Loss Of Chondrocytes Research Articles

New Technique for Cell-Seeded Collagen Matrix-Supported Autologous Chondrocyte Transplantation

Autologous chondrocyte transplantation/implantation (ACT/ACI) is becoming increasingly common for the treatment of large cartilage defects in the knee joint. The traditional ACT technique involves injection of a suspension of cells into the cartilage defect, which is covered with a periosteal flap or collagen membrane. The technique requires extensive suturing to create an effective seal; however, cell leakage remains a potential problem. Matrix-induced autologous chondrocyte implantation (MACI/MACT) avoids this potential problem by using a membrane on which chondrocytes are seeded and cultured for several days, before the membrane is cut to the correct size and shape of the defect. Time-consuming extensive suturing is unnecessary. However, cutting and repeated manipulation of the seeded membrane may result in the loss of critical chondrocytes. A modified technique termed ACT-collagen membrane seeding (ACT-Cs) has been developed in which expanded chondrocytes are applied to the collagen membrane after it has been cut to size, substantially reducing the risk of viable cell loss while retaining the ease and speed of the MACI/MACT procedure. In addition, the seeding of mitotically active chondrocytes onto the membrane after expansion and immediately before transplantation allows direct application of high cell concentrations.

Differential response of porcine osteoblasts and chondrocytes in cell or tissue culture after 5-aminolevulinic acid-based photodynamic therapy

Outcome in osteochondral allografting is limited by the immunological incompatibility of the grafted tissue. Based on a resistance of chondrocytes to photodynamic therapy in cell culture it is proposed that 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) might be used to inactivate bone while maintaining viability of chondrocytes and thus immunomodulate bone selectively. Chondrocytes and osteoblasts from porcine humeral heads were either isolated (cell culture) or treated in situ (tissue culture). To quantify cytotoxic effects of 5-ALA-PDT (0-20 J/cm(2), 100 mW/cm(2)) an (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) (MTT)-assay was used in cell culture and in situ hybridization in tissue culture to assess metabolic active cells (functional osteoblasts: col alpha(1)(I) mRNA, functional chondrocytes: col alpha(1)(II) mRNA). In cell culture, survival after 5-ALA-PDT was significantly higher for chondrocytes (5 J/cm(2): 87+/-12% compared to untreated cells) than for osteoblasts (5J/cm(2): 12+/-11%). In tissue culture, the percentage of functional chondrocytes in cartilage showed a decrease after 5-ALA-PDT (direct fixation: 92+/-2%, 20 J/cm(2): 35+/-15%; P<0.0001). A significant decrease in the percentage of bone surfaces covered by functional osteoblasts was observed in freshly harvested (31+/-3%) compared to untreated tissues maintained in culture (11+/-4%, P<0.0001), with no further decrease after 5-ALA-PDT. Chondrocytes were more resistant to 5-ALA-PDT than osteoblasts in cell culture, while in tissue culture a loss of functional chondrocytes was observed after 5-ALA-PDT. Since osteoblasts - but not chondrocytes - were sensitive to the tissue culture conditions, devitalized bone with functional cartilage might already be achieved by applying specific tissue culture conditions even without 5-ALA-PDT.

Thermoforming of tracheal cartilage: Viability, shape change, and mechanical behavior

Trauma, emergent tracheostomy, and prolonged intubation are common causes of severe deformation and narrowing of the trachea. Laser technology may be used to reshape tracheal cartilage using minimally invasive methods. The objectives of this study were to determine: (1) the dependence of tracheal cartilage shape change on temperature and laser dosimetry using heated saline bath immersion and laser irradiation, respectively, (2) the effect of temperature on the mechanical behavior of cartilage, and (3) tissue viability as a function of laser dosimetry. Ex vivo rabbit trachea cartilage specimens were bent and secured around a cylinder (6 mm), and then immersed in a saline bath (45 and 72 degrees C) for 5-100 seconds. In separate experiments, tracheal specimens were irradiated with a diode laser (lambda = 1.45 microm, 220-400 J/cm(2)). Mechanical analysis was then used to determine the elastic modulus in tension after irradiation. Fluorescent viability assays combined with laser scanning confocal microscopy (LSCM) were employed to image and identify thermal injury regions. Shape change transition zones, between 62 and 66 degrees C in the saline heating bath and above power densities of 350 J/cm(2) (peak temperatures 65+/-10 degrees C) for laser irradiation were identified. Above these zones, the elastic moduli were higher (8.2+/-4 MPa) than at lower temperatures (4.5+/-3 MPa). LSCM identified significant loss of viable chondrocytes within the laser-irradiation zones. Our results indicate a change in mechanical properties occurs with laser irradiation and further demonstrates that significant thermal damage is concurrent with clinically relevant shape change in the elastic cartilage tissues of the rabbit trachea using the present laser and dosimetry parameters.

Chondrocyte Apoptosis: Implications for Osteochondral Allograft Transplantation

Osteochondral allograft transplantation is a useful technique to manage larger articular cartilage injuries. One factor that may compromise the effectiveness of this procedure is chondrocyte cell death that occurs during the storage, preparation, and implantation of the osteochondral grafts. Loss of viable chondrocytes may negatively affect osteochondral edge integration and long-term function. A better understanding of the mechanisms responsible for chondrocyte loss could lead to interventions designed to decrease cell death and improve results. Recent studies indicate that apoptosis, or programmed cell death, is responsible for much of the chondrocyte death associated with osteochondral allograft transplantation. Theoretically, some of these cells can be rescued by blocking important apoptotic mediators. We review the role of apoptosis in cartilage degeneration, focusing on apoptosis associated with osteochondral transplantation. We also review the pathways thought to be responsible for regulating chondrocyte apoptosis, as well as experiments testing inhibitors of the apoptotic pathway. These data suggest that key contributors to the apoptotic process can be manipulated to enhance chondrocyte survival. This knowledge may lead to better surgical outcomes for osteochondral transplantation.

Fresh Osteochondral Allografts for Posttraumatic Knee Defects: Long-term Followup

Fresh osteochondral allograft transplantation has been an effective treatment option with promising long-term clinical outcomes for focal posttraumatic defects in the knee for young, active individuals. We examined histologic features of 35 fresh osteochondral allograft specimens retrieved at the time of subsequent graft revision, osteotomy, or TKA. Graft survival time ranged from 1 to 25 years based on their time to reoperation. Histologic features of early graft failures were lack of chondrocyte viability and loss of matrix cationic staining. Histologic features of late graft failures were fracture through the graft, active and incomplete remodeling of the graft bone by the host bone, and resorption of the graft tissue by synovial inflammatory activity at graft edges. Histologic features associated with long-term allograft survival included viable chondrocytes, functional preservation of matrix, and complete replacement of the graft bone with the host bone. Given chondrocyte viability, long-term allograft survival depends on graft stability by rigid fixation of host bone to graft bone. With the stable osseous graft base, the hyaline cartilage portion of the allograft can survive and function for 25 years or more.

A type I collagen defect leads to rapidly progressive osteoarthritis in a mouse model

This study was undertaken to test the hypothesis that abnormalities of the subchondral bone can result in osteoarthritis (OA). We used a knockin model of human osteogenesis imperfecta, the Brittle IV (Brtl) mouse, in which defective type I collagen is expressed in bone. OA in individual mice was documented by micro-magnetic resonance imaging (micro-MRI) and micro-computed tomography (micro-CT). Alterations in the knee joints were confirmed by histopathologic and immunohistochemical analysis. In addition, atomic force microscopy (AFM) was used to assess the ultrastructure of the articular cartilage and subchondral bone matrix. Brtl mice had decreased integrity of bone but initially normal articular cartilage. However, by the second month of life, Brtl mice developed alterations of the cartilage that were characteristic of OA, as documented by micro-CT, micro-MRI, and histologic evaluation. In addition, chondrocyte loss and breakdown of the collagen matrix in the residual cartilage were demonstrated using AFM. The Brtl mouse model demonstrates that progressive destruction of articular cartilage characteristic of OA may be secondary to altered architecture of the underlying subchondral bone.

Surgical suturing of articular cartilage induces osteoarthritis-like changes

In clinical tissue-engineering-based approaches to articular cartilage repair, various types of flap are frequently used to retain an implanted construct within the defect, and they are usually affixed by suturing. We hypothesize that the suturing of articular cartilage is associated with a loss of chondrocytes from, and osteoarthritis-like changes within, the perisutural area. We established a large, partial-thickness defect model in the femoral groove of adult goats. The defects were filled with bovine fibrinogen to support a devitalized flap of autologous synovial tissue, which was sutured to the surrounding articular cartilage with single, interrupted stitches. The perisutural and control regions were analyzed histologically, histochemically and histomorphometrically shortly after surgery and 3 weeks later. Compared to control regions, chondrocytes were lost from the perisutural area even during the first few hours of surgery. During the ensuing 3 weeks, the numerical density of cells in the perisutural area decreased significantly. The cell losses were associated with a loss of proteoglycans from the extracellular matrix. Shortly after surgery, fissures were observed within the walls of the suture channels. By the third week, their surface density had increased significantly and they were filled with avascular mesenchymal tissue. The suturing of articular cartilage induces severe local damage, which is progressive and reminiscent of that associated with the early stages of osteoarthritis. This damage could be most readily circumvented by adopting an alternative mode of flap affixation, such as gluing with a biological adhesive.

Intra-articular Injection of Hyaluronan Diminishes Loss of Chondrocytes in a Rat Immobilized-Knee Model

Joint immobilization is a useful and common treatment modality in orthopedics. However, it also causes unfavorable outcome such as articular cartilage degeneration. Intra-articular injection of hyaluronan has been accepted as a treatment of osteoarthritis, but its effects on immobilized joint remain to be clarified. Hyaluronan is a polysaccharide, distributed ubiquitously in various tissues. In this study, we examined the effect of hyaluronan on the articular cartilage in immobilized joints. The unilateral knee joints of adult male rats were immobilized at 150 degrees in flexion with an internal plate and screws for 1, 2, 4, 6, 8, 12, or 16 weeks (n = 84). Hyaluronan or saline (50 microl/each injection) was administered intra-articularly on the day of surgery and once a week. The articular cartilage from the medial midcondylar region of the knee was obtained, and divided into non-contact, contact and transitional areas (between the non-contact and the contact areas). In each area, a degree of degeneration was evaluated by histomorphometric grading, and measurements of thickness and number of chondrocytes. Histological grading scores in the hyaluronan group were smaller at 12 and 16 weeks compared with those in the saline group. The thickness of the articular cartilage increased in the transitional area in both groups. The number of chondrocytes in the contact and transitional areas gradually decreased, but their number in the hyaluronan group was greater at 12 and 16 weeks compared with that in the saline group. Hyaluronan showed chondroprotective effects on the articular cartilage in a rat immobilized-knee model.

Preventing chondrocyte programmed cell death caused by iatrogenic injury

Cartilage repair technology is advancing at a rapid pace. However, all techniques share a common weakness—unintentional chondrocyte cell death resulting from cartilage injury that occurs during preparation of the defect site. The loss of chondrocytes at the edge of host cartilage is likely to contribute to failed integration of regenerated tissue or grafts to the surrounding cartilage. Recent studies have demonstrated that “apoptosis”, or programmed cell death (PCD), may be responsible for much of the cell death caused by cartilage injury. Theoretically, inhibitors of key pathways responsible for PCD could rescue chondrocytes and improve the results of cartilage repair surgery. The purpose of this study was to test the hypothesis that short-term, intra-articular PCD inhibitor treatment can limit chondrocyte death in vivo following simulated preparation of host cartilage for a repair procedure. A microcurette was used to create full-thickness articular cartilage injuries to the femoral condyles of adult New Zealand White rabbits. Animals received daily intra-articular injections either with a potent PCD inhibitor or with vehicle alone. Treatment with the inhibitor resulted in a significant reduction in the percentage of chondrocytes undergoing PCD compared to controls [treated=10.1±2.4%; controls=26.5±3.6%; (p=0.0013)]. These results provide proof of concept for the use of PCD inhibitors to enhance the results of cartilage repair surgeries.

In Vitro Exposure to 0.5% Bupivacaine Is Cytotoxic to Bovine Articular Chondrocytes

Intra-articular use of 0.5% bupivacaine is common in arthroscopic surgery. This study was conducted to test the hypotheses that (1) 0.5% bupivacaine is toxic to articular chondrocytes, and (2) the intact articular surface protects chondrocytes from the effects of short-term exposure to 0.5% bupivacaine. Freshly isolated bovine articular chondrocytes were prepared into alginate bead cultures and were treated with 0.5% bupivacaine solution or 0.9% saline for 15, 30 or 60 minutes, washed, and returned to growth media. Chondrocytes were recovered from alginate 1 hour, 1 day, and 1 week after bupivacaine exposure; they were fluorescently labeled to identify apoptotic and dead cells and were analyzed by flow cytometry. Twelve osteochondral cores were harvested from bovine knees. The superficial 1 mm of cartilage was removed from 6 cores (top-off). Intact and top-off cores were submerged in 0.9% saline or 0.5% bupivacaine solution for 30 minutes and then maintained in chondrocyte growth media for 24 hours. Live-cell/dead-cell fluorescent imaging was assessed using confocal microscopy. Greater than 99% chondrocyte death/apoptosis was observed in all bupivacaine-exposed alginate bead cultures compared with 20% cell death in saline-treated controls (P < .05). Osteochondral cores with intact surfaces treated with 0.5% bupivacaine showed 42% dead chondrocytes. When the articular surface was removed, 0.5% bupivacaine resulted in increased cell death, with 75% dead chondrocytes (P < .05). Results show that 0.5% bupivacaine solution is cytotoxic to bovine articular chondrocytes and articular cartilage in vitro after only 15 to 30 minutes' exposure. The intact bovine articular surface has some chondroprotective effects. Because healthy chondrocytes are important for maintenance of the cartilage matrix, chondrocyte loss may contribute to cartilage degeneration. This study shows a cytotoxic effect of 0.5% bupivacaine solution on bovine articular chondrocytes in vitro. Although these results cannot be directly extrapolated to the clinical setting, the data suggest that caution should be exercised in the intra-articular use of 0.5% bupivacaine.

Cellular events leading to chondrocyte death after cartilage impact injury

We undertook this study to test our postulate that leukocytes extend the zone of injury in cartilage after acute mechanical trauma. Fresh cadaveric canine femoral condyles were subjected to 20-25-MPa impact injury. Condyle explants or dispersed chondrocytes were cultured with autologous blood mononuclear leukocytes (MNLs). Viability of chondrocytes at varying distances from the impact site was assessed by trypan blue exclusion. Mechanical injury caused a significant loss of viable chondrocytes over 7 days, even in cartilage >10 mm from the impact site. After biomechanical stress, death of cells within 10 mm of the impact could be largely prevented by addition of N(G)-monomethyl-L-arginine to inhibit nitric oxide (NO) generation. Chondrocytes within 10 mm of the impact were also susceptible to killing by living MNLs, but not by incubation with the supernatants of endotoxin-activated MNLs. Chondrocytes in this vulnerable zone expressed intercellular adhesion molecule 1 (ICAM-1) (CD54), facilitating attachment of MNLs that localized adjacent to the chondrocytes. Leukocytes killed dispersed chondrocytes harvested from the impact zone by generation of reactive oxygen species. Leukocyte-mediated killing could be blocked by desferoxamine or by antibodies to CD18, which prevent attachment of leukocytes to ICAM-1-expressing chondrocytes. Our data suggest that after mechanical injury, chondrocytes distant from the site may be killed through the generation of NO. Inflammatory leukocytes further extend the zone of chondrocyte death by adhering to chondrocytes expressing ICAM-1 and by inducing the accumulation of free oxygen radicals in the chondrocyte cytoplasm. Patients may benefit from therapies that reduce infiltration of inflammatory leukocytes into acutely injured cartilage.

Healing of articular cartilage defects. An experimental study of vascular and minimal vascular microenvironment

The repair potential in a chondral defect with (treatment A) or without access to bone marrow elements (treatment B) at the basis of the defect sealed by a rim-sutured periosteal flap was studied using adult New Zealand rabbits (22 weeks) as an experimental model. At sacrifice, macroscopical changes, synovial fluid contents, degree of filling, thickness of the cartilage rim, and the subchondral bone were evaluated. Histomorphometric measurements of extent of filling (mainly fibrous tissue) of the defect at 36 weeks postoperatively, showed 50% filling in treatment A compared with 33% in treatment B (p = 0.011). A difference in height of the cartilage rim between the experimental groups and sham-control was measured ( p = 0.005). Cartilage degeneration was observed at the cartilage rim of the original defect, and included loss of chondrocytes and disruption of surface continuity in both experimental groups. In addition, treatment A resulted in a significantly increased thickness of the subchondral bone in the defect in comparison to treatment B at 2 weeks and at 36 weeks (p = 0.021). The increased thickness of the subchondral bone may be of concern for the bone marrow stimulation techniques regarding the long-term outcome.

TRANSPLANTATION OF OSTEOCHONDRAL ALLOGRAFTS AFTER COLD STORAGE

Background: Transplantation of fresh osteochondral allografts stored at hypothermia into knee cartilage defects is a common procedure; however, the length of time that allografts can be stored prior to transplantation is controversial and has been determined, in part, by the results of vital stain uptake by chondrocytes. This study was performed to further define the limits of allograft storage. Methods: Articular cartilage from six cadavers was stored for up to fifty-one days in tissue-culture media, and histologic sections were evaluated histomorphometrically to quantify the loss of chondrocytes. Samples of the cartilage were also placed into tissue culture to assess cell growth. Animal studies were performed in parallel on sixteen adult baboons with osteochondral allografts transplanted into the medial femoral condyle. Prior to transplantation, all allografts were stored in RPMI-1640 with 10% fetal calf serum at 4° to 6°C for up to eighty-five days. The transplants were graded on their gross and histological appearance, as well as their histochemical properties. Results: Many of the human samples stored at hypothermia in culture media for up to forty days retained some recognizable chondrocytes, but morphometry showed a gradual, significant decrease in the number of chondrocytes after nine days (p = 0.001). In addition, the cell outgrowth occurred from all specimens stored for up to fifteen days but not in samples stored for longer than thirty-four days. In animal studies, transplanted allograft cartilage that had been stored for less than eighteen days looked smooth and glistening, but grafts stored for over twenty-one days were pale, pitted, fragmented, or yellow, and chondrocytes were absent. Conclusions: Time-dependent loss of chondrocytes in articular cartilage stored at hypothermia, especially in specimens stored for longer than fifteen to twenty days, was observed in this study. Cartilage allografts transplanted into nonhuman primates after twenty-one days of storage underwent more severe degenerative changes than allografts that had been stored for less than twenty-one days. These findings suggest caution when transplanting cartilage stored at hypothermia for over twenty days. Clinical Relevance: Surgeons who perform fresh osteochondral allograft transplantation should be cognizant of the time-dependent changes associated with cold storage of these grafts.

Transplantation of Osteochondral Allografts After Cold Storage

Transplantation of fresh osteochondral allografts stored at hypothermia into knee cartilage defects is a common procedure; however, the length of time that allografts can be stored prior to transplantation is controversial and has been determined, in part, by the results of vital stain uptake by chondrocytes. This study was performed to further define the limits of allograft storage. Articular cartilage from six cadavers was stored for up to fifty-one days in tissue-culture media, and histologic sections were evaluated histomorphometrically to quantify the loss of chondrocytes. Samples of the cartilage were also placed into tissue culture to assess cell growth. Animal studies were performed in parallel on sixteen adult baboons with osteochondral allografts transplanted into the medial femoral condyle. Prior to transplantation, all allografts were stored in RPMI-1640 with 10% fetal calf serum at 4 degrees to 6 degrees C for up to eighty-five days. The transplants were graded on their gross and histological appearance, as well as their histochemical properties. Many of the human samples stored at hypothermia in culture media for up to forty days retained some recognizable chondrocytes, but morphometry showed a gradual, significant decrease in the number of chondrocytes after nine days (p = 0.001). In addition, the cell outgrowth occurred from all specimens stored for up to fifteen days but not in samples stored for longer than thirty-four days. In animal studies, transplanted allograft cartilage that had been stored for less than eighteen days looked smooth and glistening, but grafts stored for over twenty-one days were pale, pitted, fragmented, or yellow, and chondrocytes were absent. Time-dependent loss of chondrocytes in articular cartilage stored at hypothermia, especially in specimens stored for longer than fifteen to twenty days, was observed in this study. Cartilage allografts transplanted into nonhuman primates after twenty-one days of storage underwent more severe degenerative changes than allografts that had been stored for less than twenty-one days. These findings suggest caution when transplanting cartilage stored at hypothermia for over twenty days.

Long-term Viability and Mechanical Behavior Following Laser Cartilage Reshaping

To investigate the long-term in vivo effect of laser dosimetry on rabbit septal cartilage integrity, viability, and mechanical behavior. Nasal septal cartilage specimens (control and irradiated pairs) were harvested from 18 rabbits. Specimens were mechanically deformed and irradiated with an Nd:YAG laser across a broad dosimetry range (4-8 W and 6-16 seconds). Treated specimens and controls were autologously implanted into a subperichondrial auricular pocket. Specimens were harvested an average +/- SD of 208 +/- 35 days later. Tissue integrity, histology, chondrocyte viability, and mechanical property evaluations were performed. Tissue damage results were compared with Monte Carlo simulation models. All laser-irradiated specimens demonstrated variable tissue resorption and calcification, which increased with increased dosimetry. Elastic moduli of the specimens were significantly either lower or higher than controls (all P<.05). Viability assays illustrated a total loss of viable chondrocytes within the laser-irradiated zones in all treated specimens. Histologic examination confirmed these findings. Experimental results were consistent with damage profiles determined using numerical simulations. The loss of structural integrity and chondrocyte viability observed across a broad dosimetry range underscores the importance of spatially selective heating methods prior to initiating application in human subjects.

Upregulation of Apoptotic and Matrix-related Gene Expression during Fresh Osteochondral Allograft Storage

We identified changes in proapoptotic and extracellular matrix-related gene expression with prolonged storage of fresh osteochondral allografts using gene array analysis to better understand the process of graft degradation during storage. Six human distal femurs were obtained according to standard organ harvesting protocol and stored in serum-free allograft media. Each was examined at baseline (within 72 hours postmortem), 21 days (average time of implantation), and 35 days (maximum time to implantation) for proapoptotic and extracellular matrix-related gene expression using two 100-gene microarrays, cell viability using confocal microscopy, and proteoglycan synthesis via SO4 incorporation. We found numerous genes showing upregulation associated with increased storage time, including CD30, CD30 ligand, Fas, Fas ligand, tumor necrosis factor-alpha, and several caspases. Cell viability and proteoglycan synthesis also were significantly decreased with increased storage. Loss of chondrocytes via apoptosis is likely a key determinant of osteochondral allograft viability during storage, whereas extracellular matrix degeneration may occur at a later stage. These findings provide targets for future media modulation. Improved graft viability and the potential for lengthened storage periods through improved storage conditions may improve clinical outcomes and availability of fresh osteochondral allografts.

Developmental Regulation of Wnt/β-Catenin Signals Is Required for Growth Plate Assembly, Cartilage Integrity, and Endochondral Ossification

Studies have suggested that continuous Wnt/beta-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/beta-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of beta-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated beta-catenin mutant (delta-beta-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/beta-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Delta-beta-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.

Vulnerability to ROS-induced cell death in ageing articular cartilage: The role of antioxidant enzyme activity

To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H2O2) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.

The cyclin-dependent kinase inhibitor p57(Kip2) mediates proliferative actions of PTHrP in chondrocytes.

Parathyroid hormone-related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57(Kip2) is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrP's proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression.