gms | German Medical Science

Objectives: Osteoarthritis is one of the prevalent causes of disability worldwide. Due to its avascularity, and the reduced mitotic activity of adult chondrocytes together with their decreased responsiveness to anabolic growth factors, articular cartilage has limited capacity for self-repair. In this respect, adipose-derived mesenchymal stem cells (ADMSC) represent an attractive source for cartilage regeneration because of their self-renew potential, abundance and reduced donor site morbidity. Previously it was shown that FGF-2 treatment of bone marrow-derived MSCs in monolayer upregulates the expression of the chondrocyte marker alpha10 integrin and enhances their chondrogenic differentiation. In this study we used a similar approach to investigate the potential of ADMSC for cartilage repair. In particular, we examined the role of integrin alpha 10 stimulation on priming of ADMSC towards chondrogenesis.

Methods: Human ADMSCs and BMSCs were cultured in monolayer for 5 days with or without FGF-2 under normoxia or hypoxia. TGFß receptors expression and elevation of chondrogenic markers in monolayer, were proven by RT-PCR. Afterwards, MSCs were differentiated towards the chondrogenic lineage in pellet culture for 28 days in the presence or absence of TGFß1 and BMP2 (T/B). Integrin and chondrogenic gene expression levels were investigated by RT-PCR. Protein expression of integrin alpha 10 was determined by immunoblotting. Safranin-O (SO) staining for proteoglycans and immunohistochemistry (IHC) for collagen type II and aggrecan were performed to demonstrate chondrogenesis. Biomechanical properties and structure of cartilaginous matrix were examined by atomic force microscopy (AFM).

Results and Conclusion: We could successfully demonstrate that FGF-2 pretreatment of ADMSC and BMSC in monolayers, increased the expression of Itga10 and Sox9, while decreased the expression of Itga11 and Agc1, independently of the oxygen tension. Western blot analysis confirmed that also alpha 10 protein levels elevates upon FGF-2 treatement. The stronger stainings for proteoglycan, aggrecan and collagen type II, demonstrated superior chondrogenic differentiation of BMSCs with T/B administration in pellet cultures compared to ADMSCs. ADMSCs showed patches proteoglycan deposition and increased Col2a1 mRNA expression in T/B-treated pellets upon FGF-2 and hypoxic preconditioning. With the aid of AFM, we could confirm collagen fibers development in peripherally differentiated ADMSC pellets and entirely differentiated BMSC control pellets.

Our results clearly show that FGF-2 pretreatment of ADMSCs is sufficient to increase the expression of the chondrogenic markers Itga10 and Sox9 in monolayer, and subsequently prime these cells for an improved chondrogenic outcome. We suggest that modulation of Itga10 expression in ADMSCs may help to develop better cell-based therapeutic strategies to treat osteoarthritis.