Chondrocyte Clustering Research Articles

Laser scanning confocal arthroscopy of a fresh cadaveric knee joint

Osteoarthritis (OA) inflicts an enormous burden upon sufferers and healthcare systems worldwide. Continuing efforts to elucidate the aetiology of OA have indicated the need for non-destructive methods of in vivo microstructural assessment of articular cartilage (AC). In this study, we describe the first use of a recently developed laser scanning confocal arthroscope (LSCA) to image the cartilage of a fresh frozen cadaveric knee from a patient with OA. Using an adaptation of the International Cartilage Repair Society (ICRS) joint mapping protocol, the joint was divided into three discrete regions (femoral condyle, patella and tibial plateau) for grading according to the ICRS (Outerbridge) system. The LSCA was used to generate images from each area within the three regions. Following imaging, the joint was sectioned and histology was performed on the corresponding sites with histological grading (modified-Mankin). Quantitative results of ICRS, LSCA and histological OA assessment were compared using intraclass correlation (ICC) and Pearson correlation analysis. The LSCA enabled visualisation of chondrocyte morphology and cell density, with classical OA changes such as chondrocyte clustering, surface fibrillation and fissure formation evident. Obvious qualitative similarities between LSCA images and histology were observed, with fair to moderate agreement (P<0.05) demonstrated between modalities. In this study, we have shown the viability of the LSCA for non-destructive imaging of the microstructure of OA knee cartilage. LSCA technology is potentially a valuable research and clinical tool for the non-destructive assessment of AC microstructure in early to late OA.

Targeting pleiotropin to treat osteoarthritis

Pleiotropin (PTN) is a secreted heparin-binding peptide expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. Although PTN is abundant in fetal or juvenile cartilage, it is undectable in mature cartilage. However, PTN is re-expressed in chondrocytes in early stages of osteoarthritis where it is detectable in situ and in synovial fluids from patients. PTN enhances chondrogenesis by stimulation of extra-cellular matrix synthesis, reduction of degrading matrix metalloproteases and induction of their inhibitors; PTN also slightly reduces pro-inflammatory factors, such as nitric oxide and vascular endothelial growth factor. Furthermore, PTN stimulates chondrocyte clustering and proliferation. Thus, PTN appears to mediate repair and protective processes in osteoarthritic cartilage and appears to be a promising factor to treat osteoarthritis.

Assessing macroscopic and microscopic indicators of osteoarthritis in the distal interphalangeal joints: A cadaveric study

Osteoarthritis is manifested both by macroscopically visible lesions and by specific histological indicators. Although traditional views of the disease process invoke physical abrasion of joint surfaces, recent studies indicate that tissue-level changes may precede grossly visible lesions of articular cartilage. This study investigates the association between gross and histological indicators of osteoarthritis at the manual interphalangeal joints, and examines a sequence of events that may lead to the onset of cartilage degeneration. Interphalangeal joints from the hands of nine cadavers were dissected, of which 52 joints were collected and further evaluated. Gross degradation of the proximal articular surface was graded on a scale of 0-3 (with 0 representing normal cartilage with no visible lesions). Osteoarthritic lesions were found in 86% of specimens and showed no preferential occurrence between males and females or right and left hands. Histological analysis indicated that known microscopic indicators of osteoarthritis always occur in Grade 1-3 specimens, but can also be recognized in some macroscopically normal specimens. Many macroscopically normal specimens exhibited chondrocyte clustering (28.6%) and/or tide mark irregularities (57%), indicating that these features are most likely the earliest to develop in the progression of osteoarthritis. It is possible that the initiating etiology is thickening of the subchondral bone, but this was not directly observed. Results indicate significant thinning of the cartilage as macroscopic degradation progresses. Our study supports, with slight modification, a previously proposed cascade of histological changes that may ultimately lead to the physical destruction of articular cartilage.

Mechanical Behavior of Articular Cartilage after Osteochondral Autograft Transfer in an Ovine Model

Background Grafting of autologous hyaline cartilage and bone for articular cartilage repair is a well-accepted technique. Although encouraging midterm clinical results have been reported, no information on the mechanical competence of the transplanted joint surface is available. Hypothesis The mechanical competence of osteochondral autografts is maintained after transplantation. Study Design Controlled laboratory study. Methods Osteochondral defects were filled with autografts (7.45 mm in diameter) in one femoral condyle in 12 mature sheep. The ipsilateral femoral condyle served as the donor site, and the resulting defect (8.3 mm in diameter) was left empty. The repair response was examined after 3 and 6 months with mechanical and histologic assessment and histomorphometric techniques. Results Good surface congruity and plug placement was achieved. The Young modulus of the grafted cartilage significantly dropped to 57.5% of healthy tissue after 3 months (P < .05) but then recovered to 82.2% after 6 months. The aggregate and dynamic moduli behaved similarly. The graft edges showed fibrillation and, in some cases (4 of 6), hypercellularity and chondrocyte clustering. Subchondral bone sclerosis was observed in 8 of 12 cases, and the amount of mineralized bone in the graft area increased from 40% to 61%. Conclusions The mechanical quality of transplanted cartilage varies considerably over a short period of time, potentially reflecting both degenerative and regenerative processes, while histologically signs of both cartilage and bone degeneration occur. Clinical Relevance Both the mechanically degenerative and restorative processes illustrate the complex progression of regeneration after osteochondral transplantation. The histologic evidence raises doubts as to the long-term durability of the osteochondral repair.

Degradable hydrogel scaffolds for in vivo delivery of single and dual growth factors in cartilage repair

As our population ages, treatment for joint pain associated with articular cartilage damage is becoming a prevalent challenge. Accordingly, this work investigates local delivery of two regulatory proteins - transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) - to cartilage defects from degradable scaffolds as a potential strategy for improving cartilage repair. The effects of TGF-beta1 and/or IGF-1 delivery on osteochondral repair in adult rabbits were examined through histomorphometric analysis of 11 markers of osteochondral repair. Complete scaffold degradation occurred allowing for assessment of the healing response at 12 weeks post-surgery. When compared to untreated defects, higher scores were observed with IGF-1-treated defects for the six markers of neo-surface repair: neo-surface morphology, cartilage thickness, surface regularity, chondrocyte clustering, and the chondrocyte/glycosaminoglycan content of the neo-surface and the cartilage surrounding the defect. Surprisingly, the benefits of IGF-1 delivery were not maintained when this growth factor (GF) was co-delivered with TGF-beta1, despite numerous in vitro reports of the combinatory actions of these GFs. While localized delivery of IGF-1 may be a promising repair strategy, further in vivo assessment is necessary, since fibrous tissue was commonly observed in the neo-surface of all treatment groups. More importantly, this study highlights the need to rigorously examine GF interactions in the wound healing environment and demonstrates that in vitro observations do not directly translate to the in vivo setting.

Effects of pleiotrophin, a heparin-binding growth factor, on human primary and immortalized chondrocytes

Pleiotrophin (PTN) is a secreted heparin-binding peptide expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. In fetal and juvenile, but not in mature cartilage, PTN is abundant. Furthermore, PTN is re-expressed in chondrocytes in early stages of osteoarthritis (OA). Since little is known about the functions of PTN in cartilage, we investigated the occurrence of PTN receptors in human articular cartilage in situ and PTN effects on human primary and immortalized chondrocytes in vitro. Receptor expression and regulation was monitored by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. PTN effects and signal transduction were studied by electrophoretic mobility shift, Boyden chamber cell migration and proliferation assays, effects on gene expression by real time RT-PCR and that on nitric oxide (NO) by the Griess reaction. Of the putative PTN signaling receptors, immortalized and primary chondrocytes (pc) expressed the anaplastic lymphoma kinase (ALK), less the receptor-type protein tyrosine phosphatase zeta/beta (PTPzeta). ALK expression was upregulated upon ligand exposure. PTN stimulation activated the AP-1 (activator protein-1) transcription factor and altered gene expression. Prolonged stimulation induced PTN mRNA expression slightly, reduced vascular endothelial growth factor (VEGF) mRNA as well as NO production. Whereas mRNA expression of matrix metalloproteinases (MMPs) MMP-1 and MMP-13 was reduced, their inhibitors TIMP-1 and TIMP-2 were induced. Furthermore, PTN stimulated chondrocyte migration and proliferation. These results show that PTN is an autocrine growth factor in cartilage. We suggest that PTN may be involved in the clustering and proliferation of chondrocytes observed in the early stages of OA.

Safety of, and biological and functional response to, a novel metallic implant for the management of focal full‐thickness cartilage defects: Preliminary assessment in an animal model out to 1 year

Focal full-thickness cartilage lesions of the human medial femoral condyle (MFC) can cause pain and functional impairment. Affected middle-aged patients respond unpredictably to existing treatments and knee arthroplasty may be required, prompting risk of revision. This study assesses the safety of, and biological and functional response to, a metallic resurfacing implant which may delay or obviate the need for traditional arthroplasty. The anatomic contour of the surgically exposed MFC of six adult goats was digitally mapped and an 11 mm diameter full-thickness osteochondral defect was created. An anchor-based Co-Cr resurfacing implant, matching the mapped articular contour, was implanted. Each goat's contralateral unoperated femorotibial joint was used as a control. Postoperative outcome was assessed by lameness examination, radiography, arthroscopy, synoviocentesis, necropsy, and histology up to 26 (n = 3) or 52 (n = 3) weeks. By postoperative week (POW) 4, goats demonstrated normal range of motion, no joint effusion, and only mild lameness in the operated limb. By POW 26 the animals were sound with only occasional very mild lameness. Arthroscopy at POW 14 revealed moderate synovial inflammation and a chondral membrane extending centrally across the implant surface. Radiographs at POWs 14 to 52 implied implant stability in the operated joints, as well as subchondral bone remodeling and mild exostosis formation in the operated and contralateral unoperated joints of some goats. By POW 26, histology revealed new trabecular bone abutting the implant. At POWs 26 and 52 MFC cartilage was metachromatic and intact in the operated and unoperated femorotibial joints. Proximal tibiae of some operated and unoperated limbs demonstrated limited subchondral bone remodeling and foci of articular cartilage fibrillation and thinning. The chondral membrane crossing the prosthesis possessed a metachromatic matrix containing singular and clustered chondrocytes. Our data imply the safety, biocompatibility, and functionality of the implant. Focal articular damage was documented in the operated joints at POWs 26 and 52, but lesions were much reduced over those previously reported in untreated defects. Expanded animal or preclinical human studies are justified.

A cadaveric investigation into the links between macroscopic and microscopic osteoarthritic changes at the hip

Our objective was to investigate the frequency and distribution of osteoarthritic changes at the hip, including the relationship between osteoarthritic lesions on the femoral head surface and histological changes in articular cartilage, in 12 cadavers. Twelve embalmed cadavers (five males and seven females) were dissected, and the femoral head was removed from both sides (24 femoral heads). Any gross osteoarthritic changes were noted and graded (on a scale of 1-3). A circular disc was then removed from the equator of the femoral head and divided into nine regions. Out of 192 segments, 54 underwent sectioning and staining with haematoxylin and eosin to assess histological changes in cartilage. Osteoarthritis of the hip was present in all cadavers, with all males having bilateral OA and 50% having grade 2 or higher lesions (50% were grade 1), and four of the seven female specimens having bilateral OA and only 7% with grade 2 lesions (with 71% grade 1 and 21% normal). Chondrocyte clustering was most commonly observed in the deep layer of cartilage followed by the intermediate and superficial layers respectively, as the grade of the macroscopic lesion increased. Cartilage injury at the histological level precedes any visible denudation of the femoral head articular cartilage. This study supports the hypothesis that early osteoarthritic changes occur in the deep layer of cartilage near the tide mark and progress superficially concomitant with an overall increase in the osteoarthritic lesion size on the femoral head surface.

Heterogeneity in patellofemoral cartilage adaptation to anterior cruciate ligament transection; chondrocyte shape and deformation with compression

The purpose of this study was to determine if the opposing cartilages of the feline patellofemoral joint adapted differently to short-term anterior cruciate ligament transection (ACL-T) and if the magnitude of chondrocyte deformation upon tissue loading was altered under ACL-T conditions compared to contralateral controls. In situ static compression of physiological magnitude was applied to the feline patellofemoral cartilage 16 weeks post-ACL-T and cartilage and chondrocyte deformation were evaluated by histomorphometry. Six adult cats were euthanized 16 weeks after unilateral ACL-T. A peak surface pressure of 9 MPa was applied to the fully intact patella and femoral groove cartilages. After in situ fixation under compression, sections from the centre of the indent and from an adjacent unloaded area of the cartilages were analysed. Chondrocyte shape, size, clustering and volumetric fraction were quantified. Experimental patellar articular cartilage was thicker, contained larger chondrocytes that were more frequently arranged in clusters and had, on average, a larger chondrocyte volumetric fraction compared to contralateral controls. In contrast, the experimental femoral groove cartilage demonstrated little adaptation to ACL-T. The patellar articular cartilage adapts to short-term ACL-T to a greater extent than femoral groove cartilage. We speculate that differences in the histological parameters of control tissues, such as cartilage thickness and the magnitude and depth distribution of chondrocyte shape, size and volumetric fraction may contribute to predisposing patellar cartilage, and not femoral groove cartilage, to adaptation after ACL-T.

Increased concentrations of neuro-excitatory amino acids in rat anterior cruciate ligament-transected knee joint dialysates: a microdialysis study

Changes in excitatory amino acid (EAA) levels were examined in the knee joint dialysates of rats with early osteoarthritis (OA). Early OA was induced by anterior cruciate ligament (ACL) transection in one knee and the contralateral knee was used as the sham-operated control, the side for ACL transection being assigned randomly. Twenty weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high performance liquid chromatography. The rats were then sacrificed for histopathological examination. Hematoxylin/eosin and Safranin-O staining showed cartilage fibrillation, clustering of chondrocytes, and a reduction in matrix proteoglycans at week 20 in the ACL-transected knee, but not in the sham-operated knee. Levels of glutamate and aspartate in dialysates from the ACL-transected knee were significantly increased by 92 ± 20.3% or 57 ± 17.5%, respectively, compared to those in the contralateral sham-operated knee. This increase may contribute to the pathogenesis of early OA.

Neonatal Lethal Osteochondrodysplasia with Low Serum Levels of Alkaline Phosphatase and Osteocalcin

Neonatal lethal skeletal dysplasias are rare and typically involve thoracic malformations and severe limb shortening. We report on a newborn boy manifesting an osteochondrodysplasia associated with fatal respiratory insufficiency who had normal lung volumes and extremity lengths. His disorder featured aberrant skeletal patterning and defective ossification including a severely osteopenic skull, apparent absence of clavicles, and clefting of the mandible and vertebrae. Serum alkaline phosphatase and osteocalcin levels were markedly low. Biochemical studies suggested parathyroid insufficiency probably from critical illness. Histopathology at autopsy excluded impaired mineralization of skeletal matrix, but endochondral bone formation appeared disorganized with growth plate clustering of chondrocytes in hypertrophic zones and in zones of provisional calcification. Parathyroid glands were not found. Despite features of two distinctive heritable entities, hypophosphatasia and cleidocranial dysplasia, the cumulative findings did not match either condition, and no mutations were found in either the tissue nonspecific ALP isoenzyme or core-binding factor genes, respectively, or in the genes encoding osteocalcin or the osteoblast transcription factor osterix. This patient could represent the extreme of cleidocranial dysplasia (a disorder not always associated with structural mutation in core-binding factor A1), but more likely he defines a unique osteochondrodysplasia disrupting both intramembranous and endochondral bone formation.

Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor).

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage. Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2. The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo. In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro. These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.

Histological features of osteoarthritic canine cartilage after prolonged administration of carprofen

SummaryThe objective of this study was to determine whether the non-steroidal anti-inflammatory drug (NSAID) carprofen causes adverse histological changes to cartilage after prolonged administration for treatment of naturally acquired osteoarthritis.Dogs diagnosed with cranial cruciate ligament rupture and secondary osteoarthritis of their stifle joints were divided into two groups: those who had received a standard dose of carprofen for a minimum of four weeks and those who had not received a NSAID for a minimum of one month. Cartilage from the intercondylar notch was obtained when the patients underwent surgical procedures to stabilize the stifle joint. The samples were processed and evaluated for histological differences using haematoxylin and eosin and safranin-O-fast green-iron haematoxylin stains and were graded according to a Mankin scale (graded 0-14) for osteoarthritis.The histopathological findings were variable and included loss of matrix staining, irregularities to the cartilage surface, matrix fragmentation, chondrocyte clustering, matrix degeneration and vascular and synovial invasion (pannus) and were not specific to either group. There was not any significant difference between study groups (p = 0.2721) according to the Wilcoxan rank sum test.

Distribution of newly synthesized aggrecan in explant cultures of bovine cartilage treated with retinoic acid.

This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when explant cultures of articular cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In cartilage explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.

An experimental model of osteoarthrosis of the temporomandibularjoint in monkeys

The purpose of this study was to develop a model of osteoarthrosis of the temporomandibular joint in monkeys, which is remarkably similar in structure and function to that of humans. Nine juvenile monkeys, two as controls and seven as an experimental group, were used in this study. In the experimental group, the articular eminence on both sides was surgically made steeper. Two animals were killed at 1 week, four at 6 months, and one at 1 year postoperatively and the temporomandibular joints were examined macroscopically and microscopically. Typical changes of osteoarthrosis were observed in the 6-month and 1-year specimens. These comprised clustering of chondrocytes which resulted in vertical and horizontal splitting in the articular cartilage, and fibrillation of the articular surface resulting in fibrous union in the joint cavity. These degenerative changes advanced progressively over time. Slight anterior displacement and degenerative changes in the articular disc were also seen.

A rare case of polyarticular synovial osteochondromatosis

Synovial osteochondromatosis is a rare condition characterised by the production of multiple nodules of hyaline cartilage within the subsynovial connective tissue of joints, tendons and bursae. The nodules of proliferative cartilage may remain attached to the synovial membrane or detach to form loose bodies in the synovial cavity. Most reported cases have been of the monoarticular form of the disease, with the majority in the knee and hip. Polyarticular forms comprise only 5% of cases.Here we report a case of polyarticular synovial osteochondromatosis in a dissecting room cadaver (female; 76 y). Multiple, calcified, intra‐ and extra‐articular nodules (n = 14) were located bilaterally in the elbow, proximal radioulnar and shoulder joints. They had a lustrous, cobblestone‐like appearance and the largest (27 mm) was located in the anteromedial aspect of the right elbow joint. The smallest (5 mm) was deep to the annular ligament of the left proximal radioulnar joint.Signs of cartilage degeneration (fissuring and chondrocyte clustering), similar to those well documented in osteoarthritis, were observed histologically. Articular cartilage covered only a small area of the nodules while periosteum dominated the remaining surface. Thin sparsely distributed spicules of spongy bone were seen inside the nodules, separated by regions of fatty marrow.The affected joints also showed signs of osteoarthritis. This is consistent with the common view that osteoarthritis is a secondary complication of synovial osteochondromatosis. However osteoarthritic change was also evident in other joints.

Cell proliferation in human arthrotic joint cartilage

Osteoarthritic (OA) cartilage is histologically characterized by the appearance of cell clusters, which are probably generated by mitotic cell division. The aim of this study was to analyze the distribution and amount of proliferating chondrocytes in healthy and osteoarthritic cartilage systematically. 6 normal and 43 osteoarthritic cartilage/bone samples were obtained during autopsies or total knee replacements. The cartilage specimens were stained with safranin-o and scored according to the Mankin-System. Proliferating chondrocytes were identified by immunohistochemical detection of the antigen PCNA. The number of proliferating chondrocytes was determined by counting 100 chondrocytes in each cartilage layer. In normal and osteoarthritic human cartilage, proliferating chondrocytes were detected. The amount of proliferating chondrocytes increased during the progression of OA cartilage changes. Expression of PCNA was determined in single chondrocytes and clustered chondrocytes. Chondrocyte cell division was activated specifically in cartilage with severe OA changes. Osteoarthritic chondrocytes, which are known to increase the synthesis of different matrix proteins, show the phenomenon of mitotic cells division. Whether the increased amount of proliferating chondrocytes fails to compensate the rate of apoptotic chondrocytes, or whether the generation of cell clusters represents only a fruitless repair effort is a subject of recent research. However, the induction of chondrocyte proliferation is a potential target in the treatment of OA.

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.

Response of articular cartilage and subchondral bone to internal fixation devices made of poly- l-lactide: a histomorphometric and microradiographic study on rabbits

To study the tissue response of articular cartilage and subchondral bone to biodegradable fixation devices, pins and rods made of poly- l-lactide with a fibers-in-matrix texture were implanted through the articular surface of the intercondylar portion of the distal rabbit femur. The initial raw material viscosity average molecular weight of the polymer was 660,000. One pin or screw was implanted per animal. The pins were cylindrical and measured 4.5 mm in transverse diameter. The screws had a core diameter of 3.2 mm and an outer diameter of 4.5 mm. At insertion, the implants were cut flush with the articular surface. After follow-up times of 36 and 48 weeks, the specimens were examined histomorphometrically and microradiographically. The intact contralateral femur served as a control for comparison. No signs of erosion or degradation of the polymer could be seen in the specimens. A brim of reparative tissue was formed at the entrance of the implant channel. The width of the reparative tissue from the tissue–implant boundary towards the center of the entrance hole varied greatly between the specimens, from 30 to 950 μm. In most specimens this bridging tissue consisted of undifferentiated mesenchymal tissue. Only two out of 24 specimens showed a near-normal metachromatic toluidine-blue staining of the matrix. Degenerative chondrocyte clustering occurred in the pre-existing cartilage within a 400 μm wide zone from the tissue–implant interface into the recipient tissues. Some new-bone formation was seen to envelop the implant in all specimens, but the fractional osteoid formation surface of the trabeculae showed a value significantly higher than that of the intact control side only in the screw-implanted 36-week specimens. Because of the long degradation time of poly- l-lactide, the restoration process of the articular cartilage was slow, and with regard to the quality and quantity of the reparative tissue, very variable. Large implants made of poly- l-lactide may not be suitable for insertion through intra-articular surfaces.

Semiquantitative reverse transcription-polymerase chain reaction analysis of syndecan-1 and -4 messages in cartilage and cultured chondrocytes from osteoarthritic joints

Objective To determine the steady-state of messenger RNA (mRNA) levels of syndecan-1 and syndecan-4 in cartilage samples and chondrocytes derived from human osteoarthritic knee joints.Methods Steady-state levels of gene-specific mRNA (relative to β-actin) were measured by semiquantitative polymerase chain reaction (PCR).Results RT-PCR allowed detection of syndecan-1 (for the first time) and syndecan-4 in both cartilage samples and articular chondrocytes cultured as primary monolayers. The mRNA levels of syndecan-1 were reduced in cartilage tissue from heavily damaged compared to normal-looking areas whereas those of syndecan-4 were significantly increased. In contrast, the expression of syndecan-1 was higher in cultured chondrocytes derived from the fibrillated osteoarthritic cartilage than in cells obtained from intact cartilage, while the syndecan-4 message levels did not differ between the two sites.Conclusion The expression of the cell-surface syndecans 1 and 4 is altered during the osteoarthritic degradative process of the knee joint. The discoordinate syndecan gene expression, which is probably related to the chondrocyte proliferation and clustering, may contribute to the disorganization of the cartilage and the development of OA processes. Isolation and culturing the chondrocytes as monolayers dramatically change the expression of these genes and cannot reflect the in situ condition.