Statement of Purpose: Articular cartilage is a connective tissue with low self-regeneration potential due to its avascular nature and lack of access to progenitor cells ^[1]. Therefore, cartilage regeneration is still an unmet clinical need that may be solved by tissue engineering and regenerative medicine (TERM) strategies. TGF-b3 and IGF-I were identified as important proteins on the regulation of cartilage development and on the homeostasis of mature articular cartilage ^{[2, 3]}. In accordance, it is hypothesized that the availability of these two growth factors (GFs) at a biomaterial substrate would lead to the successful chondrogenic differentiation of mesenchymal stem cells (MSCs). The leading goal of this study is to develop a multi-functionalized electrospun nanofibrous mesh (NFM) with chondrogenic induction potential, through the immobilization of autologous TGF-b3 and IGF-I captured from platelet lysates (PLs).

Methods: Activation and functionalization of NFMs, followed by single or mixed antibodies (anti-TGF-b3; anti-IGF-I)  immobilization, were performed as described elsewhere ^[4]. Quantification of fluorescent secondary antibodies (Alexa Fluor^® 488 and 595) was used as an indirect method of antibodies immobilization. The GFs from PLs and recombinant-origin were incubated for 1h at RT, followed by a blocking step. The GFs not captured by the antibodies were quantified by ELISA. The chondrogenic potential of this biofunctionalized nanofibrous system (single or mixed, with TGF-b3 and/or IGF-I captured from PL or recombinant-origin) were further assessed by culturing human bone marrow-derived MSCs during 28 days in basal medium. Bare NFMs cultured with hBMSCs under standard chondrogenic differentiation medium supplemented with TGF-b3 and IGF-I were used as positive control.

Results: The antibodies against TGF-b3 and IGF-I were successfully immobilized at the surface of activated and functionalized NFM, being 4 mg/mL the maximum binding concentration of each antibody. They were also immobilized over the same substrate in a mixed fashion at 1:10 proportion (0.4 mg/mL anti-TGF-b3 : 3.6 mg/mL anti-IGF-I), which corresponds to the proportion used in the chondrogenic differentiation medium.

The bioactivity of immobilized mixed antibodies was tested by using recombinant GFs, achieving a binding efficiency of 49 ± 10 %  for TGF-b3 and 65 ± 15 % for IGF-I. Moreover, higher binding efficiency were observed for GFs captured from pool of PL by NFMs functionalized with mixed antibodies (99.3 ± 0.4 %  for TGF-b3 and 77.5 ± 2.4 %).  

Biochemical performance of hBMSCs cultured on biofunctional nanofibrous systems under basal or chondrogenic media was assessed by quantification of cells viability and proliferation, and total protein synthesis, as well as glycosaminoglycan (GAG) production. Biological data confirms the biological activity of bound TGF-b3 and IGF-I, since the biofunctional nanofibrous systems are more effective when compared to control condition (Figure 1). The relative expression of chondrogenic transcripts confirm the genotype of hBMSCs cultured on biofunctional nanofibrous systems. The typical spherical morphology of chondrocytes, as well as the Safranin O and Alcian Blue stainings, and the immunolocalization of collagen type II confirmed the formation of a cartilaginous ECM. 

Conclusions: The biological results indicate that the functionalized nanofibrous substrates are able to promote chondrogenesis, being more effective than the standard differentiation condition. We hypothesize that our substrates maximize the exposure of the hBMSCs to the immobilized GFs, by the close contact established between them at the surface of the substrate.  

The proposed biofunctional nanofibrous systems can act as an effective cartilage tissue engineering scaffold, operating as a synthetic and active ECM-like support for stimulating hBMSC growth and differentiation.

References:  ^[1]Correa, D. Lietman, Semin Cell Dev Biol. 2016; ^[2]Petrou, M. Regen Med, 2013;8:157-170. ^[3]Longobardi, L. J Bone Miner Res, 2006;21:626-636. ^[4]Oliveira, C. Biomacromolecules, 2014; 15:2196-2205.

Acknowledgments: Authors acknowledge the financial support from FCT/MCTES and FSE/POCH/PD/169/2013, for a PhD grant (No. PD/BD/113797/2015), the SPARTAN, PTDC/CTM-BIO/4388/2014 and the FRONthera, NORTE-01-0145-FEDER-0000232.