Enzymatic Isolation of Articular Chondrons: Is It Much Different Than That of Chondrocytes?

In native articular cartilage, chondrocytes (Chy) are completely capsulated by a pericellular matrix (PCM), together called the chondron (Chn). Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and Chn may be important in regenerative strategies. The current gold standard for the isolation of Chns from cartilage dates from 1997. Although previous research already showed the low cell yield and the heterogeneity of the isolated populations, their compositions and properties have never been thoroughly characterized. This study aimed to compare enzymatic isolation methods for Chy and Chns and characterizes the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-h (5H) gold standard Chn isolation method (0.3% dispase +0.2% collagenase II), an overnight (ON) Chn isolation (0.15% dispase +0.1% collagenase II), and an ON Chy isolation (0.15% collagenase II +0.01% hyaluronidase). Type VI collagen staining, fluorescence-activated cell sorting (FACS) analysis, specific cell sorting, and immunohistochemistry were performed using a type VI collagen staining, to study their isolation efficiency and quality of the PCM. These analyses showed a heterogeneous mixture of Chy and Chns for all three methods. Although the 5H Chn isolation resulted in the highest percentage of Chns, the cell yield was significantly lower compared to the other isolation methods. FACS, based on the type VI collagen staining, successfully sorted the three identified cell populations. To maximize Chn yield and homogeneity, the ON Chn enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting. Impact statement Since chondrocytes are highly dependent on their microenvironment for maintaining phenotypic stability, it is hypothesized that using chondrons results in superior outcomes in cartilage tissue engineering. This study reveals the constitution of cell populations obtained after enzymatic digestion of articular cartilage tissue and presents an alternative method to obtain a homogeneous population of chondrons. These data can improve the impact of studies investigating the effect of the pericellular matrix on neocartilage formation.

Endotrophin and C6Ma3, serological biomarkers of type VI collagen remodelling, reflect endoscopic and clinical disease activity in IBD.

In inflammatory bowel disease (IBD), the chronic inflammation deeply affects the intestinal extracellular matrix. The aim of this study was to investigate if remodeling of the intestinal basement membrane type VI collagen was associated with pathophysiological changes in Crohn's disease (CD) and ulcerative colitis (UC). Serum from IBD patients (CD: n = 65; UC: n = 107; irritable bowel syndrome: n = 18; healthy subjects: n = 20) was investigated in this study. The serological biomarkers C6Ma3 (a matrix metalloproteinase (MMP) generated fragment of the type VI collagen α3 chain) and PRO-C6, also called endotrophin (the C-terminus of the released C5 domain of the type VI collagen α3 chain) were measured by ELISAs. Serum C6Ma3 was increased in CD patients with moderate to severe and mild endoscopically active disease compared to endoscopic remission (p = 0.002, p = 0.0048), respectively, and could distinguish endoscopically active disease from remission with an AUC of 1.0 (sensitivity: 100%, specificity: 100%) (p < 0.0001), which was superior to CRP. C6Ma3 was increased in CD patients with moderate to severe clinical disease compared to mild and remission (p = 0.04; p = 0.009). Serum PRO-C6, endotrophin, was increased in CD patients in clinically remission compared to mild disease (p = 0.04) and moderate to severe disease (p = 0.065). In UC, fecal calprotectin was the only marker that alone could distinguish both clinical and endoscopic active and inactive disease. Type VI collagen degradation of the α3 chain mediated by MMPs was increased in CD patients with endoscopically active disease, measured by the serological biomarker C6Ma3, which was able to distinguish endoscopically active from inactive CD.

Prognostic value of blood-based fibrosis biomarkers in patients with metastatic colorectal cancer receiving chemotherapy and bevacizumab.

A desmoplastic colorectal cancer stroma, characterized by excess turnover of the cancer-associated fibroblast derived collagens type III and VI, can lead to reduced drug-uptake and poor treatment response. We investigated the association between biomarkers of collagen type III and VI and overall survival (OS) in patients with metastatic colorectal cancer (mCRC). Serum samples were collected from 252 patients with mCRC prior to treatment with bevacizumab and chemotherapy. Serum concentrations of biomarkers reflecting formation of collagen type III (PRO-C3) and VI (PRO-C6) and degradation of collagen type VI (C6M and C6Mα3) were determined by ELISA. The biomarkers were evaluated for associations with OS, individually, combined, and after adjusting for carcinoembryonic antigen (CEA), lactate dehydrogenase (LDH) and performance status (PS). High baseline levels (> median) of each collagen biomarker were significantly associated with shorter OS (PRO-C3: HR = 2.0, 95%CI = 1.54-2.63; PRO-C6: HR = 1.6, 95%CI = 1.24-2.11; C6M: HR = 1.4, 95%CI = 1.05-1.78; C6Mα3: HR = 1.6, 95%CI = 1.16-2.07). PRO-C3 and PRO-C6 remained significant after adjustment for CEA, LDH and PS. Weak correlations were seen between the collagen biomarkers (r = 0.03-0.59) and combining all improved prognostic capacity (HR = 3.6, 95%CI = 2.30-5.76). Collagen biomarkers were predictive of shorter OS in patients with mCRC. This supports that collagen- and CAF biology is important in CRC.

Structural and compositional diversity of fibrillin microfibrils in human tissues.

Elastic fibers comprising fibrillin microfibrils and elastin are present in many tissues, including the skin, lungs, and arteries, where they confer elasticity and resilience. Although fibrillin microfibrils play distinct and tissue-specific functional roles, it is unclear whether their ultrastructure and composition differ between elastin-rich (skin) and elastin-poor (ciliary body and zonule) organs or after in vitro synthesis by cultured cells. Here, we used atomic force microscopy, which revealed that the bead morphology of fibrillin microfibrils isolated from the human eye differs from those isolated from the skin. Using newly developed pre-MS preparation methods and LC-MS/MS, we detected tissue-specific regions of the fibrillin-1 primary structure that were differentially susceptible to proteolytic extraction. Comparing tissue- and culture-derived microfibrils, we found that dermis- and dermal fibroblast-derived fibrillin microfibrils differ in both bead morphology and periodicity and also exhibit regional differences in fibrillin-1 proteolytic susceptibility. In contrast, collagen VI microfibrils from the same dermal or fibroblast samples were invariant in ultrastructure (periodicity) and protease susceptibility. Finally, we observed that skin- and eye-derived microfibril suspensions were enriched in elastic fiber- and basement membrane-associated proteins, respectively. LC-MS/MS also identified proteins (such as calreticulin and protein-disulfide isomerase) that are potentially fundamental to fibrillin microfibril biology, regardless of their tissue source. Fibrillin microfibrils synthesized in cell culture lacked some of these key proteins (MFAP2 and -4 and fibrillin-2). These results showcase the structural diversity of these key extracellular matrix assemblies, which may relate to their distinct roles in the tissues where they reside.

Rheumatic Heart Disease and Myxomatous Degeneration: Differences and Similarities of Valve Damage Resulting from Autoimmune Reactions and Matrix Disorganization.

Autoimmune inflammatory reactions leading to rheumatic fever (RF) and rheumatic heart disease (RHD) result from untreated Streptococcus pyogenes throat infections in individuals who exhibit genetic susceptibility. Immune effector mechanisms have been described that lead to heart tissue damage culminating in mitral and aortic valve dysfunctions. In myxomatous valve degeneration (MXD), the mitral valve is also damaged due to non-inflammatory mechanisms. Both diseases are characterized by structural valve disarray and a previous proteomic analysis of them has disclosed a distinct profile of matrix/structural proteins differentially expressed. Given their relevance in organizing valve tissue, we quantitatively evaluated the expression of vimentin, collagen VI, lumican, and vitronectin as well as performed immunohistochemical analysis of their distribution in valve tissue lesions of patients in both diseases. We identified abundant expression of two isoforms of vimentin (45 kDa, 42 kDa) with reduced expression of the full-size protein (54 kDa) in RHD valves. We also found increased vitronectin expression, reduced collagen VI expression and similar lumican expression between RHD and MXD valves. Immunohistochemical analysis indicated disrupted patterns of these proteins in myxomatous degeneration valves and disorganized distribution in rheumatic heart disease valves that correlated with clinical manifestations such as valve regurgitation or stenosis. Confocal microscopy analysis revealed a diverse pattern of distribution of collagen VI and lumican into RHD and MXD valves. Altogether, these results demonstrated distinct patterns of altered valve expression and tissue distribution/organization of structural/matrix proteins that play important pathophysiological roles in both valve diseases.

Col6a1 null mice as a model to study skin phenotypes in patients with collagen VI related myopathies: expression of classical and novel collagen VI variants during wound healing.

Patients suffering from collagen VI related myopathies caused by mutations in COL6A1, COL6A2 and COL6A3 often also display skin abnormalities, like formation of keloids or "cigarette paper" scars, dry skin, striae rubrae and keratosis pilaris (follicular keratosis). Here we evaluated if Col6a1 null mice, an established animal model for the muscle changes in collagen VI related myopathies, are also suitable for the study of mechanisms leading to the skin pathology. We performed a comprehensive study of the expression of all six collagen VI chains in unwounded and challenged skin of wild type and Col6a1 null mice. Expression of collagen VI chains is regulated in both skin wounds and bleomycin-induced fibrosis and the collagen VI α3 chain is proteolytically processed in both wild type and Col6a1 null mice. Interestingly, we detected a decreased tensile strength of the skin and an altered collagen fibril and basement membrane architecture in Col6a1 null mice, the latter being features that are also found in collagen VI myopathy patients. Although Col6a1 null mice do not display an overt wound healing defect, these mice are a relevant animal model to study the skin pathology in collagen VI related disease.

The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture.

BACKGROUND AND OBJECTIVE: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. MATERIAL AND METHODS: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. RESULTS: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h. CONCLUSION: The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases.

Abnormal human chondrocyte morphology is related to increased levels of cell-associated IL-1ß and disruption to pericellular collagen type VI.

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL-1ß and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2-photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a "smooth" membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL-1ß or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno-histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL-1ß-associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r(2) = 0.988) between the number of processes/cell (0-5) and IL-1ß, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL-1ß and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration.

Distribution of extracellular matrix components in normal and degenerated canine tricuspid valve leaflets.

The aim of the present study was to investigate the composition and distribution of various extracellular matrix (ECM) components in normal canine tricuspid valves (TVs) and in TVs affected by chronic valvular disease (CVD). The parietal (pTV) and septal (sTV) leaflets of the TVs from 27 dogs were investigated immunohistochemically for expression of collagen types I, III, IV and VI, elastin, laminin, fibronectin and heparan sulphate. Normal pTV consisted mainly of elastin and collagen VI in the atrialis, fibronectin in the thin spongiosa and mixed collagens in the fibrosa. The layered structure was less distinct in sTV, with numerous adipocytes and proteoglycans in the spongiosa and collagen III predominating in the fibrosa. The earliest stages of CVD affecting the pTV were recognized in the spongiosa and progression to advanced disease was characterized by nodular accumulation of proteoglycans within the free edge of the leaflet. These nodular lesions of the pTV contained more fibronectin, elastin and collagens I and VI than those affecting the sTV. These findings contrast with those reported in CVD affecting the mitral valve (MV) in which the early lesions affect the atrialis and advanced disease involves the entire leaflet. The pathogenesis of CVD in TV may involve initial alterations of the tricuspid annulus that lead to early lesions within the spongiosa, resulting in further shear stress and proteoglycan accumulation at the free edge of the pTV.

Extracellular matrix remodelling in human diabetic neuropathy.

The extracellular matrix of peripheral nerve plays a vital role in terms of normal nerve fibre function and also in the regenerative response following nerve injury. Nerve fibre loss is a major feature of diabetic neuropathy; however, the regenerative response is limited and this may be associated with changes in the composition of the extracellular matrix. Glycoproteins and collagens are major components of the extracellular matrix and are known to be important in terms of axonal growth. This work has therefore examined whether changes in the expression of two major glycoproteins, laminin and tenascin, and three collagen types (IV, V and VI) occur in the endoneurial and perineurial connective tissue compartments of human diabetic nerve. Despite being known to have a positive effect in terms of axonal growth, laminin levels were not elevated in the diabetic nerves. However, the pattern of tenascin expression did differ between the two groups being found in association with axon myelin units in the diabetic samples only. The pattern of collagen IV expression was the same in both tissue groups and was not found to be up-regulated. However, levels of collagen V and VI were both significantly increased in the endoneurium and for collagen VI also in the perineurium.

Using proteomics, immunohistology, and atomic force microscopy to characterize surface damage to lambskins observed after enzymatic dewooling.

The effects of conventional lime sulfide depilation and enzymatic depilation on the enamel layer of pickled lamb pelts were examined using atomic force and optical microscopy, immunohistological, and proteomic techniques. Microscopy showed that the surface structure of enzymatically depilated material was visibly less organized than conventionally processed material, implying that the enzymes used for depilation were responsible for this difference. Proteomic analyses identified an absence of collagen VI in the enamel of skins that had been processed with enzymes, in contrast to the skins that had been processed using conventional methods, which was confirmed using immunolocalization studies. It is therefore possible that the destruction of collagen VI during enzymatic depilation may cause the changes to the enamel structure observed during enzyme processing and in turn affect the quality of the finished product.

A refined diagnostic algorithm for Bethlem myopathy.

OBJECTIVE: Mutations in COL6A1, COL6A2, and COL6A3, the genes that encode the extracellular matrix component collagen VI, lead to Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). Unlike UCMD, BM is difficult to diagnose because of its clinical overlap with other contractural phenotypes and the lack of sensitivity of standard muscle biopsy immunohistochemical diagnostic techniques. METHODS: We appraised two potential techniques for the diagnosis of BM: dual immunofluorescence (IF) for collagen VI and basal lamina-located perlecan in muscle, and immunofluorescent labeling of collagen VI in skin biopsy-derived fibroblast cultures, which was conducted in 40 patients by blinded investigators and correlated with genetic findings. RESULTS: Dual IF was indistinguishable from normal controls in most BM patients. However, abnormalities in the IF labeling pattern of collagen VI were detected in more than 78% of genetically confirmed BM patient fibroblast cell lines. In addition, in a group of patients with unknown diagnosis studied prospectively, the fibroblast IF technique was highly predictive of the presence of a COL6A mutation, providing a positive predictive value of 75%, a sensitivity and negative predictive value of 100%, and a specificity of 63%. CONCLUSIONS: Immunofluorescent labeling of collagen VI in fibroblast cultures is a useful addition to current diagnostic services for Bethlem myopathy (BM). It can be used to guide molecular genetic testing, the gold standard diagnostic technique for BM, in a cost-effective and time-saving manner.

Three-dimensional morphology of the pericellular matrix of intervertebral disc cells in the rat.

Intervertebral disc cells are surrounded by a pericellular matrix that is biochemically and morphologically distinct from other extracellular matrix regions. Although the function of the pericellular matrix is not fully understood, prior studies of pericellular matrix-chondrocyte regions in articular cartilage (termed 'chondrons') suggest that the size, shape, and mechanical properties of the pericellular matrix significantly influence the micromechanical environment of the contained cells. A first step in understanding the role of the pericellular matrix in the intervertebral disc is to quantify the three-dimensional morphology and zonal variations of these regions across the disc. In this study, three-dimensional reconstructions and morphometric measurements of pericellular matrix-cell regions were obtained in situ using fluorescence confocal microscopy of en bloc sections of nucleus pulposus and anulus fibrosus of the rat disc immunolabeled for type VI collagen. The morphology of the pericellular matrix and cells varied significantly across regions, with distinct pericellular matrix aspect ratios (largest/smallest diameter) showing shapes that were generally large and rounded in the nucleus pulposus (average of 1.9), and ellipsoidal and discoidal in the inner (2.4) and outer anulus fibrosus (2.8). The average pericellular matrix volume per cell was found to be significantly larger in the nucleus (6424 microm(3)) than that of inner (1903 microm(3)) and outer (1433 microm(3)) anulus. Pericellular matrix regions containing 1 or 2 cells were the dominant subgroup in the rat intervertebral disc at both 1 and 12 months of age. Multicellular pericellular matrix regions were present more often in the younger nucleus pulposus and outer anulus fibrosus. The orientation of the pericellular matrix regions further varied significantly across the disc, reflecting local collagen matrix architecture. These studies provide new information on the organization and shape of intervertebral disc cells and their surrounding pericellular matrix, which may provide new insights into the mechanisms that regulate cell-matrix interactions.

An immunohistochemical study of the triangular fibrocartilage complex of the wrist: regional variations in cartilage phenotype.

The triangular fibrocartilage complex (TFCC) transmits load from the wrist to the ulna and stabilizes the distal radioulnar joint. Damage to it is a major cause of wrist pain. Although its basic structure is well established, little is known of its molecular composition. We have analysed the immunohistochemical labelling pattern of the extracellular matrix of the articular disc and the meniscal homologue of the TFCC in nine elderly individuals (age range 69-96 years), using a panel of monoclonal antibodies directed against collagens, glycosaminoglycans, proteoglycans and cartilage oligomeric matrix protein (COMP). Although many of the molecules (types I, III and VI collagen, chondroitin 4 sulphate, dermatan sulphate and keratan sulphate, the oversulphated epitope of chondroitin 6 sulphate, versican and COMP) were found in all parts of the TFCC, aggrecan, link protein and type II collagen were restricted to the articular disc and to entheses. They were thus not a feature of the meniscal homologue. The shift in tissue phenotype within the TFCC, from a fibrocartilaginous articular disc to a more fibrous meniscal homologue, correlates with biomechanical data suggesting that the radial region is stiff and subject to considerable stress concentration. The presence of aggrecan, link protein and type II collagen in the articular disc could explain why the TFCC is destroyed in rheumatoid arthritis, given that it has been suggested that autoimmunity to these antigens results in the destruction of articular cartilage. The differential distribution of aggrecan within the TFCC is likely to be reflected by regional differences in water content and mobility on the radial and ulnar side. This needs to be taken into account in the design of improved MRI protocols for visualizing this ulnocarpal complex of the wrist.

The evolution of collagen expression in sarcomatoid renal cell carcinoma.

The development of a sarcomatoid morphotype is recognized as an extreme form of dedifferentiation in renal cell carcinoma and is associated with a poor prognosis. Although sarcomatoid renal cell carcinoma shows pronounced spindle cell morphology, clear cell renal cell carcinoma may show early spindle cell change with cellular elongation, and the prognostic significance of this is debated. To determine the relationship between sarcomatoid renal cell carcinoma and clear cell renal cell carcinoma showing early spindle cell change, we have investigated collagen expression using immunohistochemistry in these 2 tumor types. Both sarcomatoid renal cell carcinoma and early spindle cell change tumors showed pericellular interstitial expression of collagen types I and III, whereas sarcomatoid renal cell carcinoma also showed cytoplasmic expression of these collagen types. Expression of these collagen types in typical clear cell renal cell carcinoma was, in occasional cases, limited to faint and patchy staining in a pericellular interstitial distribution. Tumor cells did not stain for collagen type IV in sarcomatoid renal cell carcinoma, early spindle cell change, or typical clear cell renal cell carcinoma. In sarcomatoid renal cell carcinoma, there was diffuse pericellular expression of collagen type V and patchy pericellular expression of collagen type VI, whereas early spindle cell change tumors showed patchy pericellular staining with antibodies to collagen type V. Collagen type VI expression in early spindle cell change was largely confined to the vascular adventitia and areas of scarring, although very occasional foci of faint interstitial staining were also seen. In typical clear cell renal cell carcinoma, staining of collagen types V and VI was limited to the vascular adventitia and foci of desmoplasia, whereas no staining of tumor cell cytoplasm were seen. This study has shown that collagen expression of sarcomatoid renal cell carcinoma differs from that of early spindle cell change and provides validating evidence that these 2 morphotypes should not be considered together for classification purposes.

Specific inhibition of nonsense-mediated mRNA decay components, SMG-1 or Upf1, rescues the phenotype of Ullrich disease fibroblasts.

Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that degrades aberrant mRNAs containing premature translation termination codons (PTCs). The essential proteins for NMD include SMG-1, a protein kinase, and Upf1, a substrate of SMG-1 with RNA helicase activity. In this study, we evaluated the effects of NMD inhibition by siRNA-mediated knockdown of SMG-1 or Upf1 on the phenotype of Ullrich disease, an autosomal recessive congenital muscular dystrophy. The patient studied showed a homozygous frameshift mutation with a PTC in the collagen VI alpha2 gene, which encodes a truncated but partially functional protein. The patient's fibroblasts showed a nearly complete loss of the triple-helical collagen VI protein and functional defects in the extracellular matrix (ECM) due to the crucial deficiency of the collagen VI alpha2 protein. We have shown that siRNA-mediated knockdown of SMG-1 or Upf1 causes the up-regulation of the mutant triple-helical collagen VI, resulting in the formation of partially functional ECM. We suggest that the inhibition of NMD may be useful as a therapeutic approach to treat some human genetic diseases exacerbated by NMD.

Zonal variations in the three-dimensional morphology of the chondron measured in situ using confocal microscopy.

OBJECTIVE: Chondrocytes in articular cartilage are surrounded by a narrow pericellular matrix (PCM), which together with the enclosed cell(s) are termed the "chondron". Although the precise function of this tissue region is unknown, previous studies provide indirect evidence that the PCM plays an important role in governing the local mechanical environment of chondrocytes. In particular, theoretical models of the chondron under mechanical loading suggest that the shape, size, and biomechanical properties of the PCM significantly influence the stress-strain and fluid flow environment of the cell. The goal of this study was to quantify the three-dimensional morphology of chondron in situ using en bloc immunolabeling of type VI collagen coupled with fluorescence confocal microscopy. METHODS: Three-dimensional reconstructions of intact, fluorescently labeled chondrons were made from stacks of confocal images recorded in situ from the superficial, middle, and deep zones of porcine articular cartilage of the medial femoral condyle. RESULTS: Significant variations in the shape, size, and orientation of chondrocytes and chondrons were observed with depth from the tissue surface, revealing flattened discoidal chondrons in the superficial zone, rounded chondrons in the middle zone, and elongated, multicellular chondrons in the deep zone. CONCLUSIONS: The shape and orientation of the chondron appear to reflect the local collagen architecture of the interterritorial matrix, which varies significantly with depth. Quantitative measurements of morphology of the chondron and its variation with site, disease, or aging may provide new insights into the influence of this structure on physiology and the pathology of articular cartilage.

Identification, content, and distribution of type VI collagen in bovine tendons.

Tendon composition changes according to differentiation, mechanical load, and aging. In this study, we attempted to identify, localize, and quantify type VI collagen in bovine tendons. Type VI collagen was identified by the electrophoretic behavior of the alpha chains and Western blotting, and by rotary shadowing. Type VI collagen was extracted from powdered tendon with three sequential 24-h extractions with 4 M guanidine-HCl. The amount of type VI collagen was determined by enzyme-linked immunosorbent assay for purely tensional areas and for the compressive fibrocartilage regions of the deep flexor tendon of the digits, for the corresponding fetal and calf tendons, and for the extensor digital tendon. The distal fibrocartilaginous region of the adult tendon was richer in type VI collagen than the tensional area, reaching as much as 3.3 mg/g (0.33%) of the wet weight. Calf tendons showed an accumulation of type VI at the fibrocartilage site. Immunocytochemistry demonstrated that type VI collagen was evenly distributed in the tensional areas of tendons but was highly concentrated around the fibrochondrocytes in the fibrocartilages. The results demonstrate that tendons are variable with regard to the presence and distribution of type VI collagen. The early accumulation of type VI collagen in the region of calf tendon that will become fibrocartilage in the adult suggests that it is a good marker of fibrocartilage differentiation. Furthermore, the distribution of type VI collagen in tendon fibrocartilage indicates that it organizes the pericellular environment and may represent a survival factor for these cells.

Immunolocalization of matrix proteins in different human cartilage subtypes.

Cartilage exerts many functions in different tissues and parts of the body. Specific requirements presumably also account for a specific biochemical composition. In this study, we investigated the presence and distribution pattern of matrix components, in particular collagen types in the major human cartilages (hyaline, fibrous, and elastic cartilage) by histochemical and immunohistochemical means. Macroscopically normal articular cartilages, menisci, disci (lumbar spine), epiglottal, and tracheal tissues were obtained from donors at autopsy. Aurical and nasal cartilages were part of routine biopsy samples from tumor resection specimens. Conventional histology and immunohistochemical stainings with collagen types I, II, III, IV, V, VI, and X and S-100 protein antibodies were performed on paraformaldehyde-fixed and paraffin-embedded specimens. The extracellular matrix is the functional component of all cartilages as indicated by the low cell densities. In particular major scaffold forming collagen types I (in fibrous cartilage) and II (in hyaline and elastic cartilages) as well as collagen type X (in the calcified layer of articular cartilages, the inner part of tracheal clips, and epiglottis cartilage) showed a specific distribution. In contrast, the "minor" collagen types III, V, and VI were found in all, collagen type IV in none of the cartilage subtypes. In this study, we present a biochemical profile of the major cartilage types of the human body which is important for understanding the physiology and the pathophysiology of cartilages.

Characterisation of collagen VI within the islet-exocrine interface of the human pancreas: implications for clinical islet isolation?

BACKGROUND: To optimize the methods used for human islet isolation for transplantation, it is important to improve our understanding of the structure of the islet-exocrine interface. In this study, the composition of collagen subtypes in the interface have been characterized and quantified in human pancreas. METHODS: Human adult pancreases were retrieved from older (mean age 55.7+/-3.0 yrs) and young donors (mean age 21.8+/-3.2 yrs). Tissue from the body of each pancreas was examined by quantitative immunohistochemistry. Collagen within the islet-exocrine interface was identified by immunolabeling for collagen I, IV, V or VI and islets identified either morphologically or by immunolabeling for insulin. Collagen subtypes were quantified and data expressed as collagen area at the interface relative to the islet area. Statistical analysis was by ANOVA or Mann Whitney U test. RESULTS: In older pancreases, collagen IV, V and VI were present throughout the islet-exocrine interface, whereas collagen I was more variable. The mean peri-islet collagen VI proportion was significantly greater than that of collagen I or IV. Mean islet area and the proportional collagen VI content in specimens from younger subjects were not significantly different to those in older subjects. CONCLUSIONS: Collagen VI is a major component of the islet-exocrine interface of the adult pancreas, the content being more than double that of collagen I or IV. However, the proportional collagen VI content was not dependent on the age of the donor. These data may facilitate the design of new collagenases, targeting major substrates such as collagen VI in order to improve clinical islet isolation.