Relapsing polychondritis. Pathological and immunological findings in an autopsy case

Relapsing polychondritis (RP) is a disorder of unknown etiology characterized by the destruction of cartilage. A case of RP in a 59-year-old male was autopsied, and systemic inflammation of various cartilages was confirmed. We demonstrated the circulating antibodies to Type II (cartilage) collagen. No antibodies to other collagen types were demonstrated. The presence of granular deposits of immunoglobulins, fibrinogen, and the C3 component of complement at the chondrofibrous junction was also demonstrated. From these findings, this case suggested that the pathogenesis of RP is related to an immune mechanism.     

Elastofibroma dorsi: An immunochemical study of collagen content

The types of collagen present in a case of elastofibroma dorsi were determined using type specific, characterized collagen antibodies. The presence of type II collagen (normally present only in articular cartilage and in selected ocular structures) is discussed with regard to the pathogenesis of this lesion, and the use of collagen antibodies is discussed with regard to their potential value in better characterizing and classifying mesenchymal tumors.     

Relapsing polychondritis developing in a patient with Graves' disease: a possible association with propylthiouracil]

A 31-year-old woman presented 1993 with fever, painful swelling of cartilaginous portions of the ears and the bridge of nose, polyarthralgia including costochondroral pains, and episcleritis. She has been taking propylthiouracil since 1991 when she was diagnosed as Graves' disease. Laboratory evaluations revealed an elevated erythrocyte sedimentation rate (ESR) of 133 mm/h, a high CRP level of 13.2 mg/dl and positive antinuclear antibodies and anti-type II collagen antibodies. Histopathological findings of the biopsy specimen from the auricular cartilage included chondrocyte degeneration, matrix destruction and inflammatory cell infiltration. She was diagnosed as RP and treatment with 30 mg/day of prednisolone dramatically improved all symptoms and signs, accompanied by a fall in ESR, CRP and autoantibodies. When prednisolone was tapered to 5 mg/day, a clinical relapse occurred. After discontinuation of propylthiouracil, she has been well without prednisolone. Propylthiouracil-induced SLE-like syndrome or antineutrophil cytoplasmic antibodies (ANCA) related angitis has been reported previously. In addition, recent studies demonstrated that about 20% of sera from patients with relapsing polychondritis are P-ANCA positive. This is the first report suggesting a possible association between the development of relapsing polychondritis and propylthiouracil.     

Light and electron microscopic study of ear cartilage in a case of relapsing polychondritis evolving under corticoid treatment

Summary Light and electron microscope studies of the ear cartilage in a patient with relapsing polychondritis (RP) under corticoid treatment are reported. Unilateral auricular deformation evolved without inflammatory episodes and the lesions consisted mainly of marginal erosions filled with fine collagen fibrils and containing degenerating perichondrial cells in their basal parts. Degenerative cells were scattered throughout the perichondrium, but cartilage erosions only occured when numerous cells were affected in a same area. Cartilage outside the eroded zones did not seem to be modified. Cartilage lesions thus appear to be a result of a chondrocyte renewal defect leading to loss of proteoglycans and elastic fibers, with only collagen remaining. These data suggest that inflammation is probably not the initial pathogenic process responsible for cartilage injury in RP, but that a metabolic defect in perichondrial cells might be involved.This work was carried out with a grant from the INSERM, CRL No 79. 1 218 4     

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.     

IgA nephropathy in relapsing polychondritis

Relapsing polychondritis (RP) is a multisystem autoimmune disease characterized by the presence of antibodies to type II collagen. This collagen is found predominantly in cartilaginous tissues, vitreous humor, aorta and notochord. Involvement of the kidney is rare, only 7 cases having been recorded, and there is no type II collagen in glomeruli. Six of the previous cases had crescentic glomerulonephritis. We report here two cases of biopsy proven RP in which IgA nephropathy was seen, the first examples recorded. Both patients had hematuria and slight proteinuria, with mild impairment of renal function. The histological and immunofluorescence pattern on both biopsies was in keeping with IgA nephropathy. Both patients received steroids with diminution/disappearance of hematuria and proteinuria. In view of the potentially progressive nature of glomerular disease with RP, the renal status should be investigated in all patients with RP.     

Differential detection of type II collagen N-terminal and C-terminal denaturation epitopes in degrading cartilage.

AIMS: To investigate the relative stability of collagen metabolites in degrading cartilage. METHODS: New antipeptide antibodies to denaturation epitopes located in the N-terminal and C-terminal regions of the type II collagen helix have been made and characterized. Type II collagen fragments in the conditioned medium from cultures of degrading bovine nasal cartilage were detected by immunoblotting with the new antisera as well as by N-terminal sequencing. The antibodies were also used in immunohistochemical studies of normal and osteoarthritic human cartilage. RESULTS: Type II collagen fragments with an apparent molecular mass of approximately 30 kDa were detected in cartilage conditioned media using antibody AH12L3, which recognizes N-terminal epitope AH12. The N-terminal sequence of one of these fragments matched exactly a sequence in the N-terminal region of type II collagen. Antibody AH9L2, which recognizes C-terminal epitope AH9, did not bind to any protein bands in the immunoblotted culture medium. In immunohistochemical studies, antibody AH12L3 detected extensive regions of degraded collagen in osteoarthritic cartilage and a more restricted pattern of staining in nonarthritic cartilage. Far less immunostaining was apparent in all cartilage specimens with antibody AH9L2. CONCLUSIONS: These results indicate that the N-terminal region of type II collagen is more resistant to proteolysis than the C-terminal region, an observation that has important implications for the choice of epitopes that are likely to be good markers of damage to cartilage collagen in patients with arthritis.     

Differential detection of type II collagen N-terminal and C-terminal denaturation epitopes in degrading cartilage.

AIMS: To investigate the relative stability of collagen metabolites in degrading cartilage. METHODS: New antipeptide antibodies to denaturation epitopes located in the N-terminal and C-terminal regions of the type II collagen helix have been made and characterized. Type II collagen fragments in the conditioned medium from cultures of degrading bovine nasal cartilage were detected by immunoblotting with the new antisera as well as by N-terminal sequencing. The antibodies were also used in immunohistochemical studies of normal and osteoarthritic human cartilage. RESULTS: Type II collagen fragments with an apparent molecular mass of approximately 30 kDa were detected in cartilage conditioned media using antibody AH12L3, which recognizes N-terminal epitope AH12. The N-terminal sequence of one of these fragments matched exactly a sequence in the N-terminal region of type II collagen. Antibody AH9L2, which recognizes C-terminal epitope AH9, did not bind to any protein bands in the immunoblotted culture medium. In immunohistochemical studies, antibody AH12L3 detected extensive regions of degraded collagen in osteoarthritic cartilage and a more restricted pattern of staining in nonarthritic cartilage. Far less immunostaining was apparent in all cartilage specimens with antibody AH9L2. CONCLUSIONS: These results indicate that the N-terminal region of type II collagen is more resistant to proteolysis than the C-terminal region, an observation that has important implications for the choice of epitopes that are likely to be good markers of damage to cartilage collagen in patients with arthritis.     

Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.     

Gene expression and matrix proteinase activity in osteochondrosis dessicans

Introduction Osteochondrosis dessicans (OCD) is a disorder of unknown aetiology where often a fragment of cartilage and subchondral bone separates from the articular surface. Previous studies have shown histological changes in glycosaminoglycan content in OCD cartilage compared to normal cartilage ( Koch et al. 1997 ). It has also been shown in equine OCD cartilage that there is excessive degradation of type‐II collagen compared to normal cartilage ( Laverty et al. 2002 ). The present study was undertaken to examine the gene expression in human OCD cartilage compared to its normal autologous articular cartilage and human osteoarthritic (OA) cartilage. Methods Cartilage from five OCD patients (18–34 years) was obtained at the time of surgery. Pieces of cartilage were either snap‐frozen (in preparation for RNA isolation) or the proteoglycans extracted with 4 m GuHCl. Total RNA was isolated from the cartilage using RNeasy minicolumns and reagents (Qiagen) according to the manufacturer's protocol. RT‐PCR was performed using an RNA PCR kit (Perkin‐Elmer) using a number of oligonucleotide primers. GuHCl‐extracted proteoglycan fragments were analysed using Western blotting with a number of antibodies to aggrecan metabolites, collagen metabolites and the small leucine‐rich proteoglycans. Results and discussion When OCD cartilage was compared to normal and human OA cartilage, there was an increase in aggrecan, collagen type‐II and collagen type‐X RNA expression. There was no change in RNA expression of link protein or type‐I collagen. The RNA expression of the aggrecanases (ADAMTS enzymes) was also different in the three different cartilage samples. Neither ADAMTS‐1, ‐4 or ‐5 was present in the normal cartilage. In contrast, in the OCD cartilage, there was expression of both ADAMTS‐1 and ‐4, whereas in the OA cartilage, there was expression of ADAMTS‐4 and ‐5. In the case of MMP RNA expression, MMP‐3 was decreased and MMP‐13 increased in OCD cartilage compared to both normal and OA samples. In addition, the expression of all three TIMP isoforms was increased in the OCD cartilage. Although inflammatory components are not expected in OCD pathology, expressions of inflammatory mediators such as COX‐2, IL‐1‐α and TNF‐α were all increased in the OCD cartilage when compared to normal, but expression of these mRNAs in the OA cartilage was higher. Analysis of proteoglycan fragments in the OCD cartilage by Western blotting showed the presence of aggrecan fragments containing the G1 domain, interglobular domain and the C‐terminal neoepitope generated by aggrecanase cleavage. There was also immunoreactivity for biglycan and link protein. Conclusion These results suggest that the phenotypic expression of chondrocytes at the site of the OCD lesion are markedly different from ‘normal’ articular cartilage and also pathological OA cartilage. Interestingly, the expression patterns of matrix proteinases and their natural inhibitors were also markedly different in OCD cartilage, again suggesting that there are specific biochemical expression patterns in OCD pathology, which may potentially be biomarkers of the disease process. Further studies are necessary to elucidate how the differences in gene expression and matrix protease activity may be involved in the aetiology of OCD.     

Site-specific ultrasound reflection properties and superficial collagen content of bovine knee articular cartilage

Previous quantitative 2D-ultrasound imaging studies have demonstrated that the ultrasound reflection measurement of articular cartilage surface sensitively detects degradation of the collagen network, whereas digestion of cartilage proteoglycans has no significant effect on the ultrasound reflection. In this study, the first aim was to characterize the ability of quantitative 2D-ultrasound imaging to detect site-specific differences in ultrasound reflection and backscattering properties of cartilage surface and cartilage-bone interface at visually healthy bovine knee (n = 30). As a second aim, we studied factors controlling ultrasound reflection properties of an intact cartilage surface. The ultrasound reflection coefficient was determined in time (R) and frequency domains (IRC) at medial femoral condyle, lateral patello-femoral groove, medial tibial plateau and patella using a 20 MHz ultrasound imaging instrument. Furthermore, cartilage surface roughness was quantified by calculating the ultrasound roughness index (URI). The superficial collagen content of the cartilage was determined using a FT-IRIS-technique. A significant site-dependent variation was shown in cartilage thickness, ultrasound reflection parameters, URI and superficial collagen content. As compared to R and IRC, URI was a more sensitive parameter in detecting differences between the measurement sites. Ultrasound reflection parameters were not significantly related to superficial collagen content, whereas the correlation between R and URI was high. Ultrasound reflection at the cartilage-bone interface showed insignificant site-dependent variation. The current results suggest that ultrasound reflection from the intact cartilage surface is mainly dependent on the cartilage surface roughness and the collagen content has a less significant role.     

Neoepitopes as biomarkers of cartilage catabolism

Progressive degradation of articular cartilage is a central feature of arthritis and a major determinant of long term joint dysfunction. There are no treatments able to halt the progression of cartilage destruction presently available, and monitoring the benefit of potential therapies is hampered by our inability to measure the "health" of articular cartilage. Serial radiographic assessment of joint space narrowing, the current gold standard, requires measurements over a prolonged time (1-5 years) and is prone to technical difficulties. Other strategies for evaluating cartilage degradation are needed to enable both short and long term monitoring of disease progression and response to therapy. One avenue that holds promise is the use of biomarkers that accurately reflect the degradative state of the articular cartilage. Antibodies that recognise terminal amino acid sequences generated by proteolysis at specific sites in the core protein of both aggrecan and type II collagen (neoepitope antibodies) have become available in recent years. These antibodies have been invaluable for identifying the proteinases responsible for cartilage breakdown both in vitro and in vivo. The presence of neoepitope sequences generated by specific metalloenzyme cleavage of aggrecan and type II collagen correlates well with the progression of cartilage degeneration, both in vitro and in mouse models of arthritis. Preliminary results with quantitative assays of type II collagen neoepitopes suggest that they may be useful markers of joint disease in humans. Long term studies correlating neoepitope concentration with clinical and radiographic disease are now required to validate the utility of neoepitopes as surrogate markers of cartilage degeneration and joint disease.     

Patterns of autoreactivity to collagen type II in autoimmune MRL/l mice.

The kinetics and mechanisms for secretion of antibodies against native and denatured collagen type II have been studied in spontaneously arthritic MRL/l mice. Circulating antibodies were quantified by an ELISA assay and frequencies of specific antibody secreting spleen cells by an ELISPOT assay. The degree of humoral immunity to collagen type II increased at late stages of the disease (6 months of age) whereas severe synovitis was seen earlier (5 months of age). Both the appearance of anti-collagen II producing cells and development of synovitis was preceded by and not correlated with a general state of polyclonal B cell activation. In MRL/l mice, collagen II specific antibodies appeared spontaneously and titres were largely unaffected by collagen II immunization. The levels of circulating anti-collagen II antibodies in MRL/l mice were lower, and the antibodies displayed lower avidities and different specificities as compared with the antibodies generated in collagen II high responder DBA/l mice after immunization with collagen II. It is suggested that the antibody response in MRL/l mice against collagen type II does not need MHC-restricted T cell help and that induction of antibody production to collagen II in MRL/l mice is triggered by joint cartilage destruction and subsequent collagen II release.     

MRI and histologic analysis of collagen type II sponge on repairing the cartilage defects of rabbit knee joints

There are limited treatment options for cartilage defects in clinical practice because of the lack of suitable biomaterials. Here, we evaluated the effects of collagen type II sponge on the articular cartilage repairing process using a cartilage injury of a rabbit knee joint model. We showed that the home-made collagen type II sponges appeared to have a suitable pore size of 93.26 ± 38.4 μm for chondrocyte growth. MRI with H&E staining results demonstrated that the effusion absorption in the collagen type II sponge treated group was quicker than that of the control group. Moreover, sporadic cartilage signals first appeared at 6 weeks in the collagen type II sponge treated group. Safranin O staining and immunohistochemical analysis confirmed that the newly formed cartilage expresses glycosaminoglycan and type II collagen matrix. Using Sirius red polarized light staining, we showed that the newly formed cartilage-like areas from the collagen type II treated group are significantly greater than those of the control group. Taken together, our data demonstrated that the home-made collagen type II sponge is able to promote cartilage repair in the cartilage injury of a rabbit knee joint model.     

Airway involvement in relapsing polychondritis

Relapsing Polychondritis (RP) is an uncommon, chronic, and potentially life-threatening multisystem disorder characterized by recurrent inflammatory episodes affecting the cartilaginous tissues of the external ears, nose, peripheral joints, larynx and tracheobronchial tree, sometimes leading to their destruction. RP can also inflame other proteoglycan-rich structures, such as the eye, heart, blood vessels and inner ears. Systemic symptoms are common, and vasculitis affecting skin or internal organs may occur. The etiology of RP is still unknown, but the pathogenetic role of the autoimmunity is suggested by frequent overlaps with various autoimmune diseases, and by the presence of autoantibody against cartilage in the serum of patients with RP. Although several reports have demonstrated the clinicopathologic manifestations and radiologic findings of RP, there are no specific features of RP. Therefore, it is difficult to show the diagnosis of RP. Airway involvements are major causes of morbidity and mortality, and they have accounted for most of the deaths due to RP. To suppress the inflammation of airway mucosa and cartilage is extremely important in the successful treatment for RP. Above all, earlier diagnosis would lead to better outcomes.     

Cartilage canals in human thyroid cartilage characterized by immunolocalization of collagen types I, II, pro-III, IV and X

In this study the collagenous composition of cartilage canals in human thyroid cartilage, which are perichondral invaginations of blood vessels and connective tissue, and the surrounding cartilage matrix were investigated by immunolabelling with specific antibodies against type I, II, pro-III, IV and X collagen. During childhood and early adolescence no cartilage canals were detected in thyroid cartilage, and immunolabelling for type IV collagen was restricted to basal lamina components of blood vessels in the perichondrium. First immunolabelling for type IV collagen, belonging to blood vessels in cartilage canals, in both sexes was detected about the end of the second decade; it was localized in the dorsal part of the thyroid cartilage plate. At this time thyroid cartilage has already reached its final form and size. As revealed by von Kossa staining, vascularization preceded mineralization and ossification. In contrast to the male thyroid cartilage plate, no immunostaining for type IV collagen and no ossification was detected in the ventral half of female thyroid cartilage even in advanced age. The extracellular matrix of cells in cartilage canals showed positive immunostaining for collagen types I and pro-III as well as for collagen type II, indicating that the cells in the canal possess fibroblastic and chondrogenic properties. The extracellular matrix of hypertrophic chondrocytes adjacent to cartilage canals showed strong immunoreactivity for type X collagen. First mineralization was detected close to cartilage canals, suggesting that mineralization in human thyroid cartilage starts in the extracellular matrix adjacent to cartilage canals.     

Immunohistological study on collagen in cartilage-bone metamorphosis and degenerative osteoarthrosis

Summary Synthesis of collagen by chondrocytes was studied by immunofluorescence using antibodies specific for type I, II and III collagen. The following tissues and culture conditions were chosen for this immunohistological study: normal articular cartilage, epiphyseal growth cartilage, cartilage undergoing osteoarthrotic degeneration, suspension culture and monolayer culture. While type II collagen is the unique collagen all over hyaline cartilage, type I collagen is produced by hypertrophic chondrocytes in the growth plate. In addition, chondrocytes in osteoarthrotic areas of articular cartilage synthesize type I collagen. Under in vitro culture conditions, chondrocytes initially produce type II collagen and synthesize later on type I collagen. The change of synthesis from type II to type I collagen is more rapid in monolayer than in suspension culture. It is concluded that the presence of matrix compounds and the cellmatrix interaction as well are necessary to maintain synthesis of type II collagen in chondrocytes. Alterations in the cell-matrix interactions are shown to occur in the hypertrophic zone of the epiphyseal growth plate, in cartilage undergoing osteoarthrotic degeneration as well as in chondrocytes grown in culture. Thus, change in the control of gene activity may subsequently lead to change in collagen synthesis. It is possible that the synthesis of type I collagen, which cannot fulfil the physiological function of a structural element in cartilageneous tissue, is a crucial factor in the process of osteoarthrosis.

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Abbreviations EDTA Ethylendiaminetetraacetate - FITC Fluoresceine isothiocyanate This investigation was supported by grants of the Deutsche Forschungsgemeinschaft, Mu 378/4, Re 388/1 and SFB 51     

Independent appearance of anti-thymocyte and anti-RNA antibodies in NZB/NZW F1 mice.

Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. alpha[1(I)]2alpha2. Artefactual triplehelical structures of the composition [alpha1(I)]3 or [alpha2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen culd already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 56 degrees and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [alpha1(II)]3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.     

Problems in the immunolocalization of type IX collagen in fetal calf cartilage using a monoclonal antibody

Monoclonal antibodies were prepared against the pepsin-resistant fragments (X1-X3) of bovine type IX collagen. One of the five hybridomas that gave a positive reaction in an enzyme-linked immunosorbent assay was selected (H1a) for structural analysis and immunolocalization of type IX collagen. The location of the epitope for H1a was deducted from immunoblots and electron microscopic observations after rotary shadowing. The H1a antibody binds to one end of the longest X2, X3, X4 molecules, and preferentially 40-55nm from one end of X1 molecules thus, on or near the noncollagenous domain, NC2. Different immunolocalizations of type IX collagen in the superficial, middle and deep zones of fetal calf epiphyseal cartilage were observed depending on the thickness of the section and on hyaluronidase digestion conditions. In the middle and deep zones, staining with H1a throughout the matrix was obtained only with thin sections (5 microns) and digestion for 1 h at 37 degrees C. With thick sections (15 microns) or with digestion for 1 h at 24 degrees C, staining was restricted to the pericellular regions. Staining throughout the matrix was obtained in the superficial zone under all experimental conditions. Without hyaluronidase treatment, no immunofluorescent staining was seen with either H1a or polyclonal antibody to type II collagen, indicating that type IX collagen is present throughout the matrix in the different zones of fetal calf cartilage. This result is in good accordance with the recent demonstration of common cross-links between type II and type IX collagen in chicken and bovine cartilage. However, the preferential unmasking of type IX collagen antigenic sites in the pericellular regions of middle and deep zones of fetal calf cartilage does not preclude the presence in that region of a special pericellular organization of the collagenous network.     

基质金属蛋白酶和胶原在创伤关节软骨组织中的表达

背景：研究发现,基质金属蛋白酶和胶原参与关节软骨组织机体生理重建及病理破坏。 目的：观察膝关节骨软骨缺损及表面软骨缺损动物模型关节软骨组织中胶原及基质金属蛋白酶的表达变化。 方法：雌性SD大鼠48只随机分为3组：骨软骨缺损组在双膝关节制作骨软骨缺损模型,表面缺损组在双膝关节制作表面软骨缺损,对照组双膝关节制作关节囊切开。分别于术后4、8、12周取股骨髁标本,行苏木精-伊红染色,免疫组化检测Ⅰ型胶原、Ⅱ型胶原、基质金属蛋白酶3的表达。 结果与结论：骨软骨缺损组术后4周缺损中有少量新生组织生成,8及12周可见到纤维组织填充,修复组织细胞外基质Ⅰ型胶原免疫组化染色阳性,Ⅱ型胶原免疫组化染色阴性,关节软骨组织中基质金属蛋白酶3表达增高。表面缺损组表面软骨缺损4及8周未见修复迹象,12周可见微量纤维组织填充,细胞外基质Ⅰ型胶原免疫组化染色阳性,Ⅱ型胶原免疫组化染色阴性,术后表面缺损组关节软骨组织基质金属蛋白酶3表达增高。对照组关节软骨组织Ⅰ型胶原免疫组化染色阴性,Ⅱ型胶原免疫组化染色阳性,基质金属蛋白酶3低表达,无形态学异常改变。说明机械性损伤可以导致关节软骨细胞外基质成分发生改变,丧失其原有的生物学特性而退变,基质金属蛋白酶3在损伤后的软骨组织中表达增高,使细胞外基质的降解增加,是导致关节软骨退变的重要因素。