The objective of this experiment was to determine if the reduced biomechanical forces due to microgravity impair the ability of chondrocytes to maintain healthy articular cartilage, leading to increased cartilage breakdown; specifically whether the reduced mechanical forces associated with microgravity lead to osteoarthritis. Although the effects of microgravity on bone are well known and appear to be reversible, the effects of reduced mechanical forces on the cartilage surfaces of articulating joints are less well understood. Studies in humans and animals clearly demonstrate that osteoarthritis results from abnormal (both increased and decreased) mechanical forces on cartilage. Based on these findings, the hypothesis is that appropriate mechanical forces are critical for the health of articular cartilage. Following on from this, a further hypothesis is that the absence of significant biomechanical forces affects that ability of the chondrocyte to maintain healthy functioning cartilage. This may lead to accelerated cartilage breakdown and osteoarthritis.
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For the STS-131 mission, animals were subjected to 15 days of microgravity and the joints harvested three hours following landing. Femoro-tibial joints from four flight and four ground control animals were paraffin-embedded, sectioned and stained for proteoglycan using toluidine blue. Proteoglycan degradation is a key indicator of cartilage breakdown and one of the first stages in osteoarthritis. Stained sections were scored using a modified Mankin histological semi-quantitative grading system (see Research Plan for description of scoring system).
For RNA expression analyses, joint articular cartilage total RNA from the four flight and ground control samples were isolated. Since the function of microRNAs (miRNAs) in cartilage has received substantial research attention and several appear essential to normal development and homeostasis each of the eight samples were subjected to miRNA microarray analysis using the Affymetrix Genechip miRNA array. Samples were also hybridized to the Affymetrix Murine Gene 1.0 ST GeneChip microarray for mRNA expression.
miRNA data: Bioinformatic analysis of the miRNA data demonstrated that 10 miRNA were upregluated (p>0.001) and 26 were downregulated in flight compared to control mice. Interestingly, miR-35, which was downregulated 1.4-fold, is a known mechanoresponsive microRNA in chondrocytes. It has been reported that miR-35 was upregluated following cyclical loading of chick sternal chondrocytes and that it plays a role in chondrocytee proliferation and differentiation. Furthermore, miR365 binds to and inhibits HDAC4, a negative regulator of chondrocyte hypertrophy. Our finding that miR-365 is downregulated in the reduced biomechanical unloading of spaceflight is consistent with a role in mechano-sensing. Reduced levels of miR-365 would be expected to reduce chondrocyte proliferation thereby contributing to an OA phenotype.
Thirteen mRNA were upregulated>1.5-fold in flight compared to the ground control animals and one downgraded>1.5 fold. Of the 13 upregulated genes, 7 have been implicated in either osteoarthritis, rheumatoid arthritis or both. There are Vdr, Nfkbia, Cited2, Traf6, Mtl, Txnip, and Cdknla. The highest upregulated gene was Vdr which encodes the vitamin D receptor. Vdr is a key genetic and biochemical marker of osteoarthritis (OA). Several genetic association studies on large populations of OA patients have linked the Vdr gene locus with knee OA. The link between Vdr and OA is further highlighted by the finding that Vdr mRNA ius upregulated in severe osteoarthritis. Consistent with this is the finding that, vitamin D, the Vdr ligand, upregulates matrix metalloprteinase (MMP) production. MMPs are the major class of matrix-degrading enzymes in cartilage.
Composition, Matrix components, articular cartilage, mouse, Immunohistochemistry
Damage, Breakdown, articular cartilage, mouse, Modified Mankin histological semi-quantitative grading
Expression, gene, Whole genome, mRNA, articular cartilage, mouse, Quantitative PCR (qPCR)
Morphology, Cell density, articular cartilage, mouse, Modified Mankin histological semi-quantitative grading
Morphology, organization, Articular cartilage, mouse, Modified Mankin histological semi-quantitative grading
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Morphology, Pericellular and interterritorial matrix proteoglycan, articular cartilage, mouse, Modified Mankin histological semi-quantitative grading