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PubMed1995

Topographical variation within the articular cartilage and subchondral bone of the normal ovine knee joint: a histological approach.

Topographical variation in the articular cartilage and subchondral bone of the normal ovine knee was examined using histological techniques. The articular cartilage was examined grossly, then histological sections were cut and the cartilage thickness and chondrocyte density were measured. Bone mineral density, thickness of the subchondral bone plate (SBP) and volume and surface histomorphometrical parameters and mineral apposition rate were calculated for the subchondral bone. It was found that the articular cartilage on the tibial plateaux was thicker, less cellular, and overlay a thicker SBP than that on the femoral condyles. Similarly, the cartilage in the medial joint compartments was thicker, less cellular and overlying a thicker less dense SBP than that in the lateral joint compartments. There was no variation in bone histomorphometric parameters or mineral apposition rate between regions. Biomechanical testing has shown that loading is not uniform throughout the normal human knee joint. The present results suggest that loading within the ovine knee is also nonuniform, with the central regions of the tibial plateaux bearing greater loads than the femoral condyles, and the medial joint compartment being loaded more than the lateral one. The articular cartilage and subchondral bone have adapted in order to best withstand these variations in loading. These histological findings, plus the topographical variations in cartilage biochemistry reported by Read et al. (Topographical variation in composition, PG-biosynthesis and swelling pressure of cartilages of loaded tibio-femoral joints (Abstract). Proceedings of the Combined Meeting of the Orthopaedic Research Societies of USA, Japan and Canada.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cryopreservation of articular cartilage. Ultrastructural observations and long-term results of experimental distal femoral transplantation.

Ultrastructural changes associated with the freeze-preservation of human articular cartilage have been investigated and related to changes in transplanted distal femoral allografts in nonhuman primates. Human osteoarticular specimens were frozen at 2 degrees/minute in the presence of 15% glycerol and kept in liquid nitrogen freezers (vapor phase) from one day to two years. Ultrastructural changes were confined primarily to chondrocytes and were related to the freezing phenomenon, not to the time of storage. The cartilage matrix was affected little, explaining why articular cartilage initially survives clinical transplantation, but later undergoes degenerative changes. Osteoarticular allografts of baboons were frozen in an identical fashion to the human articular cartilage and transplanted into adult baboons. Long-term observations (five years) on these animals showed healing and replacement of the osseous portion of cryopreserved allografts. Fractures that appeared to coincide with maximum revascularization of the graft were the principal complication. Articular surfaces of the cryopreserved allografts underwent degenerative changes over five years. These degenerative changes were also manifested radiologically and appeared similar to those observed in humans. By contrast, fresh osteoarticular allografts healed poorly through fibrous union. However, in one of two fresh allografts, the articular cartilage remained intact five years after transplantation.

Adolescent↗
PubMed2005

Effect of tissue maturity on cell viability in load-injured articular cartilage explants.

OBJECTIVE: During joint maturation, articular cartilage undergoes compositional, structural, and biomechanical changes, which could affect how the chondrocytes within the cartilage matrix respond to load-induced injury. The objective of this study was to determine the effects of tissue maturity on chondrocyte viability when explanted cartilage was subjected to load-induced injury. DESIGN: Cartilage explants from immature (4-8-week-old) and mature (1.5-2-year-old) bovine humeral heads were cyclically loaded at 0.5 hertz in confined compression with a stress of 1 or 5 megapascals for 0.5, 1, 3, 6 and 16 h. Cell death was assessed at 0, 24 and 48 h after load removal using cell viability dyes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The organization of pericellular matrix (PCM), biochemical composition and biomechanical properties of the cartilage were also determined. RESULTS: For the immature and mature cartilage, cell death began at the articular surface and increased in depth with loading time up to 6h. No increase of cell death was found after load removal for up to 48 h. In both groups, cell death increased at a faster rate with the increase of stress level. The depth of cell death in the immature cartilage was greater than the mature cartilage, despite the immature cartilage having a higher bulk aggregate modulus. A less organized PCM in immature cartilage was found as indicated by the weak staining of type VI collagen. CONCLUSION: Cells in the mature cartilage are less vulnerable to load-induced injury than those in immature cartilage.

Animals↗
PubMed2000

Effective isolation of high-quality total RNA from human adult articular cartilage.

The isolation of large quantities of good-quality RNA from human articular cartilage has been a long-standing problem for researchers working with human articular cartilage. In this paper we report a protocol which we have developed based on the Qiagen RNeasy procedure to produce high yields of purified, DNA-free RNA from normal and osteosteoarthritic human articular cartilage. The average yield of RNA was 8.39 microg/g (n = 59) for normal and 6.69 microg/g (n = 58) for osteoarthritic cartilage (average ratio OD 260/280 = 1.8-1.9). Quantitative PCR, cDNA array technology, and Northern blot analysis were used to verify the quality of the RNA.

Adult↗
PubMed1996

Ultrastructural identification of a membrane-like structure on the surface of normal articular cartilage.

Cytochemical and immunocytochemical approaches have been applied to the study of the surface of articular cartilage in humans, bovine and rats. Specimens were fixed in situ or soon after bioptic sampling with chemicals able to preserve and visualize proteins (glutaraldehyde, tannic acid), lipids (osmium tetroxide, malachite green, uranyl acetate) and proteoglycans (toluidine blue O, cuprolinic blue, cetyl pyridinium chloride). Mixtures of reagents were also used. Oriented serial thin sections were observed as such or after treatment with chemicals (chloroform-methanol, Triton X 100) or enzymes (chondroitinases, hyaluronidases, trypsin). Hyaluronan was detected by the use of glial-hyaluronate-binding-protein and antibodies against it. High concentration of osmium tetroxide or fixatives containing markers for lipid or for proteoglycans revealed that the surface of the articular cartilage, in all animal species examined, was covered by mono-multilayered discontinuous three-laminar sheets, which could be partly removed by chloroform-methanol and Triton X 100, were sensitive to hyaluronidase, chondroitinase and trypsin, and were immunopositive for hyaluronan. Each three-laminar sheet was 12-14 nm thick, was always separated from the cartilage itself and could be easily displaced. It is proposed that the surface of normal articular cartilage is covered by a discontinuous mono/multilayered pseudo-membrane, that can be better preserved by fixatives injected into the joint cavity and seems to consist of phospholipids, glycosaminoglycans and proteins. This membrane-like structure might have a protecting role in preventing direct contacts between the articular cartilage and toxic agents present in the synovial fluid and/or exert a lubricating effect within the articular joint.

Animals↗
PubMed1988

Ultrastructural localisation of alkaline phosphatase activity in osteoarthritic human articular cartilage.

The distribution of alkaline phosphatase activity in human articular cartilage from normal and osteoarthritic joints has been examined by an electron microscope technique, probably for the first time. In osteoarthritic cartilage chondrocytes and matrix vesicles close to the tidemark were positive for alkaline phosphatase activity. Large numbers of matrix vesicles were found within the extracellular matrix of osteoarthritic cartilage, and there is a specific relation between phosphatase activity, matrix vesicles, and initial mineral formation in the tidemark region of articular cartilage.

Aged↗
PubMed1999

Correlation of laminated MR appearance of articular cartilage with histology, ascertained by artificial landmarks on the cartilage.

The object of this study was to correlate the laminae of articular cartilage on magnetic resonance (MR) imaging with histologic layers. T1- and fast spin-echo T2-weighted images of articular cartilage with artificial landmarks were obtained under high gradient echo strength (25 mT/m) conditions and a voxel size of 78 x 156 x 2000 microm. Images were also obtained with a) changed frequency-encoding directions; b) changed readout gradient strength; and c) a varied number of phase-encoding steps. T2 mapping was performed with angular variations. Artificial landmarks allowed accurate comparison between the laminae on MR images and the histologic zones. No alterations of the laminae were noted by changing the frequency gradient direction. Altering readout gradient strengths did not show a difference in the thickness of the laminae, and increasing the phase-encoding steps resulted in a more distinct laminated appearance, ruling out chemical shift, susceptibility, and truncation artifacts. The T2 mapping profile showed an anisotropic angular dependency from the magic angle effect. In conclusion, the laminated appearance of articular cartilage on spin-echo and fast spin-echo MR images correlated with the histologic zones rather than MR artifacts.

Animals↗
PubMedDate not supplied

Immunolocalization of transforming growth factor-beta s and type I and type II receptors in rat articular cartilage.

The activities of TGF-beta s and their receptors (T beta R) in the articular cartilage have not been fully elucidated as yet. Temporal and spatial expression of TGF-beta s and their receptors were examined immunohistochemically in the articular cartilage of growing rats to clarify their role in chondrogenesis. TGF-beta 1, -beta 2 and -beta 3 were strongly expressed from 6 to 50 weeks in the superficial, transitional, and least mature zones. The expression of TGF-beta s in hypertrophic chondrocyte was weak except TGF-beta 1. T beta R-I and T beta R-II were co-expressed with the ligand in the superficial, transitional, and least mature zones throughout the growth phase. This study revealed that TGF-beta s in the articular cartilage plays a major role in the modulation of endochondral ossification ensuring the growth and maintenance of the chondrocyte lineage in articular cartilage.

Activin Receptors, Type I↗
PubMed1993

The effects of methotrexate on normal and osteoarthritic lapine articular cartilage.

OBJECTIVE: The effects of methotrexate (MTX) on articular cartilage and its influence on the development of osteoarthritis (OA) lesions were tested in a lapine partial medial meniscectomy model. METHOD: Animals were divided into groups consisting of unoperated and operated rabbits that either received or did not receive MTX treatment. After 8 weeks knee articular condylar cartilage was examined for gross and histologic anatomy, active and total neutral metalloproteinases, tissue inhibitor of metalloproteinase (TIMP), DNA, uronic acid and hydroxyproline content. RESULTS: Carbon black retention and histologic scores revealed moderately severe changes in the OA animals with a tendency to less severe changes in OA animals receiving MTX. Unoperated animals receiving MTX had abnormal cartilage that displayed pitting and elevations in histologic score. Active and total neutral metalloproteinase and TIMP were elevated in both untreated and treated OA animals when compared to either unoperated or unoperated and treated animals. CONCLUSION: Articular cartilage with lesser amounts of neutral metalloproteinase and high amounts of TIMP levels often seen with other therapeutic modalities for OA, were not observed with MTX therapy. Our data suggest that MTX may have limited value in the treatment of OA.

Animals↗
PubMed1993

Tenascin is associated with articular cartilage development.

The roles of tenascin in cartilage development and function remain unclear. Based on the observation that tenascin is particularly abundant at the epiphyseal extremities of developing cartilaginous models of long bones in chick and mouse embryo, we tested the hypothesis that tenascin is involved in articular cartilage development. Immunofluorescence analysis revealed that tenascin was first localized in the cell condensation region of Day 4 chick embryo limb buds, where the cartilaginous models form. With further development, tenascin gene expression became indeed restricted to the articular cap of the models. Tenascin persisted in the articular cartilage of postnatal chickens but appeared to decrease with age. The protein was also abundant in embryonic and adult tracheal cartilage rings which, like articular cartilage, persist throughout postnatal life. Similar patterns of tenascin expression were seen in mouse. Using monoclonal antibodies to avian tenascin variants, we found that the bulk of articular cartilage contained the shortest tenascin variant (Tn190), whereas the largest variant (Tn230) was present in tissues associated or interacting with articular cartilage (ligaments and meniscus). The protein and its mRNA, however, were undetectable in growth plate cartilage undergoing maturation and endochondral ossification. This inverse correlation between chondrocyte maturation and tenascin production was corroborated by the finding that tenascin gene expression decreased markedly during maturation of chondrocytes in culture and during formation of a secondary ossification center within the articular cap in vivo. Thus, tenascin is intimately associated with the development of articular cartilage and other permanent cartilages whereas absence or reduced amounts of this matrix protein characterize transient cartilages which undergo maturation and are replaced by bone.

Animals↗
PubMed1992

The effect of storage on the biomechanical behavior of articular cartilage--a large strain study.

The transplantation of stored shell osteochondral allografts is a potentially useful alternative to total joint replacements for the treatment of joint ailments. The maintenance of normal cartilage properties of the osteochondral allografts during storage is important for the allograft to function properly and survive in the host joint. Since articular cartilage is normally under large physiological stresses, this study was conducted to investigate the biomechanical behavior under large strain conditions of cartilage tissue stored for various time periods (i.e., 3, 7, 28, and 60 days) in tissue culture media. A biphasic large strain theory developed for soft hydrated connective tissues was used to describe and determine the biomechanical properties of the stored cartilage. It was found that articular cartilage stored for up to 60 days maintained the ability to sustain large compressive strains of up to 40 percent or more, like normal articular cartilage. Moreover, the equilibrium stress-strain behavior and compressive modulus of the stored articular cartilage were unchanged after up to 60 days of storage.

Animals↗
PubMed1995

Degenerative mechanism of articular cartilage induced by low stress. A morphological study.

Immobilization leads not only to diminished joint movement but also low stress of articular cartilage. The present investigation was undertaken to observe the morphological changes which arose in articular cartilage with low stress. The joint motion remained intact. Articular cartilage from the left knees of 66 rats whose left calcaneal tendons had been transected was examined under transmission electron microscope and light microscope. The degenerative changes were observed: decreased functional activity of chondrocytes progressively degenerated cartilage and lack of compensatory proliferation of chondrocytes at the early stage. We propose that the degeneration of articular cartilage induced by immobilization is the result of combination of low stress and lack of joint motion. The following degenerative mechanism begins with chondrocytes. Chondrocyte and matrix influence each other in a vicious cycle. Low stress may restrain the repair activities.

Animals↗
PubMed1977

Glycosaminoglycans in human articular cartilage of the lower femoral epiphysis in osteoarthrosis.

Biopsy specimens of human articular cartilage were taken from the caudal summits and from the posterior part of both epicondyles of the right lower femoral epiphysis of 20 individuals within 12 hours post mortem. 12 articular surfaces were normal and 8 included changes attributable to osteoarthrosis. In one individual with osteoarthrotic changes the articular cartilage surface was subjected to a more detailed study. CPC- and ECTEOLA-cellulose column chromatography of glycosaminoglycans was performed and the content and collagen contents were determined. A reduced ratio of chondroitin to keratan sulphates in articular cartilage was found to be related to age. In osteoarthrosis this change was more pronounced and both chondroitin sulphate and keratan sulphate were of lower molecular weight and/or sulphate content. On approaching eburnized cartilage surfaces, decreasing concentrations of glycosaminoglycans were found, concomitant with a corresponding increase in non-collagenous proteins. No change in hyaluronic acid concentration was found with advancing age or in osteoarthrosis.

Adolescent↗
PubMed1978

Effects of intra-articularly administered corticosteroids and salicylates on the surface structure of articular cartilage.

This study was designed to evaluate the effects of repeated intra-articular corticosteroid and salicylate injections on the surface structure of articular cartilage in the rabbit, using the scanning electron miscroscope. We have shown that the normal surface of the rabbit's articular cartilage presents shallow depressions and mound-like elevations and that corticosteroid-treated articular cartilage exhibits progressive lesions, including fissuring and fraying. The effects of salicylates require further investigation.

Animals↗
PubMed2005

A new biotechnology for articular cartilage repair: subchondral implantation of a composite of interconnected porous hydroxyapatite, synthetic polymer (PLA-PEG), and bone morphogenetic protein-2 (rhBMP-2).

OBJECTIVE: Articular cartilage repair remains a major obstacle in tissue engineering. We recently developed a novel tool for articular cartilage repair, consisting of a triple composite of an interconnected porous hydroxyapatite (IP-CHA), recombinant human bone morphogenetic protein-2 (rhBMP-2), and a synthetic biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol (PLA-PEG)] as a carrier for rhBMP-2. In the present study, we evaluated the capacity of the triple composite to induce the regeneration of articular cartilage. METHODS: Full-thickness cartilage defects were created in the trochlear groove of 52 New Zealand White rabbits. Sixteen defects were filled with the bone morphogenetic protein (BMP)/PLA-PEG/IP-CHA composite (group I), 12 with PLA-PEG/IP-CHA (group II), 12 with IP-CHA alone (group III), and 12 were left empty (group IV). The animals were killed 1, 3, and 6 weeks after surgery, and the gross appearance of the defect sites was assessed. The harvested tissues were examined radiographically and histologically. RESULTS: One week after implantation with the BMP/PLA-PEG/IP-CHA composite (group I), vigorous repair had occurred in the subchondral defect. It contained an agglomeration of mesenchymal cells which had migrated from the surrounding bone marrow either directly, or indirectly via the interconnecting pores of the IP-CHA scaffold. At 6 weeks, these defects were completely repaired. The regenerated cartilage manifested a hyaline-like appearance, with a mature matrix and a columnar organization of chondrocytes. CONCLUSIONS: The triple composite of rhBMP-2, PLA-PEG, and IP-CHA promotes the repair of full-thickness articular cartilage defects within as short a period as 3 weeks in the rabbit model. Hence, this novel cell-free implant biotechnology could mark a new development in the field of articular cartilage repair.

Animals↗
PubMed2000

Effects of aging and dietary restriction on the structural integrity of rat articular cartilage.

The objectives of this study were to investigate the effects of aging and diet restriction on the biomechanical properties of articular cartilage, using a well-controlled rat model (Fischer 344). This animal model is recommended by the National Institute of Aging specifically to study aging and diet issues. The intrinsic biomechanical properties of articular cartilage were obtained using a creep indentation approach. The ages chosen (6, 12, 18, 24 months of age) correspond to approximate human ages of 20 to 80 years old. The diet regimen employed in this study used either an ad libitum fed group or a group fed 60% of the mean food intake of the ad libitum group. The results demonstrate that, unlike bone, rat articular cartilage biomechanical properties are not affected in a discernible manner by diet restriction, despite the fact that diet-restricted animals were significantly lighter in terms of body weight. Age effects on biomechanical properties are found only at 6 and 12 months probably due to developmental reasons, but not at later ages. It appears that aging and diet restriction have profoundly different effects on articular cartilage and bone. Another significant result of this study was to establish the rat as a suitable animal model to study cartilage biomechanical properties. Thus, the rat can be added to the list of animals that can be used to study structure-function and pathophysiological relationships in articular cartilage.

Aging↗
PubMed1992

N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage.

Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1' position.

Aggrecans↗
PubMed1994

A "coupled" subchondral bone-articular cartilage tissue culture system for the study of cartilage proteoglycan metabolism.

Current evidence suggests that interactions between the subchondral bone and the articular cartilage of mammalian diarthrodial joints may occur through the action of bone-associated peptide factors. However, there is no suitable organ culture model for studying these interactions. This study defines a long-term tissue culture system where the articular cartilage is coupled to the adjacent subchondral bone obtained from the proximal ends of bovine metacarpals. Autoradiography done over 3 mo., by utilizing [35S]SO4 incorporation into cartilage proteoglycan (PG) and a procedure for cutting non-decalcified bone, demonstrated similar numbers of silver grains over chondrocytes in all cartilage zones, including the bone-cartilage interface. Newly synthesized PG (NSPG) from the cartilage of the "coupled" system over a 3-wk period was primarily of large hydrodynamic size (Kav of 0.34). Comparable bovine articular and nasal cartilage slice systems, incubated for short periods of time, yielded similar and somewhat larger NSPG, respectively. Labeled chondroitin sulphate PG accumulating in the medium of primary chondrocyte monolayer cultures, derived from the cartilage of the coupled system at 0, 1, 2, and 3 wk, revealed two polydisperse subpopulations (Kav of 0.30 to 0.38 and 0.51 to 0.68). We conclude that this coupled bone-cartilage system is viable for prolonged periods, is suitable for studies on the metabolism of articular cartilage PGs, and seems to have some advantages over the cultured articular cartilage slice system.

Animals↗