Injurious Mechanical Compression of Bovine Articular Cartilage Induces Chondrocyte Apoptosis

Center for Biomedical Engineering, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge 02139, USA.
Archives of Biochemistry and Biophysics (Impact Factor: 3.02). 09/2000; 381(2):205-12. DOI: 10.1006/abbi.2000.1988
Source: PubMed


A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.

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Available from: Marc E Levenston, Jul 09, 2014
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    • "Parts of the remaining demaged chondrocytes are prone to apoptosis (12,52). A number of studies have shown that the rate of chondrocyte apoptosis is increased in OA cartilage and have authenticated the role of apoptosis in the pathogenesis of OA (9,11,52–55). In the present study, chondrocyte viability significantly decreased and the percentage of apoptotic chondrocytes increased, separately, in a time-dependent manner following the exposure of chondrocytes to mechanical pressure. "
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    ABSTRACT: MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR‑146a binding sequence in the 3'-untranslated region (3'-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR‑146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest that miR-146a is involved in human chondrocyte apoptosis in response to mechanical injury, and may contribute to the mechanical injury of chondrocytes, as well as to the pathogenesis of OA by increasing the levels of VEGF and damaging the transforming growth factor (TGF)-β signaling pathway through the targeted inhibition of Smad4 in cartilage.
    International Journal of Molecular Medicine 06/2014; 34(2). DOI:10.3892/ijmm.2014.1808 · 2.09 Impact Factor
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    • "In these studies, chondrocyte apoptosis was detected by caspase-3 expression and TUNEL, and confirmed by electron microscopic examination. In in vitro studies, chondrocyte apoptosis can be induced by exposing the normal cartilage explants or chondrocyte cultures either to biological [14,19,24] (e.g., nitric oxide (NO), collagenase, anti-CD95) or mechanical factors [10,11,25,26] (e.g., shear strain, loading strain). This effect can be inhibited by treatment with caspase inhibitor [24,27,28] (e.g., z.VAD.fmk), "
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    ABSTRACT: Osteoarthritis (OA) is the most common joint disease characterised by degradation of articular cartilage and bone remodelling. For almost a decade chondrocyte apoptosis has been investigated as a possible mechanism of cartilage damage in OA, but its precise role in initiation and/or progression of OA remains to the determined. The aim of this study is to determine the role of chondrocyte apoptosis in spontaneous animal models of OA. Right tibias from six male Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs were collected at 10, 16, 24 and 30 weeks of age. Fresh-frozen sections of tibial epiphysis were microscopically scored for OA, and immunostained with caspase-3 and TUNEL for apoptotic chondrocytes. The DH strain had more pronounced cartilage damage than BS2, especially at 30 weeks. At this time point, the apoptotic chondrocytes were largely confined to the deep zone of articular cartilage (AC) with a greater percentage in the medial side of DH than BS2 (DH: 5.7%, 95% CI: 4.2-7.2), BS2: 4.8%, 95% CI: 3.8-5.8), p > 0.05). DH had a significant progression of chondrocyte death between 24 to 30 weeks during which time significant changes were observed in AC fibrillation, proteoglycan depletion and overall microscopic OA score. A strong correlation (p ≤ 0.01) was found between chondrocyte apoptosis and AC fibrillation (r = 0.3), cellularity (r = 0.4) and overall microscopic OA scores (r = 0.4). Overall, the rate of progression in OA and apoptosis over the study period was greater in the DH (versus BS2) and the medial AC (versus lateral). Chondrocyte apoptosis was higher at the later stage of OA development when the cartilage matrix was hypocellular and highly fibrillated, suggesting that chondrocyte apoptosis is a late event in OA.
    International Journal of Molecular Sciences 09/2013; 14(9):17729-17743. DOI:10.3390/ijms140917729 · 2.86 Impact Factor
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    • "A possible scenario for cartilage degeneration under these circumstances might look as follows: sub-maximal long term muscle loading of joints causes selective chondrocyte death, thereby reducing the capacity of cells for maintaining matrix homeostasis, resulting in matrix breakdown, leading to OA. This scenario, together with evidence that load-induced chondrocyte apoptosis can occur even if loading is not enough to create matrix damage or biochemical and biomechanical changes to the tissue (Loening et al., 2000), suggests that cell death may precede structural breakdown of the extracellular matrix. However, while chondrocyte death may be an early response to cartilage loading, our results do not provide evidence that cell death precedes matrix degeneration. "
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    ABSTRACT: Background: Excessive chronic loading is thought to be one factor responsible for the onset of osteoarthritis. For example, studies using treadmill running have shown an increased risk for osteoarthritis, thereby suggesting that muscle-induced joint loading may play a role in osteoarthritis onset and progression. However, in these studies, muscle-induced loading was not carefully quantified. Here, we present a model of controlled muscular loading which allows for the accurate quantification of joint loading. The aim of this study was to evaluate the effects of long-term, cyclic, isometric and dynamic, muscle-induced joint loading of physiologic magnitude but excessive intensity on cartilage integrity and cell viability in the rabbit knee. Methods: 24 rabbits were divided into an (i) eccentric, (ii) concentric, or (iii) isometric knee extensor contraction group (50 min of cyclic, submaximal stimulation 3 times/week for four weeks=19,500 cycles) controlled by the stimulation of a femoral nerve cuff electrode on the right hind limb. The contralateral knee was used as a non-loaded control. The knee articular cartilages were analysed by confocal microscopy for chondrocyte death, and histologically for Mankin Score, cartilage thickness and cell density. Findings: All loaded knees had significantly increased cell death rates and Mankin Scores compared to the non-loaded joints. Cartilage thicknesses did not systematically differ between loaded and control joints. Interpretation: Chondrocyte death and Mankin Scores were significantly increased in the loaded joints, thereby linking muscular exercise of physiologic magnitude but excessive intensity to cartilage degeneration and cell death in the rabbit knee.
    Clinical biomechanics (Bristol, Avon) 05/2013; 28(5). DOI:10.1016/j.clinbiomech.2013.04.009 · 1.97 Impact Factor
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