Response of the donor and recipient cells in mesenchymal cell transplantation to cartilage defect

To facilitate the repair of articular cartilage defects, autologous mesenchymal cells from bone marrow or periosteum were transplanted in a rabbit model. Two weeks after the transplantation of the mesenchymal cells, the whole area of the original defect was occupied by cartilage. From the deep area of the reparative cartilage, which contacted with host bone, chondrocytes became hypertrophic and the invasion of bone with vasculature started, until the replacement reached the natural junction of the host cartilage and the subchondral bone about 4 weeks after transplantation. Twelve weeks after the transplantation, the repair cartilage in the defect became a little thinner than the adjacent normal cartilage, which became a little thinner 24 weeks after the transplantation (the longest observation period in the study). Large, full-thickness defects of the weight-bearing region of the articular cartilage were repaired with hyaline-like cartilage after implantation of autologous mesenchymal cells. The repair process by mesenchymal cell transplantation was explained as follows: The donor transplanted cell differentiated into cartilage and the defects were completely filled with cartilage. Then, mesenchymal cells that entered the chondrogenic lineage rapidly progressed through this lineage to the hypertrophic state, which was then the target for erosion and vascular invasion. Although this vasculature and the newly formed bone were considered to be host-derived, there was no evidence to that effect. To prove this, suitable experimental marking of these donor cells is needed. In the case of chondrocyte transplantation, the repair cartilage maintained its thickness to the full depth of the original defect; the tissue derived from the implanted chondrocytes was not invaded by vessels or replaced by subchondral bone.     

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.     

Influence of surgical preparation on the in-vivo response of osteochondral defects

Cartilage has an extremely poor capacity to heal, which has lead to intensive research into biomaterials and tissue engineering for the purpose of regenerating cartilage in vivo. Many of these techniques have shown great promise in vitro; however, the results do not always carry across to the in-vivo scenario. Healthy cartilage autografts often do not integrate with the adjacent cartilage, suggesting that cartilage is rarely capable of healing even under ideal conditions. It is hypothesized in this study that the surgical creation of defects in cartilage causes significant damage to the adjacent tissues, leading to further degradation of the cartilage and poor outcome for the repair in general. This study compares the healing response of osteochondral defects created with either a punch or a drill in the weight-bearing region of the sheep knee at 4 and 26 weeks following surgery. The use of a drill to create the defect creates a more aggressive inflammatory response at 4 weeks compared with a punch. However, by 26 weeks, defects created with a punch scored higher on the O'Driscoll cartilage grading scale. Tissue damage at the time of surgery plays an important part in the sequence of events for healing of cartilage defects. This knowledge will help to characterize and refine the ovine model for cartilage regeneration and may have an influence on surgical technique and instrumentation for clinical cartilage repair.     

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.     

Analysis of sections of implanted macroporous calcium phosphate bone substitutes by proton-induced x-emission method and energy-dispersive spectrometry

The osseointegration of porous calcium phosphate ceramics once implanted evolves in several stages. The mechanism of integration of such material usually is evaluated by histologic analysis. The trace elements present in bone can be detected in the ceramic and help to provide a semiquanti-tative evaluation of osseointegration. Two different methods of microanalysis, energy-dispersive spectrometry (EDS) and proton induced x-emission (PIXE) were used in this study to determine the appearance of trace elements (Zn, Sr, and Fe) present in bone at the implantation site containing the ceramic. Porous HA-ceramic cylinders were implanted in the cortical bone of sheep femurs for periods ranging from 2 to 36 weeks. Thick sections of the implant-containing bone were made at the end of the implantation period. A scanning line with proton or electron impacts 0.5 mm apart was plotted from the edges of the cortical bone across the implanted ceramic and the resulting x-ray spectra were determined. Following EDS analysis, the sections were surface-stained, observed under a light microscope, and the pore volume occupied by bone tissue was measured. The spectra obtained by PIXE method showed two regions for each element characterising either the bone tissue or the ceramic. Zinc and strontium present in the bone tissue, but absent from the ceramic, appeared 8 and 12 weeks after implantation, respectively. The concentration of iron present in the implant decreased with time. EDS showed no significant level of either element in the bone or the ceramic. Histologic observation revealed that immature bone invaded the pores of the outer layer of the ceramic as early as 2 weeks after implantation. The ceramics were totally osseoin-tegrated 20 weeks after implantation, although ceramic degradation continued for longer. In this experiment, the PIXE method was apparently sufficiently sensitive for monitoring the amount of trace element appearing in bone-implanted material.     

Stem cells for tissue engineering of articular cartilage

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.     

A new role for chondrocytes as non-professional phagocytes. An in vitro study

Chondrocytes are capable of engulfing latex particles, cell detritus, and necrotic and apoptotic remains in vitro. It is conceivable that chondrocytes might be involved in the clearance by phagocytosis of different materials within the cartilage. In fact, so far there is no evidence for the presence of "professional phagocytes" (macrophages and neutrophils) in this tissue. Chondrocyte suspensions obtained from rat knees and hips were cultured to assess phagocytosis of latex particles (1 microm), articular cartilage detritus, and necrotic and apoptotic chondrocyte remains (induced by VP-16 1 mM). We observed that chondrocytes phagocytosed latex particles as evaluated by confocal microscopy and flow cytometry. In addition, we observed that chondrocytes phagocytosed articular cartilage detritus and necrotic and apoptotic VP-16 induced-chondrocytes, as observed by bright field microscopy and transmission electron microscopy.     

In-situ engineering of cartilage repair: a pre-clinical in-vivo exploration of a novel system

This investigation explores a new cartilage repair technique that uses a novel method to secure a non-woven multifilamentous scaffold in the defect site after microfracture. The hypothesis is that a scaffold provides a larger surface area for attachment and proliferation of the mesenchymal stem cells that migrate from the bone marrow. Two in-vivo studies were undertaken in an ovine model. The first study, which lasted for 8 weeks, aimed to compare the new technique with microfracture. Chondral defects, 7 mm in diameter, were created in both femoral medial condyles of five ewes. One defect was treated with the new technique while the contralateral knee was treated with microfracture alone. The results revealed that the quantity of repair tissue was significantly greater in the defects treated with the new system. The second study had two time points, 3 and 6 months, and used 13 ewes. In this study, both defects were treated with the new technique but one received additional subchondral drilling in order to stimulate extra tissue growth. The majority of the implants had good tissue induction, filling 50-100 per cent of the defect volume, while the compressive modulus of the repairs was in the range of 40-70 per cent of that for the surrounding cartilage. In addition, hyaline-like cartilage was seen in all the repairs which had the additional drilling of the subchondral bone.     

Immunogold detection of types I and II chondrocyte collagen fibrils: an in situ atomic force microscopic investigation

Chondrocyte tissue engineering is a major challenge in the field of cartilage repair. The phenotype of chondrocytes consists of cartilage specific proteoglycan and type II collagen. During serial passages, chondrocytes dedifferentiate into cells, presenting a fibroblast-like phenotype consisting predominately of type I collagen synthesis. Observation of native collagen fibers could be visualized by atomic force microscope. Here, we developed an original and useful atomic force microscopy-based immunogold technique allowing biochemical distinction between types I and II collagen fibers. Imaging of 40-nm gold particles staining collagen fibers was performed in tapping mode. Rat 1 fibroblasts and human chondrosarcoma cells were used as positive models for types I and II collagen, respectively. As demonstrated by our data, primary rat chondrocytes adhering for 48 h on a glass substrate synthesize type II collagen native fibers. This technique allows analyses of local areas of the extracellular matrix of fixed cells, providing complementary data about cartilage phenotype. This simple approach could be of major interest for the biologist community in routine laboratory investigations, to localize in situ, macromolecules of the extracellular matrix.     

Tissue engineering of the synovial joint: the role of cell density

The ultimate goal in the tissue engineering of the synovial joint is to fabricate biologically derived analogues that can replace severely degenerated or traumatized synovial joint components. A number of challenges must be addressed before reaching this ultimate goal. In this report, the relevance of cell seeding density in the synthesis of chondrogenic and osteogenic matrices from human mesenchymal stem cells is explored. Human mesenchymal stem cells (hMSCs) were differentiated into chondrogenic cells and osteogenic cells ex vivo and encapsulated in poly(ethylene glycol) diacrylate (PEGDA) hydrogel at densities of 5 x 106 cells/ml, 40 x 10(6) cells/ml, and 80 x 10(6) cells/ml, in addition to a cell-free poly(ethylene glycol) (PEG) control group (0 x 10(6) cells/ml). Cell-seeded or cell-free PEG constructs were separately incubated in vitro for 4 weeks or implanted in vivo in the dorsum of immunodeficient rats for 4 weeks. In-vitro data demonstrated that hMSC-derived chondrocytes or hMSC-derived osteoblasts maintained their lineages per Safranin O and von Kossa staining after incubation for 4 weeks. The general pattern of initial cell seeding densities of 5 x 10(6) cells/ml, 40 x 10(6) cells/ml, and 80 x 10(6) cells/ml were preserved following in-vitro cultivation. Similarly, in-vivo data revealed that hMSC-derived chondrocytes and hMSC-derived osteoblasts maintained their respective lineages and the pattern of cell-seeding densities. An attempt was made to fabricate a composite construct with PEGDA hydrogel and polycaprolactone (PCL) with designed internal porosity for an osteochondral graft. Various cell-seeding densities as delineated in this report can be realized in the composite PEG-PCL graft. The findings demonstrate that cell-seeding density is likely a key parameter to consider in tissue-engineering design. The source of cells can either be transplanted cells or internally recruited cells.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell-substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

Confocal laser scanning microscopy of chondrocytes in vitro: Cytoskeletal changes after quinolone treatment

The use of quinolone antibiotics would be significant for chronically diseased children (e.g., cysticfibrosis) as a prophylactic long-term treatment. However, quinolones were shown to cause cartilage damage in experimental animals when administered during certain developmental stages. In the present study, the effect of quinolones on chondrocytes was studied in a cell culture model in order to avoid animal experiments, to investigate the influence of single factors, and to open up the possibility to test human tissue. Chondrocytes were obtained from hipjoint cartilage of 3 to 4-weeks-old rats and cultured in control medium or quinolone-supplemented medium. It was shown that quinolones heavily disturbed adhesion of chondrocytes to the culture dish, accompanied by changes in cell shape and cytoskeletal morphology. Reduction of filamentous actin (stress fibers) and disintegration of vimentin fibers was demonstrated by immunofluorescence and evaluated by con-focal laser scanning microscopy. In contrast, distribution and amount of the adhesion molecule integrin α did not change. Results of the present study indicate that quinolones disturb the adherence mechanism of chondrocytes and lead to cytoskeleton changes.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell–substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

An inexpensive device for freeze drying and plastic embedding tissues at low temperatures

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl® and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.     

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.     

The role of the surface amorphous layer of articular cartilage in joint lubrication

Articular cartilage is a complex soft tissue that performs multiple functions in the joint. In particular, the amorphous layer that covers the surface of articular cartilage is thought to play some role in lubrication. This study aimed to characterize the surface amorphous layer (SAL) using a variety of techniques, including environmental scanning electron microscopy, transmission electron microscopy, white light interferometry, and biochemical analysis of its composition. Friction tests were conducted to investigate the role of the SAL in lubrication. A protocol to remove successfully the SAL without damaging the underlying cartilage was developed and the material removed from healthy cartilage was found to contain approximately equal quantities of glycosaminoglycan (GAG), protein, and lipid. Cartilage-on-cartilage friction tests were conducted on fresh, healthy cartilage with and without the SAL, under both dynamic and static operating conditions. Removal of the SAL was not found to change the friction coefficient. However, subsequent staining of specimens indicated that the SAL had replenished during the test following loading. The replenished SAL was characterized and found to contain lipids and sulphated GAGs with undetectable protein. This study revealed experimental evidence of surface layer replenishment in articular cartilage. It was postulated that the surface layer regeneration mechanism was purely mechanical and associated with movement of GAGs and lipids through the cartilage matrix during deformation, since the experimental set-up did not contain any means of biochemical activation.     

Alteration of cartilage matrix morphology with histological processing

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neontal rats was seen by light microscopy to: (1) consist of elements, 0·5 μm thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.     

Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture

Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce tissue from the limited number of cells that can be extracted from a single individual. Relatively few cell expansion methods exist without the problems of dedifferentiation and/or loss of potency. Previously, it has been shown that mechanical vibrations can enhance chondrocyte proliferation in monolayer culture. Thus, it was hypothesized that chondrocytes grown in high-density culture would respond in a similar fashion while maintaining phenotypic stability. Isolated bovine articular chondrocytes were seeded in high-density culture on Millicell filters and subjected to mechanical vibrations 48 h after seeding. Mechanical vibrations enhanced chondrocyte proliferation at frequencies above 350 Hz, with the peak response occurring at a 1g amplitude for a duration of 30 min. Under these conditions, the gene expression of cartilage-specific and dedifferentiation markers (collagen II, collagen I, and aggrecan) were unchanged by the imposed stimulus. To determine the effect of accumulated extracellular matrix (ECM) on this proliferative response, selected cultures were stimulated under the same conditions after varying lengths of preculture. The amount of accumulated ECM (collagen and proteoglycans) decreased this proliferative response, with the cultures becoming insensitive to the stimulus after 1 week of preculture. Thus, mechanical vibration can serve as an effective means preferentially to stimulate the proliferation of chondrocytes during culture, but its effects appear to be limited to the early stages where ECM accumulation is at a minimum.     

A new bioreactor for the controlled application of complex mechanical stimuli for cartilage tissue engineering

Mechanical stimuli have been shown to enhance chondrogenesis on both animal and human chondrocytes cultured in vitro. Different mechanical stimuli act simultaneously in vivo in cartilage tissue and their effects have been extensively studied in vitro, although often in a separated manner. A new bioreactor is described where different mechanical stimuli, i.e. shear stress and hydrostatic pressure, can be combined in different ways to study the mechanobiology of tissue engineered cartilage. Shear stress is imposed on cells by forcing the culture medium through the scaffolds, whereas a high hydrostatic pressure up to 15 MPa is generated by pressurizing the culture medium. Fluid-dynamic experimental tests have been performed and successful validation of the bioreactor has been carried out by dynamic culture of tissue-engineered cartilage constructs. The bioreactor system allows the investigation of the combined effects of different mechanical stimuli on the development of engineered cartilage, as well as other possible three-dimensional tissue-engineered constructs.     

Tumor necrosis factor-alpha induced DNA cleavage in human articular chondrocytes may involve multiple endonucleolytic activities during apoptosis

Apoptosis has been documented in chondrocytes both in the growth plates of young, healthy cartilages and in osteoarthritic cartilages; little, however, is known about apoptosis in chondrocytes of normal adult articular cartilage. For the current study, apoptosis in adult chondrocytes was evaluated by labeling DNA fragments using the ISEL in situ end labeling of 3'-recessed strand breaks) or TUNEL (5'-recessed or blunt-ended strand breaks with terminal deoxynucleotidyl transferase-mediated nick end labeling) techniques in primary cultures of chondrocytes in monolayer. Apoptosis was induced in the chondrocytes by either Tumor Necrosis Factor alpha (TNF alpha), Interleukin 1-beta (IL-1 beta), or anti-Fas antibody but only after 48 hours in culture. At 4 and 24 hours, there was no detectable DNA fragmentation. With TNF alpha, IL1 beta, and anti-Fas antibody, chondrocytes show evidence of at least two types of DNA strand breaks within the same cell (as assessed by simultaneous labeling with ISEL and TUNEL). Therefore, some pathways leading to apoptosis in chondrocytes appear to involve more than one type of endonuclease activity. When the chondrocytes were cultured as explants with the articular matrix intact (ex vivo), neither IL-1 beta, TNF alpha, the anti-Fas antibody, nor fibronectin fragments were able to induce apoptosis in the chondrocytes. In normal human adult cartilage that was untreated and uncultured (in situ), DNA fragmentation was undetectable; however, a significant number of chondrocytes in osteoarthritic cartilage did contain strand breaks. These data suggest that apoptosis occurs in chondrocytes in which the matrix has been disrupted experimentally or destroyed by the osteoarthritic disease process. The results of these studies suggest that the ECM may be an essential survival factor for chondrocytes.