Enhanced Osteogenic Differentiation of BMSCs in vitro on A Genipin-chitosan Conjugation Scaffold Based on Surface Hydroxyapatite Nanostructure as Local Delivery of BMP-2

摘要

Introduction:The surface characteristics of biomaterials, especially regarding the sustained delivery of bone morphogenetic protein-2 (BMP-2), maybe provide a novel and effective drug delivery system that could enhance osteogenesis. In this study, we evaluated the effect of the surface biomimetic apatite nanostructure of a new hydroxyapatite (Hap) coated genipin-chitosan conjugation scaffold (HGCCS) on BMP-2 adsorption and release, and the resulting osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Materials & Methods: HGCCS was constructed using a nontoxic cross-linker (genipin) and a nano-crystallon induced biomimetic mineralization method. After immersing in 500 l of sterile PBS at 37 for 3 h, the amount of BMP-2 directly adsorbed in the control sample (genipin cross-linked chitosan framework, GCF) and HGCCS was quantified using a human BMP-2 enzyme-linked immunosorbent assay (ELISA, R&D Systems, USA) kit. Subsequently, the kinetics of BMP-2 release from GCF and HGCCS were determined by ELISA at various time points. To evaluate the bioactivity of BMP-2 released from GCF and HGCCS in vitro, phenotype observation was assessed by SEM and CLMS, alkaline phosphatase (ALP) activity was measured, and quantitative real time PCR (RT-PCR) analysis was performed for mRNA expression of osteogenic differentiation makers, including Runx2, osteopontin (OPN), and osteocalcin (OCN). Results & Discussion: HGCCS displayed a loading efficiency of 65% compared to genipin cross-linked chitosan framework (GCF) for 28% (p＜ 0.01) as quantified by ELISA. We also found that the release of BMP-2 from HGGCS was sustained for at least 14 days compared to GCF consisting of bust releases on days 1 and 3. Furthermore, the BMP-2 release from HGCCS induced an increase in ALP activity as an indicator of osteogenic differentiation for 14 days. Based on RT-PCR, HGCCS also stimulated highest mRNA expression of osteogenic differentiation makers, Runx2 by 14 days, OPN by 3 days and OCN by 14 days, respectively.The results of this study suggest that the surface biomimetic apatite nanostructure of HGCCS as a delivery system of BMP-2 is capable of promoting osteogenic differentiation in vitro. These findings open a new research avenue to regulating the release of the growth factor as a possible bone regeneration strategy and provide a promising scaffold for bone tissue engineering.