gms | German Medical Science

Objectives: Bone marrow-derived mesenchymal stromal cells (BMSCs) are still under investigation as promising cell source for cartilage regeneration. Insulin-like growth factor I (IGF-I) is widely acknowledged to play a central role in chondrogenesis, however, its use in chondrogenic differentiation of BMSCs is surprisingly rare and poorly understood. Hence, the aim of this study was to investigate effects of IGF-I during chondrogenic differentiation of BMSCs in standard pellet culture and hyaluronic acid (HA)-based hydrogels used for cartilage engineering. Special attention was paid to the influence of IGF-binding proteins (IGFBPs) on IGF-I action and bioavailability.

Methods: BMSCs were chondrogenically differentiated in pellet culture (2x105 cells per pellet) and HA-based hydrogels (8x105 / 40 µl) for 21 days in the presence of transforming growth factor-beta1 (TGF-beta1). The culture medium was supplemented with IGF-I in different concentrations (10 - 1000 ng/ml). Alternatively, des(1-3)IGF-I was applied (10 - 1000 ng/ml), a truncated IGF-I analogue that lacks the three N-terminal amino acids, binds to the IGF-I receptor with similar affinity as IGF-I, but has a strongly reduced binding to IGFBPs. As a further tool to investigate the influence of IGFBPs, the small molecule inhibitor NBI-31772 was utilized (0.1 - 10 µM) that disrupts IGF-I binding to IGFBPs. For all conditions, chondrogenesis was analyzed using histology, immunohistochemistry, quantitative biochemical assays, and qRT-PCR.

Results and Conclusion: Quantitative biochemical assays revealed that both IGF-I and des(1-3)IGF induced glycosaminoglycan (GAG) and collagen synthesis in a dose-dependent manner, however, up to 10-fold lower concentrations of des(1-3)IGF were needed to achieve chondrogenic responses similar to IGF-I in both pellet culture and HA hydrogels. These observations were well reflected by histological and immunohistochemical staining for GAG and collagen type II. The secretion of IGFBP-3 by BMSCs could be demonstrated by ELISA. Remarkably, in the IGF-I-supplemented cultures, the disruption of IGF-I binding to IGFBPs using the small molecule inhibitor NBI-31772 restored GAG synthesis in a dose-dependent manner, whereas NBI-31772 had no effect in cultures supplemented with des(1-3)IGF-I. Similarly, qRT-PCR analysis revealed a strong upregulation of the chondrogenic marker gene COL2A in the IGF-I group after NBI-31772 exposure.

Our results indicate that, compared to des(1-3)IGF-I, distinctly higher doses of IGF-I are necessary to induce chondrogenic differentiation of BMSCs owing to reduced bioavailability of IGF-I due to enhanced binding to IGFBPs. The study corroborates, in two independent culture systems, the important role of the IGF-I/IGFBP system during chondrogenesis of BMSCs.