciate ligaments and removing the meniscus. At various times post surgery, articular cartilage was examined for CII degradation using monoclonal antibody 18:6:D6. This antibody reacts to an epitope that is exposed when CII is degraded as the result of protease cleavage. Proteoglycans (PG) were localized using Safranin-O/Fast Green. Staining intensities were quantitated using image analysis software. RESULTS: In the joints with surgically induced OA, degradation of CII was seen as early as 3 weeks with the majority of the degradation localized in zones I and III. At 14 weeks the destruction of CII was more pronounced, but there was a surprising lack of degradation in zone II. There were also several other unexpected findings. The sham-operated joints, for example, were intended to serve as controls yet CII degradation was observed in rabbits killed 3 weeks after surgery. It was also expected that greater CII degradation would occur in cartilage from medial condyles, but after 14 weeks there was no significant difference between medial and lateral condyles. Finally, the loss of staining for PG correlated with the degradation of CII except in zone III where limited PG loss was observed. CONCLUSION: Differences were observed between the pattern of articular cartilage destruction in this model of secondary OA and that of primary OA. Further investigation of the mechanical and biochemical processes underlying the development of both types of OA needs to be conducted.

Immunological detection of type II collagen degradation: use in the evaluation of anti-arthritic therapies.

Propagated Swaram rat chondrosarcoma cells, rabbit chrondrocytes (from articular cartilage of knee, shoulder and hip joints), and bovine nasal cartilage explant cultures were studied. Type II collagen (CII) and its peptide fragments were quantitated in cell medium and cell layer separately, using two previously developed assays; one assay employed a monoclonal antibody, C4F6, that reacts specifically with triple helical CII and the other assay used an antibody, EIE5, that reacts specifically with a peptide of CII. A time-dependent increase in the content of CII and CII-derived peptides was observed in both rat and rabbit cultures. In both culture systems the majority of the native type II collagen is found associated with the cell layer (97% in rat cultures and 73% in rabbit cultures), while the major part of the CII peptides is found in the media (73% in rat cultures, 88% in the rabbit cultures). The concentration of peptides in the media reaches approximately 2 micrograms mL-1 in both chondrocyte monolayer cultures after 4 days. The CII peptide assay employing E1E5 was well suited to quantitate articular cartilage collagen degradation in explant culture. thus it can be used to evaluate potential therapeutic agents that can modify or inhibit cartilage degradation. The assay has the added potential that it could be used in-vivo to evaluate the effectiveness of potential metalloproteinase inhibitors in animal models of osteoarthritis or in clinical trials.

Remodeling of articular step-offs. Is osteoarthrosis dependent on defect size?

One of the major unresolved questions in trauma repair concerns the degree of step-off that can be accepted in a joint surface. In answer to this question, a new rabbit model of femoral step-off was developed. Osteoarthrotic changes in cartilage and bone and the failure of repair responses were seen at 20 weeks in the presence of a 3-mm wide sagittal defect displaced 5 mm from the joint surface and spanning the anteroposterior extent of the medial femoral condyle. This study examined the effects of a similar defect, displaced 2 mm from the joint surface, to determine whether the development of osteoarthrosis is dependent on the size of the step-off. Defects were created in 18 New Zealand white rabbits. In a second group, the medial joint surface was osteotomized, but was not displaced. In contrast to th first study, cartilaginous and bony repair resulted in closure of the surgical defect and restoration of femoral congruity. Histologic and biochemical parameters did not differ significantly between groups. The results indicate that cartilage and bone possess the ability to remodel small articular step-offs and to restore joint congruity. Furthermore, the combined data suggest that the development of osteoarthrosis requires significant articular incongruity.

Effects of certain antiarthritic agents on the synthesis of type II collagen and glycosaminoglycans in rat chondrosarcoma cultures.

Cartilage destruction is a characteristic feature of osteoarthritis. Treatment with certain nonsteroidal anti-inflammatory drugs could exacerbate cartilage destruction by impairing the synthesis of cartilage matrix proteins, type II collagen and proteoglycan. In order to monitor the changes occurring in cartilage collagen synthesis, we developed a type II collagen specific ELISA. The effects of antiarthritic agents on type II collagen and glycosaminoglycan synthesis were examined in rat chondrosarcoma cultures. Drugs were added to the monolayer cultures and 4 days later the total type II collagen, as determined by the type II collagen ELISA, and glycosaminoglycan content, as measured by dimethyl-methylene blue dye binding assay, was measured. All drugs except tiaprofenic acid decreased type II collagen synthesis by at least 40% at 100 micrograms/ml. Tiaprofenic acid at 1 microgram/ml increased type II collagen content by 54% of the controls. Glycosaminoglycan synthesis was decreased by acetylsalicylic acid, diclofenac and tiaprofenac acid, at 50 micrograms/ml or above. Indomethacin, naproxen and dexamethasone had no effect. Interestingly, tenidap stimulated the glycosaminoglycan synthesis by 32% at 100 micrograms/ml. We show that the combination of chondrosarcoma cultures, type II collagen specific ELISA and dimethylmethylene blue dye binding assay serves as a useful model for screening the effects of agents capable of modulating type II collagen and glycosaminoglycan synthesis.

Production of type II collagen specific monoclonal antibodies.

Immunoassays are used for the specific measurement of type II collagen, a major cartilage protein, which is lost in osteoarthritic joints. Poor immunogenicity and species dependent immune response to type II collagen make it difficult to obtain specific antibodies required for immunoassay development. In addition, type II collagen antibodies exhibit reactivity to structurally dissimilar antigens such as actin, myoglobin, thyroglobulin and ssDNA, complicating the isolation of specific antibodies. It is therefore necessary to characterize the antibody reactivity against both noncollagenous antigens and different collagen types. In this study, immune response to type II collagen was improved by conjugation to carrier proteins, KLH and BSA. Hybridomas were generated by fusions of lymphocytes derived from lymph nodes or spleens with X63-653-Ag8 myeloma cells. Compared to spleens, the utilization of lymph nodes as a source of lymphocytes resulted in a 23% higher number of hybridomas secreting type II collagen antibodies. Hybridomas secreting polyreactive antibodies were identified based on their reactivity to thyroglobulin and eliminated. Extensive testing of the remaining monoclonal antibodies with other structurally dissimilar antigens and various types of collagen for reactivity, allowed us to isolate specific monoclonal antibodies to type II collagen. We emphasize the importance of characterization of the reactivity of type II collagen monoclonal antibodies before employing them for immunoassays.

Quantitative immunoassays for type II collagen and its cyanogen bromide peptides.

This paper describes the development of quantitative immunoassays utilizing mouse monoclonal antibodies which are monospecific to type II collagen. The monoclonal antibodies were characterized and tested extensively for reactivity against a panel of antigens including actin, myoglobin, thyroglobulin, ssDNA, tetanus toxin and different types of collagens including their CnBr-derived peptides. Four monoclonal antibodies having strict monospecificity to type II collagen were selected. Quantitative immunoassays developed with these antibodies can measure either type II collagen in its native conformation or type II collagen-derived cyanogen bromide peptides. The assay conditions such as coating concentration of antigen, monoclonal antibody concentration, second antibody concentration and incubation times were optimized to obtain maximum possible sensitivity. These quantitative immunoassays can be employed to measure type II collagen or type II collagen-derived peptides in low amounts ranging from 20 to 100 ng/ml. The assays can be applied to chondrocyte cultures without interference from serum components or other collagen types.

An experimental model of femoral condylar defect leading to osteoarthrosis.

The acute treatment of articular step-off injuries is based largely on reduction criteria, because the presence of residual incongruity has been correlated with the development of posttraumatic arthrosis (PTA). However, this association has not been demonstrated on a prospective basis. Using the rabbit femoral condyle, we developed a surgical model of articular condylar defect without sacrificing the axial alignment or inherent stability of the knee joint. Twenty weeks after the creation of 5-mm femoral condylar defects, progressive osteoarthritic changes were confirmed by radiographic, histological, and biochemical parameters. Osteophytes were observed on the medial aspect of operated knee joints in 67% of cases. Femoral and tibial articular cartilage at the site of the condylar defect exhibited fibrillation, hypocellularity, and severe loss of safranin-O staining. Focal areas of cartilage were denuded or replaced by pannus. In no case was femoral congruity restored by cartilage repair. Statistically significant decreases in proteoglycan content were demonstrated for cartilage sampled from the weight-bearing region of the condylar defect and from the tibial surface directly beneath it. These changes resemble those arising from previously reported models of osteoarthrosis. We present the model as a valid tool for the study of articular condylar defect and its role in the development of PTA.

Morphological analysis of abnormal digital chondrogenesis in the brachypod (bpH) mouse limb in organ culture.

Brachypod (bpH/bpH), an autosomal mutation in mice, is characterized by a shortening of the long bones and paws, and a delay or absence of ossification in some of the distal limb elements. The present study represents a detailed description of the brachypod phenotype in day 12 hindlimb buds maintained for 6 days in a submerged, serum-free organ culture system. Using this in vitro system, the proximal-to-distal effect on the severity of cartilage reduction was intensified in the brachypod explants with an intermediate expression in the heterozygotes. Immunofluorescent staining of the brachypod cartilage revealed a deficiency in and an abnormal distribution of the proteoglycans. Although there was no recognizable difference in the immunofluorescent staining for type II collagen between the mutant and wild-type, electron micrographs showed the presence of thick fibrils in the matrix. Other atypical structures in the brachypod cartilage included pleomorphic nuclei, reduced intracellular glycogen granules and profuse intercellular contacts. It is proposed that with the use of this in vitro system which supports the autonomous development of the individual limb elements, experiments concerning the pathogenesis of skeletal mutations such as brachypod should be more feasible.

Evidence for polyreactivity seen with monoclonal antibodies produced against type II collagen.

Specific type II collagen monoclonal antibodies are needed for the quantification of articular cartilage collagen. In this study we produced and characterized 29 type II collagen monoclonal antibodies. Hybridomas were generated from mice immunized with rat type II collagen, selected for high antibody production against type II collagen using ELISA. Antibodies from selected and cloned hybridoma cells were purified by affinity chromatography and their reactivity tested by ELISA against a panel of antigens including actin, thyroglobulin, and single stranded DNA, all of which have been used to characterize the 'naturally occurring antibodies.' It was found that many of the anti-type II collagen monoclonal antibodies reacted to more than one antigen. The monospecific antibodies had higher affinity to type II collagen than the antibodies which demonstrated multireactivity. Because of the prevalence of polyreactive anti-type II collagen antibodies, it is advisable to employ highly selective methodologies to isolate high affinity monospecific antibodies to type II collagen.

The generation of hybridomas secreting human monoclonal antibodies reactive with type II collagen.

Human monoclonal antibodies reactive with type II collagen were obtained from patients with rheumatoid arthritis and relapsing polychondritis by fusion of cells with the human-mouse myeloma analogue HMMA2.11 TG/0. Direct fusion of peripheral blood or bone marrow mononuclear cells was unsuccessful in obtaining antibody producing hybridomas although fusion efficiency was high (1 hybridoma per 25,400 mononuclear cells fused). Polyclonal, Epstein Barr Virus transformed B cell lines derived from peripheral blood and bone marrow mononuclear cells after direct transformation, in vitro secondary immunization, and/or CD8 depletion were found to produce antibodies that reacted with type II collagen. Antibody producing EBV transformed B cells were fused with the human-mouse myeloma analogue HMMA2.11 TG/0 and six separate, stable IgM antibody producing hybridomas obtained. These results demonstrate the difficulty in obtaining human monoclonal autoantibodies, particularly of IgG isotypes, using readily accessible sources of cells in patients with chronic autoimmune diseases without expansion and preselection.

A histochemical method for the demonstration of calcifying cartilage.

The demonstration of histochemical characteristics in calcifying cartilage is fraught with methodological difficulties including the distinction of mineralized from unmineralized cartilage and the demonstration of cell detail in relatively hard tissue. This study uses the decalcified bone matrix-induced enchondral (endochondral) ossification system to demonstrate a technique of methylmethacrylate embedding, thin sections, and a combination of histochemical stains that distinguishes mineralized from unmineralized cartilage while preserving excellent cell detail. These techniques are applicable to other areas of enchondral ossification and are exemplified by the staining of growth plates.

Production and specificity of antibodies against the central region of type II collagen.

Collagen type-specific antibodies as well as antibodies to particular portions of the molecule are extremely useful tools especially for the quantification of collagens and for immunohistochemical examinations in developing embryos. Quantification of collagens in CNBr-solubilized tissue samples presupposes the production of antibodies against CNBr-derived collagen fragments. For the first time, as antigens for the immunization of rabbits, cyanogen-bromide derived fragments of collagen type II were used, obtained by direct digestion of tissue (Swarm chondrosarcoma from rat) and separation by gel filtration chromatography. Antisera were applied to affinity chromatography and the eluted antibodies were characterized by ELISA, immunoblotting, inhibition studies and immunohistochemistry. The antibodies from five different rabbits show high specificity for type II collagen and are directed against sequential determinants in the central portion of the type II collagen molecule. The easy way of obtaining the fragments directly from tissue, combined with their immune response in rabbits, gives the possibility of producing type II collagen-specific, fragment-directed antibodies in a convenient and rapid way.

The biosynthetic response of the mature chondrocyte in early osteoarthritis.

Metabolic studies in early experimental osteoarthritis (OA) have shown that the rate of proteoglycan synthesis in the diseased articular cartilage may be markedly enhanced relative to normal; elevated rates of synthesis were however accompanied by increased release of new molecules from the tissue so that the response was apparently non-reparative. Described here are experiments with immature and mature chondrocytes in culture which show that aging of chondrocytes in vivo is accompanied by a marked fall in the capacity of these cells to synthesize link protein and to assemble a proteoglycan-rich matrix. It is suggested that poor deposition of proteoglycan by mature chondrocytes in OA may result from insufficient synthesis of link protein for stabilization of aggregates.

The basement membrane of the persisting maternal blood vessels in the placenta of Callithrix jacchus.

Formation and morphology of the thickened basement membrane-like layer around the persisting maternal vessels of the Callithrix jacchus placenta were investigated from day 45 until term (day 142) using light, electron and immunofluorescence microscopy. Thickening occurs with the establishment of contacts between the vessels and the syncytiotrophoblast (day 48). Final thickness is reached at about day 100. The course of the vessels shows wide gaps where the maternal blood flows freely into the intertrabecular spaces. As revealed by electron microscopy, the extracellular sheath around the maternal vessels consists of an inner subendothelial basement membrane (3-6 microns) and an outer fibril-containing layer (2-4 microns). Cell debris is seen between the two layers and in the basement membrane. Plaques of granular and fine-filamentous material are incorporated into the fibril-containing layer. The synthesis of the basement membrane material is localized in the endothelial cells. Immunofluorescence microscopy reveals collagen types IV and V, laminin and heparan sulfate proteoglycan (BM-1) in the sheath around the persisting vessels. Fibronectin occurs only in certain areas or in the form of dots. Collagen types I and III are not seen in the region of the vascular wall. It can, therefore, be assumed that the subendothelial layer represents a genuine basement membrane; the fibrils consist of collagen type V and the plaques contain fibronectin. The existence of the thick perivascular sheath is attributed to the persistence and stability of the maternal vessels.

[Development of connective tissue in mouse skeletal muscle]. / Zur Entwicklung des Bindegewebes im Skelettmuskel der Maus.

The development of the connective tissue in the dorsal forelegs of mouse embryos and sucklings was investigated with the aid of immunofluorescence and electron microscopy. Collagen type III is visible earlier in the muscular connective tissue than collagen type I. However, it seems that collagen does not play a fundamental role in the organization of the muscular tissue. The occurrence of collagen is rather an indication of the mechanical development of the muscles. In early embryos (day 13 to day 15) some desmosome-like cell contacts are visible between fibroblasts and myoblasts, as well as myotubes.

Preparation of a specific antiserum against elastin of the human aorta.

Elastin was isolated from the human aorta by three different extraction methods. Immunization was carried out in sheep. The presence of antibody against each elastin preparation in the sheep sera was confirmed by immunofluorescence. However, antiserum against elastin isolated by collagenase/guanidine dithioerythritol showed the most specific reactions in the cryostat sections. No cross-reactivity to type I, III and IV collagen, fibronectin, laminin and proteoglycan BM 1 was observed.

Isolation and purification of cyanogen bromide-derived peptides of type II collagen directly from tissue (Swarm chondrosarcoma).

A preparative procedure is described for isolating type II collagen-fragments directly from tissue. Swarm chondrosarcoma from rat, a cartilagenous tissue rich in type II collagen, was digested by cyanogen bromide in 70% formic acid. The resulting crude extract was desalted (G 25 column chromatography) and lyophylized. The yield of peptide mixture was about 1 250 mg obtained from 100 g tissue. The method of purification commonly used for type II collagen prior to cyanogen bromide-cleavage yielded 20 mg peptides from 100 g tissue. Separation of the cyanogen bromide-derived fragments was performed by gel filtration. The column was run at 43 degrees C (denaturing-temperature of collagens) to avoid fibril formation, and a volatile buffer was used (ammonium formate buffer, pH 7.5) so that the effluent fractions could be easily lyophylized. Two-dimensional gel electrophoresis of the main peaks of the column profile demonstrated that this purification step resulted in a good separation of the fragment mixture, although additional steps may be necessary for complete separation of the peptides. The most striking advantages of the method for direct digestion of tissue outlined here are the increase in yield (about 60-fold) and the reduction of purification steps (avoiding type II collagen purification).