Modification of osteoarthritis in the guinea pig with pulsed low-intensity ultrasound treatment.

OBJECTIVE: The Hartley guinea pig develops articular cartilage degeneration similar to that seen in idiopathic human osteoarthritis (OA). We investigated whether the application of pulsed low-intensity ultrasound (PLIUS) to the Hartley guinea pig joint would prevent or attenuate the progression of this degenerative process. METHODS: Treatment of male Hartley guinea pigs was initiated at the onset of degeneration (8 weeks of age) to assess the ability of PLIUS to prevent OA, or at a later age (12 months) to assess the degree to which PLIUS acted to attenuate the progression of established disease. PLIUS (30 mW/cm(2)) was applied to stifle joints for 20 min/day over periods ranging from 3 to 10 months, with contralateral limbs serving as controls. Joint cartilage histology was graded according to a modified Mankin scale to evaluate treatment effect. Immunohistochemical staining for interleukin-1 receptor antagonist (IL-1ra), matrix metalloproteinase (MMP)-3, MMP-13, and transforming growth factor (TGF)-beta1 was performed on the cartilage to evaluate patterns of expression of these proteins. RESULTS: PLIUS did not fully prevent cartilage degeneration in the prevention groups, but diminished the severity of the disease, with the treated joints showing markedly decreased surface irregularities and a much smaller degree of loss of matrix staining as compared to controls. PLIUS also attenuated disease progression in the groups with established disease, although to a somewhat lesser extent as compared to the prevention groups. Immunohistochemical staining demonstrated a markedly decreased degree of TGF-beta1 production in the PLIUS-treated joints. This indicates less active endogenous repair, consistent with the marked reduction in cartilage degradation. CONCLUSIONS: PLIUS exhibits the ability to attenuate the progression of cartilage degeneration in an animal model of idiopathic human OA. The effect was greater in the treatment of early, rather than established, degeneration.

Developmental basis for hind-limb loss in dolphins and origin of the cetacean bodyplan.

Among mammals, modern cetaceans (whales, dolphins, and porpoises) are unusual in the absence of hind limbs. However, cetacean embryos do initiate hind-limb bud development. In dolphins, the bud arrests and degenerates around the fifth gestational week. Initial limb outgrowth in amniotes is maintained by two signaling centers, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA). Our data indicate that the cetacean hind-limb bud forms an AER and that this structure expresses Fgf8 initially, but that neither the AER nor Fgf8 expression is maintained. Moreover, Sonic hedgehog (Shh), which mediates the signaling activity of the ZPA, is absent from the dolphin hind-limb bud. We find that failure to establish a ZPA is associated with the absence of Hand2, an upstream regulator of Shh. Interpreting our results in the context of both the cetacean fossil record and the known functions of Shh suggests that reduction of Shh expression may have occurred approximately 41 million years ago and led to the loss of distal limb elements. The total loss of Shh expression may account for the further loss of hind-limb elements that occurred near the origin of the modern suborders of cetaceans approximately 34 million years ago. Integration of paleontological and developmental data suggests that hind-limb size was reduced by gradually operating microevolutionary changes. Long after locomotor function was totally lost, modulation of developmental control genes eliminated most of the hind-limb skeleton. Hence, macroevolutionary changes in gene expression did not drive the initial reduction in hind-limb size.

Overview of studies comparing human normal cartilage with minimal and advanced osteoarthritic cartilage.

A major area under study in the osteoarthritis (OA) research field is the characterization of specific molecular and biochemical changes that distinguish advanced diseased cartilage from less involved or normal tissue. This information is important to better define the pathogenic mechanisms that are operating during OA progression and to identify disease-specific markers. This review describes recent studies that have addressed changes in chondrocyte gene expression, proliferation, and apoptosis in "experimental" (more advanced OA cartilage) versus "control" (less involved or non-OA cartilage). Included is a comprehensive listing of recently published studies in this area with general findings. The review also includes a discussion of study design and the strengths and weaknesses of the various approaches. In addition, specific strategies to deal with some of the important issues are discussed. One particular model utilizing minimal and advanced OA cartilage obtained from the same patient is described in more detail.

Cartilage viability after repetitive loading: a preliminary report.

OBJECTIVE: To assess matrix changes and chondrocyte viability during static and continuous repetitive mechanical loading in mature bovine articular cartilage explants. METHODS: Cartilage explants were continuously loaded either statically or cyclically (0.5 Hz) for 1-72 h (max. stress 1 megapascal). Cell death was assessed using fluorescent probes and detection of DNA strand breakage characteristic of apoptosis. Cell morphology and matrix integrity were evaluated using histology and transmission electron microscopy. RESULTS: Repetitive loading of articular cartilage at physiological levels of stress (1 megapascal) was found to be harmful to only the chondrocytes in the superficial tangential zone (STZ) and depended on the characteristics (static vs cyclic) and duration (1-72 h) of the applied load. The chondrocytes in the middle and deep zone remained viable at all times. Static loads caused cell death at an early time (3 h) as compared with cyclic loads (sinusoidal, 0.5 cycles per s for 6 h). The amount and extent of cell death peaked at 6 h of cyclic loading, and did not change in subsequent experiments run for longer periods of time (up to 72 h). There was no indication of fragmented nuclear DNA but there was evidence of injurious cell death (necrosis) by electron microscopy. Morphological analysis of cartilage repetitively loaded for 24 h showed matrix damage only in the uppermost superficial layer at the articular surface, reminiscent of the early stages of osteoarthritis. CONCLUSIONS: Cell death in mature cartilage explants occurred after 6 hours of continuous repetitive load or 3 h of static load. Cell death was directly related to the mechanical load, as control (free-swelling) explants remained viable at all times. The excessive, repetitive loading conditions imposed are not physiological, and demonstrate the deleterious effects of mechanical overload resulting in morphological and cellular damage similar to that seen in degenerative joint disease.

Response of engineered cartilage tissue to biochemical agents as studied by proton magnetic resonance microscopy.

OBJECTIVE: To test the hypothesis that magnetic resonance imaging (MRI) results correlate with the biochemical composition of cartilage matrix and can therefore be used to evaluate natural tissue development and the effects of biologic interventions. METHODS: Chondrocytes harvested from day-16 chick embryo sterna were inoculated into an MRI-compatible hollow-fiber bioreactor. The tissue that formed over a period of 2-4 weeks was studied biochemically, histologically, and with MRI. Besides natural development, the response of the tissue to administration of retinoic acid, interleukin-1beta (IL-1beta), and daily dosing with ascorbic acid was studied. RESULTS: Tissue wet and dry weight, glycosaminoglycan (GAG) content, and collagen content all increased with development time, while tissue hydration decreased. The administration of retinoic acid resulted in a significant reduction in tissue wet weight, proteoglycan content, and cell number and an increase in hydration as compared with controls. Daily dosing with ascorbic acid increased tissue collagen content significantly compared with controls, while the administration of IL-1beta resulted in increased proteoglycan content. The water proton longitudinal and transverse relaxation rates correlated well with GAG and collagen concentrations of the matrix as well as with tissue hydration. In contrast, the magnetization transfer value for the tissue correlated only with total collagen. Finally, the self-diffusion coefficient of water correlated with tissue hydration. CONCLUSION: Parameters derived from MR images obtained noninvasively can be used to quantitatively assess the composition of cartilage tissue generated in a bioreactor. We conclude that MRI is a promising modality for the assessment of certain biochemical properties of cartilage in a wide variety of settings.

The transcription factor deltaEF1 is inversely expressed with type II collagen mRNA and can repress Col2a1 promoter activity in transfected chondrocytes.

The regulation of Col2a1, which encodes type II collagen, likely results from a balance of both positive and negative proteins. Here we present evidence that the transcription factor deltaEF1 participates in the negative regulation of Col2a1 transcription. A deletion analysis suggested that a region between -100 and -307 of the rat Col2a1 gene was required for activity in differentiating chick limb bud mesenchymal cells; however, mutation of a conserved E2 box site in this region actually increased promoter activity. Supershift analysis demonstrated that deltaEF1, a known transcriptional repressor, bound to the E2 box in a sequence-dependent manner. Chick limb bud mesenchymal cells, which do not express type II collagen, expressed abundant deltaEF1 mRNA, but, following differentiation in micromass culture, deltaEF1 mRNA expression was lost. Primary embryonic chick sternal chondrocytes, which express abundant type II collagen, displayed minimal levels of deltaEF1 mRNA. The inhibition of Col2a1 transcription following treatment of chick sternal chondrocytes with growth factors was accompanied by increased deltaEF1 expression. Overexpression of deltaEF1 in differentiated chondrocytes resulted in decreased expression of a reporter construct containing a collagen II promoter/enhancer insert; however, this negative regulation was not dependent on the proximal E2 box. This is the first report of a specific transcription factor involved in the negative regulation of Col2a1.

Bcl-2 regulates chondrocyte morphology and aggrecan gene expression independent of caspase activation and full apoptosis.

Bcl-2 is widely expressed in a variety of cell types and is known to block apoptosis through a conserved pathway. However, recent reports have demonstrated that Bcl-2 regulates cell behavior independent of its control of apoptosis. Chondrocytes express a unique set of matrix proteins, including the proteoglycan aggrecan, and have been widely used to study the relationship between trophic factors and apoptosis. In this article, we report that Bcl-2 affects the morphology and regulates the expression of aggrecan in a rat chondrocyte cell line (IRC). Endogenous Bcl-2 and aggrecan mRNA were both down-regulated in response to serum withdrawal in parental IRC cells, while constitutive expression of Bcl-2 maintained aggrecan levels under conditions of serum withdrawal. In addition, expression of anti-sense Bcl-2 resulted in decreased aggrecan mRNA and produced a fibroblastic morphology compared with parental cells. The caspase inhibitor ZVAD-fmk effectively blocked full apoptosis of IRC cells in response to serum withdrawal or anti-sense Bcl-2 but did not prevent the down-regulation of aggrecan expression from either signal. These results suggest a novel role for Bcl-2 in regulating the differentiated phenotype of chondrocytes and the expression of a differentiation-specific gene independent of its control of apoptosis.

Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment.

The regulation of chondrocyte apoptosis in articular cartilage may underlay age-associated changes in cartilage and the development of osteoarthritis. Here we demonstrate the importance of Bcl-2 in regulating articular chondrocyte apoptosis in response to both serum withdrawal and retinoic acid treatment. Both stimuli induced apoptosis of primary human articular chondrocytes and a rat chondrocyte cell line as evidenced by the formation of DNA ladders. Apoptosis was accompanied by decreased expression of aggrecan, a chondrocyte specific matrix protein. The expression of Bcl-2 was downregulated by both agents based on Northern and Western analysis, while the level of Bax expression remained unchanged compared to control cells. The importance of Bcl-2 in regulating chondrocyte apoptosis was confirmed by creating cell lines overexpressing sense and antisense Bcl-2 mRNA. Multiple cell lines expressing antisense Bcl-2 displayed increased apoptosis even in the presence of 10% serum as compared to wild-type cells. In contrast, chondrocytes overexpressing Bcl-2 were resistant to apoptosis induced by both serum withdrawal and retinoic acid treatment. Finally, the expression of Bcl-2 did not block the decreased aggrecan expression in IRC cells treated with retinoic acid. We conclude that Bcl-2 plays an important role in the maintenance of articular chondrocyte survival and that retinoic acid inhibits aggrecan expression independent of the apoptotic process.

Chondrocyte apoptosis in development, aging and disease.

There is increasing evidence that chondrocyte apoptosis plays an important role in cartilage development, aging and disease. A variety of morphological and biochemical studies have identified apoptotic chondrocytes in both growth plate and articular cartilage of a variety of species. In addition, there is an ever increasing list of diverse stimuli that can induce chondrocyte apoptosis in vitro. A feedback loop regulating chondrocyte apoptosis in the growth plate has been described that includes Indian Hedgehog, parathyroid hormone-related protein and Bcl-2. The molecular mechanism regulating apoptosis in articular cartilage is still under investigation. Future studies should elucidate more fully how abnormal regulation of chondrocyte apoptosis may contribute to the development of chondrodysplasias and chondrosarcomas. Also, it will be of importance to define the relationship between chondrocyte apoptosis and the regulation of chondrocyte-specific gene expression.

In vivo cartilage formation from growth factor modulated articular chondrocytes.

Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.

Cytokine inducible matrix metalloproteinase expression in immortalized rat chondrocytes is independent of nitric oxide stimulation.

The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1beta and tumor necrosis factor alpha induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming growth factor beta had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.

Chondrocyte apoptosis increases with age in the articular cartilage of adult animals.

BACKGROUND: Apoptosis in vivo has been identified in developing cartilage from embryonic chick sterna and avian and murine growth plates. To date, no evidence exists that chondrocytes in articular cartilage undergo apoptosis. METHODS: We examined the distribution of cells demonstrating fragmented DNA in the articular knee cartilage of C57BL/6 mice (aged 11, 18, 24, and 30 months) and Wistar rats (aged 6, 12, and 24 months) using a DNA end-labeling technique. RESULTS: Control experiments utilizing retinoic acid-induced apoptosis in a chondrocyte cell line, established that DNA end-labeling correlated with DNA ladder formation. In vivo, apoptotic cells were detected in articular cartilage tissue in both species examined. The percentage of apoptotic cells increased significantly (P < 0.05 with age) for all joint surfaces in both species. No significant difference was found between the medial and lateral or femoral and tibial joint surfaces of the knee. Apoptotic cells were observed in both the calcified and uncalcified regions of the articular cartilage of C57 mice. In the rat, only the calcified region of articular cartilage contained apoptotic cells. CONCLUSIONS: These results suggest that apoptosis plays a role in some aspect of maintenance, remodeling, or turnover of mature articular cartilage. In addition, the increase in apoptosis associated with aging could contribute to the greater risk for cartilage degeneration.

Cartilage formation in a hollow fiber bioreactor studied by proton magnetic resonance microscopy.

The ideal in vitro system for investigating the regulation of cartilage formation and maintenance would allow for three-dimensional tissue growth, a wide range of biochemical interventions, and non-destructive evaluation. We have developed a hollow fiber bioreactor (HFBR) system which meets these criteria. After injection with embryonic chick sternal chondrocytes, neocartilage is elaborated around the hollow fibers, reaching a thickness of up to a millimeter after four weeks of growth. This process was monitored over time with nuclear magnetic resonance (NMR) microimaging and correlative biochemical and histologic analyses. Tissue volume and cellularity increased greatly during development. This was accompanied by changes in magnetic resonance properties consistent with increased macromolecular content. Further, tissue heterogeneity, observed as regional variations in cell size in histologic sections, was also observed in quantitative NMR images.

An association between an aggrecan polymorphic allele and bilateral hand osteoarthritis in elderly white men: data from the Baltimore Longitudinal Study of Aging (BLSA).

OBJECTIVE: The aggrecan proteoglycan is a major component of articular cartilage and supports the biomechanical function of this tissue. A variable number tandem repeat (VNTR) polymorphism has been discovered recently in a region of the human aggrecan gene that codes for the chondroitin sulfate attachment sites. We examined whether alleles of this polymorphism displayed a non-random association with bilateral hand or knee osteoarthritis (OA) in men from the Baltimore Longitudinal Study of Aging (BLSA). DESIGN: DNA was obtained from 93 Caucasian men, aged 60 and above, who had bilateral hand and standing knee radiographs read for changes of OA. The DNA was analyzed by polymerase chain reaction (PCR) and/or Southern blotting for the presence of the VNTR alleles. RESULTS: Bilateral hand OA and knee OA were present in 46 and 30% of the men respectively. The following distribution of alleles was observed: allele 33 (0.5%), 29 (2.2%), 28 (31.7%), 27 (43.0%), 26 (16.7%), 25 (3.2%), 22 (2.2%) and 19 (0.5%). This distribution was similar to that detected in a random population of individuals from a separate study. In multiple logistic regression analysis, adjusting for age and body mass index, the presence of allele 27 was associated with bilateral hand OA with an odds ratio (OR) = 3.23 (95% confidence intervals (CI): 1.24-8.41). No other alleles showed an association with bilateral hand OA and the association between allele 27 and bilateral knee OA was not statistically significant (OR = 1.14; 95% CI: 0.45-2.88). CONCLUSIONS: These data demonstrate the first association between a human aggrecan gene polymorphic allele and hand OA. This finding supports the concept that genetic factors may play a role in the development and/or progression of some forms of age-onset OA.

Expression of mutant amyloid precursor proteins induces apoptosis in PC12 cells.

The cause of neuronal loss in Alzheimer disease is unknown. We investigated the effects on survival of PC12 cells expressing A692G, E693Q, and V717F mutant amyloid precursor proteins (APP). Differentiated cells expressing mutant APPs exhibited somal shrinkage, followed by cell detachment from the plates. Increased levels of oligonucleosome-sized DNA ladders and TUNEL-positive nuclei were observed, and electron microscopy revealed extensive plasma membrane blebbing, margination of condensed chromatin, and well-preserved organelles in these transfectants. The levels of TUNEL-positive cells, analyzed by a flow-cytometric method, were increased by four- to sevenfold in mutant APP transfectants, but less than twofold in wild-type APP transfectants relative to untransfected cells. Our results provide evidence that expression of mutant APPs in differentiated PC12 cells induces cell death via an apoptotic pathway.

Mesenchymal cell chondrogenesis is stimulated by basement membrane matrix and inhibited by age-associated factors.

During development of the embryonic limb, differentiation of mesenchymal progenitor cells into chondrocytes is regulated by cell shape, extracellular matrix, and growth and differentiation factors. In this study, reconstituted basement membrane (Matrigel) prepared from mouse Englebreth-Holm-Swarm tumor tissue was found to stimulate mesenchymal cell chondrogenesis in vitro and the production of cartilage at ectopic sites in athymic mice. The rate of chondrogenesis of mesenchymal cells from chick limb bud was increased four-fold by the addition of 400 micrograms/ml Matrigel to the media of micromass cultures, and this activity was not blocked by neutralizing antibodies to transforming growth factor-beta (TGF-beta) or fibroblast growth factor (FGF). Mesenchymal cells cultured on Matrigel, but not laminin or collagen type I or IV, formed spheres of condensed cells which stained with Alcian blue. Chick limb-bud mesenchymal cells suspended in Matrigel prepared from tumors grown in C57 mice aged 3, 12, or 26 months formed disks of hyaline cartilage within 2 weeks with wet weights of 59.1 mg, 35.7 mg, and 21.4 mg, indicating that the Matrigel from the old animals was less biologically active. In agreement with the in vivo data, Alcian blue staining of proteoglycan was over two-fold higher in micromass cultures supplemented with the Matrigel from young animals than in cultures treated with the Matrigel from old mice. A high-salt wash preparation of Matrigel from tumors grown in old mice increased the rate of chondrogenesis and cartilage production, suggesting that an inhibitor of chondrogenesis is produced by the old host. Thus, Matrigel contains chondrogenic activity distinct from TGF-beta or FGF. The aged host may produce factors that are inhibitory to mesenchymal cell differentiation and adversely affect cartilage formation and repair.

Evidence for apoptotic cell death in Alzheimer's disease.

We provide evidence for apoptosis in Alzheimer's disease using the in situ labeling technique TUNEL (terminal transferase-mediated dUTP-biotin nick end labeling). The technique specifically detects apoptotic cells by utilizing terminal transferase to incorporate biotinylated nucleotides into the fragmented DNA of apoptotic cells. The labeled cells are visualized by reaction with avidin peroxidase and a suitable substrate. Sections from the hippocampus of Alzheimer-diseased (AD) brains and non-AD brains were examined for apoptosis. While considerable variation in the quantity of apoptotic cells was observed among individual samples, the incidence of apoptosis in AD brains was elevated in comparison to age-matched, non-AD brains in specific regions of the hippocampal formation. Immunostaining indicated that both neurons and astrocytes were undergoing apoptosis, although the majority of the TUNEL-positive cells appeared to be glial, based on the location of the stained cells. These data suggest that apoptosis may be involved in both the primary neuronal cell loss and in the glial response that is a component of AD.

Comparison of age-associated degeneration of articular cartilage in Wistar and Fischer 344 rats.

We have carried out a comparison of cartilage degeneration that develops spontaneously with age in the Wistar and Fischer 344 rats. Young adult (6-month-old) Wistar rats were essentially free of any relevant lesion in the articular cartilage of the knee. Aged (24-month-old) Wistar rats had minimal to mild lesions found mainly in the medial tibial plateau. These lesions were indicated by a focal loss of toluidine blue staining and limited fibrillation. Approximately 68% of the old Wistar rats had some degree of pathologic change. In comparison, 100% of the Fischer 344 rats examined, both young and old, had cartilage degeneration. However, the severity of the lesions increased with age. These two rat strains have variable degrees of spontaneous age-associated cartilage degeneration and would be suitable for testing treatments to prevent or repair the cartilage damage.