INTRODUCTION

Reconstruction of large bone defects still remaining a major clinical orthopedic challenge, since the repair of a bone defect comprises not only the process of new bone formation, but also the formation of new blood vessels - angiogenesis1. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are involved on cell differentiation and bone vascularization to develop viable osseous tissue2,3. Accordingly, herein is hypothesized that the synergistic effect of autologous BMP-2 and VEGF, parallel bound over a single nanofibrous substrate, can lead to a successful osteogenic and angiogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

METHODS

Specific antibodies for BMP-2 and VEGF were immobilized over an electrospun nanofibrous mesh (NFMs), in a side-by-side design, at a maximum immobilization capacity. The BMP-2 and VEGF derived from platelet lysate (PL) were bound to the biofunctional nanofibrous substrate. The unbound protein solution was quantified by ELISA. The osteogenic and angiogenic potential of this engineered biofunctionalized system was assessed by culturing hBM-MSCs during 21 days, without exogenous induction.

RESULTS AND DISCUSSION

The antibodies against BMP-2 and VEGF were parallel immobilized at maximum concentration of 4 μg/mL each, over the same nanofibrous substrate, trying to recreate the vasculature of a bone tissue (Figure 1A-C). Higher binding efficiencies were observed for BMP-2 and VEGF captured from a pool of PL by the biofunctionalized nanofibrous substrate.

(FIGURE)

Figure 1. Spatial distribution of immobilized antibodies over the same nanofibrous substrate: anti-BMP-2 (A); anti-VEGF (B); merged view (C). Immunoexpression of an osteogenic (Collagen Ia; D) and angiogenic (CD31; E) markers in an engineered biofunctional systems, and a bare NFM (F), after 21 days of culture.

Biochemical performance of hBM-MSCs cultured on the engineered biofunctional system, without exogenous induction, confirms the biological activity of bound BMP-2 and VEGF. The quantification of angiogenic and osteogenic transcripts reveled that hBM-MSCs respond according to the growth factor bound to the engineered biofunctional system. The immunolocalization of osteocalcin and CD31 confirmed the osteogenic and angiogenic phenotype of differentiated hBM-MSCs (Figure 1D-F).

CONCLUSION

The proposed engineered biofunctional system is able to promote spatial angiogenesis and osteogenesis of hBM-MSCs, targeting an effective vascularized bone tissue engineering approach.

REFERENCES

1 S. Almubarak, et al., Bone, 2016;
2 D. Barati, et al., J Control Release 223, 2016; 3 M. Bouyer, et al., Biomaterials, 2016;

ACKNOWLEDGMENTS

Authors acknowledge the financial support from FCT/MCTES and FSE/POCH/PD/169/2013, for a PhD grant (PD/BD/113797/2015), a post-doctoral grant (SFRH/BPD/96197/2013), a IF grant (IF/00376/2014), and the projects SPARTAN (PTDC/CTM-BIO/4388/2014) and FRONthera (NORTE-01-0145-FEDER-0000232).