Abstract
IntroductionOsteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis. We hypothesized that signals from the subchondral bone may modulate production of matrix components, alter chondrogenic differentiation potential of cocultured bone marrow-derived mesenchymal stem cells (BMSC) and induce a phenotypic shift in differentiated OA chondrocytes.MethodsWe established a novel coculture model between BMSC, mixed cultures (BMSC and chondrocytes) and chondrocytes embedded in fibrin gel with OA and normal subchondral bone explants (OAB and NB). Tissues and cells were either derived from OA or trauma patients. In addition, we used adipose-derived stem cells (ASC) from liposuction. With gene expression analysis, biochemical assays, immunofluorescence and biomechanical tests we characterized the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiating BMSC cocultured with OAB or NB in comparison with monocultures (cultures without bone explants).ResultsOverall, gene expression of collagens of OAB and NB cocultured cells was reduced compared to monocultures. Concomitantly, we observed significantly lower collagen I, II and III and glycosaminoglycan (GAG) production in OAB cocultured cell lysates. In parallel, we detected increased concentrations of soluble GAGs and basic fibroblast growth factor (bFGF), interleukin (IL)-6 and IL-8 in supernatants of OAB and NB cocultures mainly at early time points. IL-1ß concentration was increased in supernatants of OAB cocultures, but not in NB cocultures. Cell-free NB or OAB explants released different amounts of IL-1ß, bFGF and soluble GAG into cell culture supernatants. In comparison to cocultures, monocultures exhibited higher Young’s modulus and equilibrium modulus. Stimulation of monocultures with IL-1ß led to a downregulation of aggrecan (ACAN) gene expression and in general to induced matrix metalloprotease (MMP)2, MMP3 and MMP-13 gene expression while IL-6 and IL-8 stimulation partly reduced ACAN, MMP3 and MMP-13 gene expression.ConclusionsOur results suggest an alteration of molecular composition and mechanical properties of the newly formed ECM in subchondral bone cocultures. We suggest that soluble factors, that is interleukins and bFGF, released in cocultures exert inhibitory effects on collagen and temporary effects on proteoglycan production, which finally results in a reduction of mechanical strength of newly formed fibrillar networks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0453-9) contains supplementary material, which is available to authorized users.
Highlights
Osteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis
Culture models We set up different culture conditions to analyze the influence of factors from subchondral bone on multipotent bone marrow-derived mesenchymal stem cells (BMSC) with respect to extracellular matrix (ECM) formation and chondrogenic differentiation
As we found release of basic fibroblast growth factor (bFGF) from OA subchondral bone (OAB) and Normal subchondral bone tissue (NB) explants cultured without cells, we assume that this adds to the bFGF detected in cocultures. bFGF and other growth factors might be released together with the GAGs they were bound to during pathophysiological situations, that is after tissue injury or during chronic inflammation [34]
Summary
Osteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis. For long-term repair and regeneration of focal cartilage defects, chondrocytes are implanted at the site of injury, not much attention has been paid to the microenvironmental effects of neighboring cartilage/subchondral bone. This is evident in diseases affecting diarthrodial joints such as osteoarthritis (OA), which is an age-related and/or trauma-induced multifactorial, slowly progressing and primarily noninflammatory degenerative disorder of the synovial joints culminating in the irreversible destruction of the articular cartilage [1,2]. Therapies using adult bone marrow-derived mesenchymal stem cells (BMSC) have a promising future to facilitate regenerative musculoskeletal tissue repair. BMSC are pluripotent cells that inherit the capacity to differentiate into cartilage, bone, fat, and other tissue types after appropriate in vitro induction [8]
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