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PubMed1983

[Ultrastructural studies of healing process of injured articular cartilage].

This study was undertaken to investigate the healing process and the nature of the repaired tissue of injured articular cartilage. Full-thickness defects were made in the articular cartilage of the femoral condyles of 48 immature rats, and were examined by using light and transmission electron microscopes from one to 48 weeks after the injury. Under the light microscope, toluidine blue metachromasia was demonstrated in the deep matrix at 4 weeks after the injury. The repaired tissue was similar to the articular cartilage in controls at 12 weeks after the injury, but decreased number of cells and irregular surface were recognized in the specimen taken at 48 weeks after the injury. By electron microscopic examinations, at 12 weeks after the injury, cells in the repaired tissue were indistinguishable from chondrocytes, and the tidemark showed a fine structure similar to that in the articular cartilage in controls where many electron dense particles were observed. Collagen fibrils in the superficial layer were arranged in parallel to the articular surface. Three dimensional meshwork of collagen fibrils was also recognized in the intermediate and deep layers, but was inferior to that in the articular cartilage in controls. At 48 weeks after the injury, the surviving cells in the repaired tissue contained deposits of glycogen, large lipid droplets and vacuoles. Amianthoid change and fibrous long-spaced collagen were occasionally observed in the matrix. Collagen fibrils in the areas with poor cell population were smaller in diameter and loosely arranged. The above findings indicate that the anomalous arrangement of collagen fibrils is a main cause for the weakness of the repaired tissue to the mechanical load, resulting the degeneration.

Animals↗
PubMed2007

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9.

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.

Adenoviridae↗
PubMed1977

The localization of articular cartilage proteoglycan by electron microscopy.

Rabbit articular cartilage was fixed with glutaraldehyde containing Ruthenium Red or Safranin O proteoglycan localization is easily obtained. Ruthenium Red stained proteoglycan is easily visualized by electron microscopy. Previous digestion with papain prevented staining by either technique confirming that the material was indeed proteoglycan. Using these methods of proteoglycan identification the sites of attachment to collagen are shown as well as the deposition of proteoglycan about cells of differing vitality and the relationship of proteoglycan to cell membranes.

Animals↗
PubMedDate not supplied

Involvement of prostaglandins from rheumatoid synovium in inhibition of articular cartilage metabolism.

Short term incubations of articular cartilage in media from cultures of rheumatoid synovial tissue result in marked inhibition of 3H-glycine and Na235SO4 incorporation into trichloroacetic acid (TCA) insoluble macromolecules by the cartilage segments with little effect on degradation of prelabeled cartilage matrix. This inhibition is time dependent and not observed in cartilage segments incubated in media derived from rheumatoid synovia cultured in the presence of indomethacin (a specific inhibitor of prostaglandin synthesis). In addition, no indomethacin-sensitive cartilage inhibitory activity was detectable in media from cultures of normal synovia. The ether solubility of the majority of the inhibitory activity and the indomethacin sensitivity suggest that the inhibitor(s) are prostaglandins.

Animals↗
PubMed1984

Changes in proteoglycan biosynthesis following leukocyte elastase treatment of bovine articular cartilage in culture.

Treatment of bovine articular cartilage in culture with a low molecular weight elastase purified from rabbit polymorphonuclear leukocytes resulted in degradation and release of proteoglycans from the tissue, coupled with a prolonged inhibition of proteoglycan biosynthesis. These observations are consistent with those seen in experimental arthritis induced in rabbits. Comparison of the size of the proteoglycan degradation products extracted from elastase-treated cartilage in culture with that from arthritic cartilage showed marked similarities. The results strongly suggest that leukocyte elastase is a contributing factor in proteoglycan degradation and inhibition of synthesis in inflammatory joint disease.

Animals↗
PubMed2006

Comparison of the effects of mechanical and osmotic pressures on the collagen fiber architecture of intact and proteoglycan-depleted articular cartilage.

One of the functions of articular cartilage is to withstand recurrent pressure applied in everyday life. In previous studies, osmotic pressure has been used to mimic the effects of mechanical pressure. In the present study, the response of the collagen network of intact and proteoglycans (PG)-depleted cartilage to mechanical and osmotic pressures is compared. The technique used is one-dimensional (2)H double quantum filtered spectroscopic MRI, which gives information about the degree of order and the density of the collagen fibers at the different locations throughout the intact tissue. For the nonpressurized plugs, the depletion had no effect on these parameters. Major differences were found in the zones near the bone between the effects of the two types of application of pressure for both intact and depleted plugs. While the order is lost in these zones as a result of mechanical load, it is preserved under osmotic pressure. For both intact and PG-depleted plugs under osmotic stress most of the collagen fibers become disordered. Our results indicate that different modes of strain are produced by unidirectional mechanical load and the isotropic osmotic stress. Thus, osmotic stress cannot serve as a model for the effect of load on cartilage in vivo.

Animals↗
PubMed2005

Repair of porcine articular cartilage defect with autologous chondrocyte transplantation.

Articular cartilage is known to have poor healing capacity after injury. Autologous chondral grafting remains the mainstay to treat well-defined, full-thickness, symptomatic cartilage defects. We demonstrated the utilization of gelatin microbeads to deliver autologous chondrocytes for in vivo cartilage generation. Chondrocytes were harvested from the left forelimbs of 12 Lee-Sung pigs. The cells were expanded in monolayer culture and then seeded onto gelatin microbeads or left in monolayer. Shortly before implantation, the cell-laden beads were mixed with collagen type I gel, while the cells in monolayer culture were collected and re-suspended in culture medium. Full-thickness cartilage defects were surgically created in the weight-bearing surface of the femoral condyles of both knees, covered by periosteal patches taken from proximal tibia, and sealed with a porcine fibrin glue. In total, 48 condyles were equally allotted to experimental, control, and null groups that were filled beneath the patch with chondrocyte-laden beads in gel, chondrocytes in plain medium solution, or nothing, respectively. The repair was examined 6 months post-surgery on the basis of macroscopic appearance, histological scores based on the International Cartilage Repair Society Scale, and the proportion of characteristic chondrocytes. Tensile stress-relaxation behavior was determined from uniaxial indentation tests. The experimental group scored higher than the control group in the categories of matrix nature, cell distribution pattern, and absence of mineralization, with similar surface smoothness. Both the experimental and control groups were superior to the null group in the above-mentioned categories. Viable cell populations were equal in all groups, but the proportion of characteristic chondrocytes was highest in the experimental group. Matrix stiffness was ranked as null > native cartilage > control > experimental group. Transplanted autologous chondrocytes survive and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.

Animals↗
PubMed1989

Stress in collagen fibrils of articular cartilage calculated from their measured orientations.

Articular cartilage may be considered as a form of pressure vessel in which the internal swelling pressure is balanced by tensile stress in the collagen fibrils. This stress is calculated by analysing the tissue as a series of microscopically small pressure vessels. The previously measured orientations of the collagen fibrils describe the structure necessary for this calculation. The stresses and strains developed in the fibrils are shown to be well within physiological limits.

Animals↗
PubMed1980

Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages.

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536--544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.

Animals↗
PubMed1975

Breakdown of proteoglycan and collagen induced in pig articular cartilage in organ culture.

Explants of articular cartilage from young pigs were maintained in organ culture for 10--16 days, and degradation of matrix was induced by retinol or complement-sufficient antiserum. The percentage breakdown of proteoglycan and collagen (as hydroxyproline release) was measured. The response of the cartilage depended on whether or not the explants were cut so as to include some of the invading marrow ('invasion zone'). In media containing retinol, cartilage lost up to three-quarters of its proteoglycan whether the invasion zone was present or not, but very little of its collagen unless this region was included. In the presence of complement-sufficient anti-serum, however, cartilage without the invasion zone was virtually unaffected, but both proteoglycan and hydroxyproline were released when invasion zone was included; here proteoglycan release began almost immediately, but there was a time-lag of 6--8 days before a substantial amount of hydroxyproline appeared in the medium. Histological examination of sample explants from the experiments supported the biochemical findings. The possible significance of the results in relation to rheumatoid arthritis is discussed.

Animals↗
PubMed1977

Effect of age on thickness of adult patellar articular cartilage.

The thickness of left patellar articular cartilages after formalin fixation was studied in a series of autopsies on 82 subjects aged 25-96 years. For each specimen the minimal uncalcified cartilage thickness in a transverse patellar slab was determined separately for a 'lateral' segment and a 'central and medial' segment. In the women the cartilage from subjects more than 50 years old showed progressive thinning with increasing age. This was due to lesions causing disintegration of the tissue and not to matrix shrinkage. It had a strong potential to progress to full-thickness cartilage loss in the older women, and to give an appearance indistinguishable from osteoarthrosis as seen in surgical excision specimens. This progression towards patellofemoral osteoarthrosis in the elderly affects the female population generally, and not just a special subgroup; however, the incidence of clinical symptoms from this cause is not known. In men progressive thinning with age of patellar cartilage in subjects more than 50 years old was less severe, especially so in the case of the 'lateral' segment; a site of full-thickness uncalcified cartilage loss on the left patella at autopsy was seen only occasionally in the older men.

Adult↗
PubMed2006

Periodic rewetting enhances the viability of chondrocytes in human articular cartilage exposed to air.

Desiccation of articular cartilage during surgery is often unavoidable and may result in the death of chondrocytes, with subsequent joint degeneration. This study was undertaken to determine the extent of chondrocyte death caused by exposure to air and to ascertain whether regular rewetting of cartilage could decrease cell death. Macroscopically normal human cartilage was exposed to air for 0, 30, 60 or 120 minutes. Selected samples were wetted in lactated Ringer's solution for ten seconds every ten or 20 minutes. The viability of chondrocytes was measured after three days by Live/Dead staining. Chondrocyte death correlated with the length of exposure to air and the depth of the cartilage. Drying for 120 minutes caused extensive cell death mainly in the superficial 500 microm of cartilage. Rewetting every ten or 20 minutes significantly decreased cell death. The superficial zone is most susceptible to desiccation. Loss of superficial chondrocytes likely decreases the production of essential lubricating glycoproteins and contributes to subsequent degeneration. Frequent wetting of cartilage during arthrotomy is therefore essential.

Adolescent↗
PubMed1989

Biochemical and morphological studies of steady state and lipopolysaccaride treated bovine articular cartilage explant cultures.

Explants of bovine articular cartilage were cultured for up to 50 days in 20% fetal calf serum in the presence or absence of the endotoxin lipopolysaccharide (LPS); or in various protocols involving different treatment times with LPS followed by recovery times in the absence of LPS. Cultures were measured in terms of rates of proteoglycan synthesis (incorporation of [35S]sulfate), proteoglycan contents and collagen contents. Histological sections were prepared for both light and electron microscopy. In fetal calf serum, the rates of synthesis and contents of proteoglycans per collagen remained constant, while for LPS treated cultures both parameters decreased. For recovery groups, the rates of proteoglycan synthesis increased during the time of recovery if the LPS treatment times were relatively short (2 weeks or less) and if the tissue was obtained from younger animals; net increase in proteoglycan contents occurred infrequently if at all during recovery protocols. Histological examinations revealed that chondrocytes in cultures maintained in fetal calf serum appeared normal with large stores of glycogen. In LPS treated cultures, chondrocytes were depleted of glycogen stores and contained numerous lipid droplets. In recovery cultures, chondrocytes replenished their glycogen contents, but the lipid droplets remained. For both LPS treated and recovery groups the extracellular matrix was depleted of proteoglycans with time in culture. The results provide further evidence for the ability of this explant culture system to maintain steady state metabolic parameters for proteoglycan metabolism over long time periods and for its utility to study reagents which regulate or perturb these parameters.

Aging↗
PubMed2003

X-ray diffraction of the molecular substructure of human articular cartilage.

The molecular substructure of human articular cartilage has been difficult to study because of its complex composition and high degree of hydration. Using newly available small-angle X-ray diffraction (SAX) instrumentation that allows very short exposure times (0.1 to 10 sec), we have obtained spatially resolved information concerning the disposition of collagen fibers in the matrix of cartilage from the normal and osteoarthritic ankle and knee joints of human cadavers. Surprisingly, in zones of cartilage damage, such as in preosteoarthritic lesions or in the severely degenerated cartilage of osteoarthritic joints, collagen fibers of the deeper layers tended to be reoriented from the vertical. The SAX technique represents a nondestructive method of analyzing the collagen network in cartilage. Taken together, the data suggest a rigid control mechanism for the fiber network and an extensive passive reorganization of the collagen fiber orientation in diseased joint cartilage.

Ankle Joint↗
PubMed2005

[Surgery on articular cartilage: from debridement to tissue engineering].

Articular cartilage is a complex tissue with a limited endogenous repair capacity. Surgical strategies to stimulate repair of damaged cartilage are presently evolving extensively. New techniques based on tissue-engineering principles with cultured cells and scaffolds are challenging established techniques based on generating a repair response from the bone marrow. This paper reviews treatments such as debridement, marrow-stimulating techniques, osteochondral transplantation and autologous chondrocyte transplantation. Debridement and marrow stimulation can provide significant relief of symptoms. Recently, randomized studies have uncovered some of the clinical effects of the new biotechnology-based surgical methods. However, no significant advances over the established surgical techniques have been developed.

Cartilage, Articular↗
PubMed1984

Application of new techniques to separation of proteoglycan aggregates from normal and destabilized rabbit articular cartilages.

Proteoglycans were prepared from rabbit articular cartilages by classical techniques employing 4.0 M guanidine. HCl by transport ultracentrifugation techniques on the purified proteoglycans, present. A new method of extracting the cartilage with 0.4 M guanidine. HCl in the presence of highly purified collagenase is presented. The same yield of proteoglycans on extraction of normal cartilage was obtained as with the classical technique, but a larger proportion of intermediate and large aggregates was obtained with the new than with the classical methodologies. The osteoarthritic cartilage was obtained from 6 month old animals, 3 months after a partial medial meniscectomy had been performed. The profile of proteoglycans from osteoarthritic cartilage consisted predominately of monomers, and a small content of aggregates spread over intermediate and large size ranges. It is postulated that by the methods of extraction, the profile of proteoglycan aggregates present in vivo is more faithfully reproduced than obtained by the classical methodologies.

Animals↗
PubMed1984

The effect of bacterial lipopolysaccharides on the biosynthesis and release of proteoglycans from calf articular cartilage cultures.

Organ cultures of bovine articular cartilage from metacarpophalangeal joints of calf maintain steady state metabolism of cartilage proteoglycans over the course of several weeks. Bacterial lipopolysaccharides (LPS) depress biosynthesis of proteoglycans in such cultures to approximately 60% of control values after 1-2 days of treatment. A glycolipid from the Salmonella minnesota Re 595 mutant, which lacks the polysaccharide chains of LPS, also depresses proteoglycan synthesis. If LPS is removed from the medium as late as after 12 days of exposure, proteoglycan synthesis returns to control values. Proteoglycans synthesized during the first week of LPS treatment are indistinguishable from those synthesized by control cultures in terms of their hydrodynamic size and the relative amounts of disaccharides released by chondroitin lyase ABC digestion of their glycosaminoglycan chains. However, after 15-18 days of treatment, significant proportions of a smaller proteoglycan are synthesized. For cultures prelabeled with [35S]sulfate, the rate of release of 35S-labeled proteoglycans from the matrix is accelerated approximately 2-fold over control during the first week of LPS treatment. This effect is completely reversed upon removal of LPS from the medium. For cultures prelabeled with [35S]sulfate, approximately 40 and 90% of the 35S-labeled proteoglycans are lost from the matrix after 18 days in control and LPS-treated cultures, respectively. The labeled proteoglycans remaining in the matrix of the control after 18 days were indistinguishable from newly synthesized proteoglycans in terms of hydrodynamic size as were those in 7-day LPS-treated cultures when approximately 40% of the labeled proteoglycans had been lost. Even after 18 days of LPS treatment, more than 60% of the remaining labeled molecules were unchanged. LPS stimulates prostaglandin E2 synthesis in these cultures while indomethacin in the presence of LPS blocks synthesis. However, indomethacin did not alter the metabolism of proteoglycans in either control or LPS-treated cultures, indicating that prostaglandins are not directly involved in regulating proteoglycan metabolism in this system.

Animals↗
PubMed1996

The role of nitric oxide in proteoglycan turnover by bovine articular cartilage organ cultures.

Monolayer cultures of articular chondrocytes synthesize large amounts of nitric oxide (NO) following exposure to IL-1. The latter has antianabolic and procatabolic activities on these cells, but little is known about the role, if any, of NO in the integrated metabolic pathways of the chondrocyte. In the present study, the role of endogenously produced NO in both the synthesis and degradation of proteoglycans was investigated for the first time. Bovine articular cartilage slices exposed to 20 U/ml human rIL-1 beta (hrIL-1 beta) synthesized large amounts of NO for 1 to 2 days, after which production fell to a steady state level approximately 20% of the peak value for the remainder of the 14-day incubation. The NO synthase inhibitor, N-monomethyl L-arginine (L-NMA, 1 mM), blocked NO production and enhanced the acute catabolic effects of hrIL-1 beta in cartilage derived from both cartilage derived from both calves and adult animals. However, in late cultures, release of proteoglycans was reduced in the presence of L-NMA. The proteolytic activity in conditioned medium of these cultures (measured as caseinolytic activity) was enhanced by L-NMA; however, this inhibitor did not affect the rates of synthesis of proteoglycans. Although NO is widely assumed to be a mediator of cartilage catabolism, our data suggest that it may instead have an acute protective effect. Whether this effect is maintained chronically is less clear.

Animals↗