Chondrogenesis-inductive nanofibrous substrate biofunctionalized with autologous growth factors from platelet lysates for stem cell based therapies

Cartilage tissue engineering holds a remarkable potential in orthopaedics, as an alternative approach to currently available therapies. Mesenchymal stem cells (MSCs) have been used to generate engineered cartilage tissue, being the Transforming Growth Factor b3 (TGF-b3) and the Insulin Growth Factor I (IGF-I) potent regulators of chondrogenesis. Therefore, it is hypothesized that the availability of these two growth factors (GFs) at the surface of a biomaterial substrate would lead to the stable chondrogenic differentiation of MSCs. The present work aims to develop a biofunctional electrospun nanofibrous mesh (NFM) with chondrogenic inductive potential, through the immobilization of autologous TGF-b3 and IGF-I from platelet lysates (PLs).

Taking advantage of the specific and efficient interactions between an antibody and its antigen, antibodies against TGF-b3 and IGF-I were immobilized at the NFMs surface at the concentration of 4 mg/mL each. These antibodies were also immobilized over the same nanofibrous substrate at the 1:10 proportion, as used in standard chondrogenic differentiation medium. Higher binding efficiencies were observed for GFs immobilized from a pool of PL, by NFMs functionalized with mixed antibodies (99.3±0.4% for TGF-b3 and 77.5±2.4% for IGF-I). Biochemical performance of human bone marrow-derived MSCs cultured during 28 days on the biofunctional nanofibrous systems (Single or Mixed, with TGF-b3 and/or IGF-I captured from PL or from recombinant-origin (rGF)) under basal or chondrogenic differentiation media was assessed by quantification of cells viability and proliferation, and total protein synthesis, as well as glycosaminoglycans (GAGs) production. Biological data confirms the biological activity of bound TGF-b3 and IGF-I, since these biofunctional nanofibrous systems are more effective when compared to the control condition. The relative expression of chondrogenic transcripts confirms the genotype of hBMSCs cultured on the biofunctional nanofibrous systems. The typical round morphology of chondrocytes, as well as the Alcian Blue staining and the immunolocalization of Collagen Type II confirmed the formation of a cartilaginous ECM. These biological results indicate that the functionalized nanofibrous substrates are able to promote chondrogenesis. The proposed biofunctional nanofibrous system can act as an effective cartilage tissue engineered scaffold, operating as a synthetic and bioactive ECM-like support of hBMSCs growth and differentiation.

Acknowledgments: We acknowledge the financial support from FCT/MCTES and FSE/POCH/PD/169/2013,for the PhD grant of MCN (PD/BD/113797/2015), and the projects SPARTAN (PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-0000232).