Development of bone substitute: poly(epsilon-caprolactone) scaffold functionalized by adhesive peptides in combination with soluble BMP-9

摘要

Introduction: Large bone defects that cannot self-repair are increasing due to the aging of the western population. Currently autografts are the gold standard to reconstruct these defects, but they had some limitations like their size. Synthetic bone substitutes are therefore of great interest. However they must be optimized to reach the properties of autografts such as osseointegration, osteoconduction and osteoinduction. For this purpose, synthetic polymers can be functionalized by adhesive peptides derived from extracellular matrix proteins. We have previously found that poly(ε-caprolactone) (PCL) films functionalized by a peptide derived from the bone sialoprotein (PCL-pBSP) promoted the adhesion of MC3T3-E1 preosteoblasts and their actin cytoskeletal organization. Furthermore, in the presence of bone morphogenetic protein-9 (BMP-9), PCL-pBSP induced their differentiation into osteoblasts. This study therefore focuses on the preparation of functionalized PCL scaffolds with defined pore size and interconnectivity that mimic the trabecular bone structure. Materials and Methods: Co-continuous blends of PCL (50 000 g/mol) and poly(ethylene oxide) PEO (100 000 g/mol) (50/50 vol%) were prepared by melt-processing (180°C, 50 rpm, 7 min) Blends were annealed during 1,2 or 4h at 180°C to let the morphology coarsen. The porous PCL scaffolds were obtained by selective extraction of PEO in water. The pore size and interconnectivity of PCL scaffolds were calculated using images obtained by micro-computed tomography scanner (Skyscan1172, Bruker). PCL was functionalized using N-maleimidocaproic acid hydrazide to graft pBSP on PCL via its thiol groups. The effect of functionalized PCL scaffold was determined on the C3H10T1/2 murine mesenchymal stem cells (MSC) attachment and cytoskeletal organization by F-actin staining. The osteoblastic lineage commitment of MSC in the presence of BMP-9 was also studied by analysing the protein levels of Runx2, osterix and alkaline phosphatase using Western blots. Sox9 and PPARy, marker of chondrogenic and adipogenic MSC differentiation respectively, were used as control. Results and Discussion: After 1 and 2h of quiescent annealing, the pore size of PCL scaffold was around 230 μm and 310 μm respectively, while it increased significantly after 4h to reach 640 μm (N=3). The scaffolds had a porosity of 45-49% with fully interconnected pores in all experimental conditions. We have also verified that the grafting of pBSP onto PCL allowed the adhesion and the cytoskeletal organization of MSCs in comparison to PCL alone. We have then verified that MSCs can answer to BMP-9 on functionalized PCL scaffold. Conclusion: We have successfully employed a reproducible method to prepare PCL scaffolds functionalized with adhesive peptides that favour their colonization by MSCs and cell response to BMP-9. This combination may be of great interest to treat large bone defects.