Controlled surface retention of osteogenic peptide-5 derived from BMP-2 on functlonalized electrospun nanofibers for guided bone regeneration

摘要

Introduction: Self-healing of bone tissue is limited in critical sized defects and able to outbreak abnormal bone regeneration because of an infiltration of undesired tissues. To overcome these clinical problems, guided bone regeneration has been upraised by using a membrane as a biomaterial with suitable pore size. Recently, bone morphogenetic protein-2 (BMP-2) has been the most frequently used biomolecule to accelerate bone formation. However, there are several side effects such as difficulty for optimization of clinically-relevant dose and unwanted cancerous signaling process. To solve these problems, we used osteogenic peptide-5 (OP5) derived from BMP-2 as an osteoinductive molecule. The peptide is expected to lead lower side effects on miss-targeted tissue and dosage limitation because they cannot perform any other cellular processes without osteogenic process. Nevertheless, delivering the peptides with materials have been challenging issue since it was hard to conjugate to other substrates. In this study, we fabricated the surfaced modified electrospun Poly(L-lactide) (PLLA) nanofibers that were coated with different amount of polydopamine which induce chemical conjugation of peptides. We examined the surface modification-dependent efficacy of the attachment of peptides and bone formation by applying the developed materials into a mouse calvarial critical sized defect model. Materials and Methods: Electrospun nanofibers were fabricated from poly(L-lactide) (PLLA). To sustain deliver the peptide to the injury, we used polydopamine chemistry to loosely conjugate the peptide on the porous membrane. In brief, the libers were coated with dopamine hydrochloride solution (pH 8.5) for 4 and 8 hours, and the polydopamine-coated PLLA nanofibers were then immersed in OP5 solution. We characterized surface properties of nanofibers by XPS and studied the relative remaining amount of OP5 for over 28 days. To confirm the osteoinductive effect of the developed material, we analyzed the proliferation, calcium minerals and gene expression (e.g. ALP, OCN, Runx2 and Colla) of human mesenchymal stem cells (hMSCs) cultured for 1 week in vitro condition with OP5 containing medium. We then implanted the membranes onto in vivo mouse calvarial defect model to confirm the bone regeneration capacity, mechanical property of the regenerated bone during 2 months, and then the values were calculated by Micro-CT, Histological staining and nano-indentation. Results and Discussion: MTT assay demonstrated that there was no cytotoxicity on hMSC treated with OP5 rangning from 0 to 100 μg/ml. It was confirmed that OP5 was retained over 28 days depending on polydopamine coating time. In vivo calvarial defect model results proved that small amount of peptide could promote the new bone forming. Furthermore, our substrates accelerated successful regeneration, and newly formed bone exhibited similar mechanical properties with native bone. Our substrates also constitued large amount of lamellar structures that was frequent on host bone tissue, while the other groups mostly formed connective tissues. Conclusion: Our data support that the presentation of OP5 on the surface of nanofibers as immobilized by using polydopamine chemistry may be an effective method to guide bone regeneration at relatively low local concentration. Furthermore, we proved that OP5 delivery system was promising enough to clinically available instead of BMP-2 delivery.