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PubMed2001

DNA cytofluorometric analysis of chondrocytes in human articular cartilages under normal aging or arthritic conditions.

OBJECTIVE: Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN). DESIGN: The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization. RESULTS: The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S- G2 index, in diseased cartilages (OA, RA and AN) was significantly high (P< 0.0001). In OA cartilage, the S-G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate. CONCLUSION: These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.

Adolescent↗
PubMed1996

The development of articular cartilage: I. The spatial and temporal patterns of collagen types.

Articular cartilage is both morphologically and biochemically heterogeneous. Its susceptibility to degenerative diseases such as arthritis and its limited repair capacity have made cartilage the focus of intense study; surprisingly, little is known of its development. Using a panel of specific antibodies, we have documented the temporal and spatial patterns of collagen types I, II, III, VI and X in the developing knee cartilage of the marsupial Monodelphis domestica from parturition to adulthood. Type I collagen was initially detected in the presumptive articular cartilage of the epiphyses in addition to the perichondrium. By 14 d postparturition, type I collagen was not detectable in the epiphyseal cartilage apart from insertion sites of ligaments and tendons of the joint. Similarly, type III collagen was detected at insertion sites of the major ligaments and tendons and within the perichondrium/periosteum but was never detected in the cartilage per se. Type II collagen was predictably distributed throughout the cartilage matrix and was also detected in the perichondrium. Type VI collagen was widely distributed throughout the cartilage matrix at parturition, but during development became restricted to a pericellular location particularly towards the presumptive articular cartilage, i.e. the epiphysis. Interestingly, generalised matrix immunopositivity was only retained in the hypertrophic cartilage of the secondary centre of ossification. After the formation of the secondary centre, type VI collagen became localised pericellularly in the deeper regions of the articular cartilage but was absent in the cartilage of the growth plate. Type X collagen showed a novel distribution pattern. In addition to being synthesised by hypertrophic chondrocytes, this collagen type was also expressed transiently by some cells at the presumptive articular surface. Furthermore, these surface chondrocytes also stained histochemically for alkaline phosphatase, suggesting that they were terminally differentiated. The fate of these terminally differentiated cells is unknown.

Alkaline Phosphatase↗
PubMed2001

Role of uppermost superficial surface layer of articular cartilage in the lubrication mechanism of joints.

The uppermost superficial surface layer of articular cartilage, the 'lamina splendens' which provides a very low friction lubrication surface in articular joints, was investigated using atomic force microscopy (AFM). Complementary specimens were also observed under SEM at -10 degrees C without dehydration or sputter ion coating. Fresh adult pig osteochondral specimens were prepared from the patellas of pig knee joints and digested with the enzymes, hyaluronidase, chondroitinase ABC and alkaline protease. Friction coefficients between a pyrex glass plate and the osteochondral specimens digested by enzymes as well as natural (undigested) specimens were measured, using a thrust collar apparatus. Normal saline, hyaluronic acid (HA) and a mixture of albumin, globulin, HA (AGH) were used as lubrication media. The surface irregularities usually observed in SEM studies were not apparent under AFM. The articular cartilage surface was resistant to hyaluronidase and also to chondroitinase ABC, but a fibrous structure was exhibited in alkaline protease enzymes-digested specimens. AFM analysis revealed that the thickness of the uppermost superficial surface layer of articular cartilage was between 800 nm and 2 microm in adult pig articular cartilage. The coefficient of friction (c.f.) was significantly higher in chondroitinase ABC and alkaline protease enzymes digested specimens. Generally, in normal saline lubrication medium, c.f. was higher in comparison to HA and AGH lubrication media. The role of the uppermost, superficial surface layer of articular cartilage in the lubrication mechanism of joints is discussed.

Animals↗
PubMed1989

Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.

Animals↗
PubMed1999

Articular cartilage joint surface reconstruction techniques.

There is renewed interest in joint surface reconstruction using a variety of new and evolving techniques for articular cartilage resurfacing. Neochondrogenesis and articular cartilage transplantation are gaining a prominent place in orthopaedic basic science research. The authors have published a reliable, repeatable, stable, and sensitive model utilizing osteochondral dowel core transplantation in an ovine model to assess various treatment and follow-up evaluation techniques for articular cartilage transplantation. As well, our laboratory has developed a handheld articular cartilage indentor for clinical assessment of biomechanical performance of joint surfaces. This article presents and reviews that model as well as a semiquantitative visual analog scale for documenting articular cartilage gross morphology. The results of magnetic resonance imaging of the osteochondral dowel transplants and the gross morphology grading are compared to the histological and histochemical grading and biochemical and biomechanical assessments to form the foundation for future work in this critical and important study area for clinical application.

Animals↗
PubMed2005

A fibril-reinforced poroviscoelastic swelling model for articular cartilage.

From a mechanical point of view, the most relevant components of articular cartilage are the tight and highly organized collagen network together with the charged proteoglycans. Due to the fixed charges of the proteoglycans, the cation concentration inside the tissue is higher than in the surrounding synovial fluid. This excess of ion particles leads to an osmotic pressure difference, which causes swelling of the tissue. The fibrillar collagen network resists straining and swelling pressures. This combination makes cartilage a unique, highly hydrated and pressurized tissue, enforced with a strained collagen network. Many theories to explain articular cartilage behavior under loading, expressed in computational models that either include the swelling behavior or the properties of the anisotropic collagen structure, can be found in the literature. The most common tests used to determine the mechanical quality of articular cartilage are those of confined compression, unconfined compression, indentation and swelling. All theories currently available in the literature can explain the cartilage response occurring in some of the above tests, but none of them can explain these for all of the tests. We hypothesized that a model including simultaneous mathematical descriptions of (1) the swelling properties due to the fixed-change densities of the proteoglycans and (2) the anisotropic viscoelastic collagen structure, can explain all these test simultaneously. To study this hypothesis we extended our fibril-reinforced poroviscoelastic finite element model with our biphasic swelling model. We have shown that the newly developed fibril-reinforced poroviscoelastic swelling (FPVES) model for articular cartilage can simultaneously account for the reaction force during swelling, confined compression, indentation and unconfined compression as well as the lateral deformation during unconfined compression. Using this theory it is possible to analyze the link between the collagen network and the swelling properties of articular cartilage.

Animals↗
PubMed1996

Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage.

The present study was designed to investigate whether or not chondrocytes in articular cartilage express type I collagen in vivo under physiological conditions. Expressions of the gene and the phenotype of type I collagen were examined in rat tibial articular cartilage in the knee joint during development. Knee joints of Wistar rats at 1, 5, and 11 weeks postnatal were fixed in 4% paraformaldehyde with or without 0.5% glutaraldehyde and decalcified in 10% EDTA. After the specimens were embedded in paraffin and serial sections made, adjacent sections were processed for immunohistochemistry and in situ hybridization for type I collagen. The epiphysis of the tibia was composed of cartilage in week- 1 rats. Formation of articular cartilage was in progress in week 5 as endochondral ossification proceeded and was completed in week 11. Anti-type I collagen antibody stained only the superficial area of the epiphysis in week 1, but the immunoreactivity was expanded into the deeper region of the articular cartilage with development in weeks 5 and 11. Hybridization signals for pro-alpha 1 (I) collagen were seen in some of chondrocytes in the epiphysis of the week-1 tibia. The most intense signals were identified in chondrocytes in week 5 and the signals appeared weaker in week 11. The present study demonstrated that chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of the articular cartilage.

Age Factors↗
PubMed1998

Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation.

The degeneration of articular cartilage as part of the clinical syndrome of osteoarthritis is one of the most common causes of pain and disability in middle-aged and older people. The strong correlation between increasing age and the prevalence of osteoarthritis, and recent evidence of important age-related changes in the function of chondrocytes, suggest that age-related changes in articular cartilage can contribute to the development and progression of osteoarthritis. Although the mechanisms responsible for osteoarthritis remain poorly understood lifelong moderate use of normal joints does not increase the risk. Thus, the degeneration of normal articular cartilage is not simply the result of aging and mechanical wear. However, high-impact and torsional loads may increase the risk of degeneration of normal joints, and individuals who have an abnormal joint anatomy, joint instability, disturbances of joint or muscle innervation, or inadequate muscle strength or endurance probably have a greater risk of degenerative joint disease. Recent work has shown the potential for the restoration of an articular surface. Currently, surgeons frequently debride joints and penetrate subchondral bone as well as perform osteotomies, with the intent of decreasing symptoms and restoring or maintaining a functional articular surface. The results of these procedures vary considerably among patients. Clinical and experimental work has shown the important influence of loading and motion on the healing of articular cartilage and joints. Experimental studies have revealed that transplantation of chondrocytes and mesenchymal stem cells; use of periosteal and perichondrial grafts, synthetic matrices, and growth factors: and other methods have the potential to stimulate the formation of a new articular surface. The long-term follow-up of small series of patients has shown that the transplantation of osteochondral autologous grafts and allografts can be effective for the treatment of focal defects of articular cartilage in selected patients. Thus far, none of these methods has been shown to predictably restore a durable articular surface to an osteoarthritic joint, and it is unlikely that any one of them will be uniformly successful. Rather, the available clinical and experimental evidence indicates that future optimum methods for the restoration of articular surfaces will begin with a detailed analysis of the structural and functional abnormalities of the involved joint and the patient's expectations for future use of the joint. On the basis of this analysis, the surgeon will develop a treatment plan that potentially combines correction of mechanical abnormalities (including malalignment, instability, and intra-articular causes of mechanical dysfunction), debridement that may or may not include hunted penetration of subchondral bone, and applications of growth factors of implants that may consist of a synthetic matrix that incorporates cells or growth factors or use of transplants followed by a postoperative course of controlled loading and motion.

Adult↗
PubMed1994

Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1991

Measurement of proteoglycan mRNA in articular cartilage--preliminary investigations.

Measurement of mRNA in adult articular cartilage could define whether treatment with nonsteroidal antiinflammatory drugs affects the rate of proteoglycan synthesis in vivo in osteoarthritis. We studied a variety of extraction and isolation methods to obtain a high yield of pure mRNA from adult articular cartilage. By combining a single step extraction procedure, precipitation in 0.8 M LiCl and poly d-T affinity isolation, we obtained pure mRNA that hybridized with a cDNA probe for the large aggregating proteoglycan. Thus, it should be feasible to assess the level of message using polymerase chain reaction technology; it is unlikely that northern blots, slot blots, or RNA protection assays can be performed directly on the small specimens of articular cartilage that are available from experimental model studies or from surgical specimens.

Animals↗
PubMed2003

Microstructural and elastic properties of the extracellular matrices of the superficial zone of neonatal articular cartilage by atomic force microscopy.

The structural and mechanical properties of the superficial zone of articular cartilage are not well understood. Most previous studies have focused on the overall properties of articular cartilage in the adult. In the present work, the extracellular matrices of the superficial zone of the jaw-joint condyle in the 7-day-old rabbit were subjected to dynamic indentation with atomic force microscopy (AFM). The surface topography of four equally divided regions of the entire articular surface lacked substantial variations, with mean roughness from 95.4 nm (+/- 28.0) to 130.1 nm (+/- 13.8). Indentations of the articular surface and the microdissected, orthogonal transverse surface revealed a narrow distribution of Young's moduli ranging from 0.92 MPa (+/- 0.12) to 1.02 MPa (+/- 0.22). These rather uniform structural and mechanical properties of the superficial zone of the neonatal articular cartilage are in contrast to our previous finding of a gradient distribution of Young's moduli of the superficial zone of adult articular cartilage from 0.95 (+/- 0.06 MPa) to 2.34 (+/- 0.26 MPa) (Hu et al.: J Struct Biol 2001:136:46-52), indicating that the mechanical properties of the articular surface are modified during development. Thus, articular cartilage's anisotropic mechanical properties may be specific to the adult, rather than the neonatal. It is further postulated that the structural and mechanical properties of the superficial zone of articular cartilage are regulated by chondrocytes in addition to their unidirectional development pathway toward subchondral bone formation.

Animals↗
PubMed2002

Elastic anisotropy of articular cartilage is associated with the microstructures of collagen fibers and chondrocytes.

Chondrocyte shape and volumetric concentration change as a function of depth in articular cartilage. A given chondrocyte shape produces different effects on the global material properties depending on the structure of the collagen fiber network. The shape and volumetric concentration of chondrocytes in articular cartilage appear to be related to the mechanical stability of the matrix. The present study was aimed to investigate, theoretically, the effects of the structural arrangement of the collagen fiber network, and the shape and distribution of chondrocytes, on the global material behavior of articular cartilage. Articular cartilage was assumed to be a four-phasic composite comprised of a matrix (associated with the properties of the proteoglycan structure), vertically and horizontally distributed collagen fibers, and spheroidal inclusions representing chondrocytes. A solution for composite materials was used to estimate the global, effective material properties of cartilage. Only the elasticity of the solid phase was investigated in the present study. Our simulations suggest that a soft, spheroidal cell inclusion in a fiber-reinforced proteoglycan matrix affects the material properties differently depending on the shape of the spheroidal inclusions. If the long axis of the inclusions is parallel to the collagen fibers, as in the deep zone, the soft inclusions increase the stiffness of the composite in the fiber direction, and reduce the stiffness of the composite in the direction normal to the fibers. Furthermore, we found that Young's modulus normal to the contact surface increases from the superficial to the deep zone in articular cartilage by a factor of 10-50, a finding that agrees well with experimental observations. Our analysis suggests that the combination of proteoglycan matrix, fiber orientation, and shape of chondrocytes are intimately related and are likely adapted to optimize the mechanical stability and load carrying capacity of the structure.

Anisotropy↗
PubMed2006

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin-1beta.

OBJECTIVE: Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin-1beta (IL-1beta), and combinations thereof. METHODS: Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17beta-estradiol, and dehydroepiandrosterone (DHEA), with or without IL-1beta. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix. RESULTS: Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 microg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration-dependent stimulation of integration (with maximum values observed with DHEA at 3 x 10(-5)M, testosterone at 10(-8)M, and 17beta-estradiol at 10(-11)M). For testosterone and 17beta-estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL-1beta at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL-1beta and sex hormones, integration of articular cartilage was reduced. CONCLUSION: This is the first study to demonstrate that steroid hormones such as 17beta-estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL-1beta. In the absence of IL-1beta or after neutralization of IL-1beta, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.

Animals↗
PubMed2006

Current concepts in the rehabilitation following articular cartilage repair procedures in the knee.

Postoperative rehabilitation programs following articular cartilage repair procedures will vary greatly among patients and need to be individualized based on the nature of the lesion, the unique characteristics of the patient, and the type and detail of each surgical procedure. These programs are based on knowledge of the basic science, anatomy, and biomechanics of articular cartilage as well as the biological course of healing following surgery. The goal is to restore full function in each patient as quickly as possible by facilitating a healing response without overloading the healing articular cartilage. The purpose of this paper is to overview the principles of rehabilitation following articular cartilage repair procedures. Furthermore, specific rehabilitation guidelines for debridement, abrasion chondroplasty, microfracture, osteochondral autograft transplantation, and autologous chondrocyte implantation will be presented based upon our current understanding of the biological healing response postoperatively.

Biomechanical Phenomena↗
PubMedDate not supplied

Local hyperthermia and cartilage breakdown: histochemical and metabolic studies on rabbit articular cartilage in vitro.

The hypothesis that local hyperthermia generated in inflamed joints may cause damage to the articular cartilage was tested in vitro using histochemical and isotopic methods. We found that this could be the case. Incubation temperatures above 38 degrees C in experiments of longer duration and 39 degrees C during shorter incubation times induced a loss of metachromasia, decreased rates of synthesis, and increased degradation of cartilage matrix macromolecules. It is postulated that local synovial hyperthermia might partly account for the cartilage degeneration that frequently occurs in rheumatoid arthritis as a result of a decreased synthesis and increased catabolism of matrix macromolecules.

Animals↗
PubMed1999

[An experimental study of viscoelastic properties of articular-cartilage of patella].

OBJECTIVE: To study the viscoelastic properties of human articular cartilages of patella in 8 cadavers, which were obtained from patients ranged in age from nineteen to fifty-one years old and died of acute head injury. METHODS: The creep and stress relaxation of articular cartilage of patella were abserved in the condition of articular cartilage loaded of 500 Pa, just as the physiological load of articular cartilage of patella in keeping human body standing position with the knee joint flexed 30 degrees, during the experiment. The deformation-time curve under certain load, the load-time curve under certain deformity and related data were obtained. RESULTS: Creep and stress relaxation showed that obvious changes occured in the first 10-15 minutes during the whole process. CONCLUSIONS: The data and curve will not only be helpful to understand the pathogenesis of chondromalacia, but also propose a feasible method to study it from biomechanical view, meanwhile provide reference data for designing the prosthesis of patella and material choosing.

Adult↗