Microenvironment of osteoarthritic cartilage and subchondral bone influences chondrogenic differentiation, extracellular matrix production and composition of bone marrow-derived stem cells and articular chondrocytes

Objective Osteoarthritis (OA) is characterized by an imbalance in cartilage and subchondral bone homeostasis, which could be potentially treated or improved by cell-based therapies. In the present study, a reproducible in vitro coculture model was established to evaluate the influence of normal and OA- cartilage or subchondral bone explants on chondrogenic differentiation of human bone marrow derived stem cells (BMSC), human adipose derived stem cells (ASC) and phenotype of OA-chondrocytes. Signals from the articular cartilage or the underlying subchondral bone were hypothesized to induce phenotypic shifts and an altered re- and differentiation potential in cocultured BMSC, ASC and differentiated chondrocytes from OA patients. To provide a chondrogenic environment, cells were embedded in fibrin gel and kept in chondrogenic medium for up to 28 days. Methods A reproducible coculture model of ASC, BMSC, mixed cultures (BMSC and chondrocytes in equal ratio) and chondrocytes embedded in fibrin gel seeded on articular cartilage or subchondral bone explants (= co- and tricultures) compared with monocultured cells in fibrin gel without explants was established. Human OA-tissues, OA-chondrocytes and BMSC were derived from patients subjected to arthroplasty. Normal (healthy) human tissues were received from knees of rare trauma affected donors (treated for sports accidents). ASC were isolated from subcutaneous fat tissue obtained from patients undergoing elective body contouring procedures. Ovine cartilage, chondrocytes and BMSC were obtained from normal (healthy) pasture sheep. Gene expression analysis, biochemical assays (ELISA, Dot-blot, DMMB, Hydroxyproline), immunofluorescence staining and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated ASC and BMSC. Results In general, all cell regimens cocultured with OA-explants exhibited reduced gene expression patterns of collagens I, II, III and X in comparison with monocultures. Significant lower levels of collagen I and II protein (BMSC) and significant lower collagen I and III protein (mixed cultures) were detected in cartilage co- or tricultures, while co- and triculture with subchondral bone inhibited collagens in general. In contrast, no changes in glycosaminoglycan (GAG) synthesis were observed for cartilage co- and tricultures, while reduced GAG production was observed in subchondral bone co- and triculture lysates. Repetition of key experiments with normal ASC confirmed inhibitory effects for OA-subchondral bone. Co- and triculture with normal cartilage or subchondral bone explants showed no or only reduced inhibitory effects on chondrogenic differentiation or collagen gene expression. In addition, biomechanical properties of the OA-cartilage or subchondral bone co- and tricultured cell-fibrin gels were affected. All co- and triculture regimens tended to exhibit lower Young´s modulus and aggregate modulus compared with monocultures. In contrast, hydraulic permeability seemed to be higher in co- and tricultures. Supernatants of cartilage and subchondral bone co- and tricultures contained significant higher IL-1β, IL-6 and IL-8 levels, as well as significant more soluble GAGs compared with controls. In general, stimulation of monocultures with IL-1β induced matrix metalloproteinase (MMP)2, and MMP3 and reduced collagens I, II and X gene expression and led to a downregulation of aggrecan gene expression. Stimulation with IL-6 reduced aggrecan, MMP3 and MMP13 gene expression and mainly reduced collagen I, II and III gene expression of BMSC and/or chondrocytes. In contrast, IL-8 stimulation of mixed and chondrocytes monocultures had only little effects, while IL-8 stimulated BMSC showed reduced collagen I, II and III gene expression. Conclusions Taken together our results suggest an inhibitory effect of factors from the microenvironment of OA-cartilage and subchondral bone on production of collagens. This indicates a distinct modulatory influence, which affects the composition of the de novo produced ECM from cocultured cells and leads to impaired mechanical strength and biochemical properties of the newly formed matrix. Experiments with ASC, normal cartilage and subchondral bone explants either might hint to disease status induced effects (OA vs. trauma) or to an effect caused by different mean age of cell and tissue donors. Soluble signal factors, i.e. pro-inflammatory cytokines (including IL-1β and IL-6), released from OA-cartilage, OA-subchondral bone, chondrocytes and osteoblasts, might partly mediate these effects on newly formed extracellular matrix properties. Thus, the microenvironment of neighbored OA-cartilage seems to provide both: promoting and inhibiting signals for BMSC differentiation and suggests that the balance of these factors determines the destiny of BMSC. This knowledge can be used to develop new strategies for cell based cartilage regeneration

Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

BACKGROUND: The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear. METHODS: Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls. RESULTS: Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC. CONCLUSION: The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds

Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes

Introduction Osteoarthritis (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. Methods We 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). Results Overall, 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. Conclusions Our 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