The Research On Preparation And Biological Properties Of Two Novel Hydroxyapatite-based Bone Biomaterials With The Function Of Long-term Controlled Drug Release

Objective and significance:the drug deli’very materials provides new strategies for the treatment of bone and joint diseases.In this paper,the preparation and biological properties of two novel HA-based composite bone materials with the function of long-term controlled drug release were studied.The grafted chitosan scaffold(CS-P24/HA)and the PLA/HA@Eu double-layer microspheres(PLA/HA(rhIGF-1)@Eu)were developed through chemical grafting and physical coating respectively.Their biological effects on mesenchymal stem cells,osteoblasts,osteoclasts or fibroblasts,as well as the therapeutic effects of these materials on the calvarial defects,bone loss or cartilage degeneration were investigated respectively.The aim of these researchs is to promote the development of drug delivery system in the bone tissue engineering and provide theoretical reference for further clinical application.Methods:(1)in this study,the CS-P24/HA biomimetic composite scaffolds for controlled delivery of P24 peptide were functionalized by the chemical grafting modification technique:firstly,P24 was conjugated with a thiolated chitosan;secondly,the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds.The effect of CS-P24/HA scaffolds on bone regeneration was evaluated,along with the underlying biological mechanisms responsible in vitro and in vivo.(2)rhIGF-1 loaded poly(lactic acid)/hydroxyapatite@Eudragit double-layer microspheres(PLA/HA(rhIGF-1)@Eu)with "core-shel" structure were formulated using HA and poly(lactic acid)by w/o/w technique and Eudragit coating process.The particle size distribution,in-vitro release profiles of rhIGF-1,the surface morphology were investigated.In vivo experiments were performed on adult male C57BL/6 mice.PLA/HA(rhIGF-1)@Eu microspheres were subcutaneously implanted into the back of mice and the effect of the microspheres on the prevention of bone loss in mice were monitored for 6 months.Results:(1)in vitro,the controlled and sustained release of bioactive P24 could last up to 90 days,furthermore,the release profiles of CS-P24/HA were linear;P24 on the scaffold significantly promoted cell adhesion,proliferation,osteodifferentiation,and mineralization with synergistic effects.Mesenchymal stem cells(MSCs)revealed spindle-shaped surface morphology,indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate.Furthermore,mRNA levels for OCN,Runx2,and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro.Similarly,the MSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds.In vivo,calvarial defects repair studies revealed that CS-P24/HA scaffolds could promote the new bone formation in the material implanted site and repair the calvarial defects of rats.This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in bone tissue engineering.(2)In vitro release of rhIGF-1 from the PLA/HA(rhIGF-1)@Eu microspheres maintained over 180 days.rhIGF-1 loaded PLA/HA(rhIGF-1)@Eu microspheres could support the cell adhesion and growth in vitro,indicating their good biocompatibility.PLA/HA(rhIGF-1)@Eu microspheres were subcutaneously implanted into the back of mice and the effect of the microspheres.In-vivo results showed that the bone mass of mice in the PLA/HA(rhIGF-1)@Eu microspheres group was significantly higher than that of the control group during the 6-month experimental period.PLA/HA(rhIGF-1)@Eu microspheres could bring beneficial effects on the bone mass of adult mice.Conclusion:(1)CS-P24/HA scaffolds can linearly and steadily release BMP-2 peptide,promote the adhesion,proliferation and osteogenic differentiation of mesenchymal stem cells,and has good repair capacity in the calvarial defects of rats.Therefore,this new CS-P24/HA scaffold is a promising bone repair material.(2)PLA/HA(rhIGF-1)@Eu double-layer microspheres could achieve the long-term release of rhIGF-1,This study is helpful to understand the biological role of IGF-1 in bone mineralization.It is a new attempt to prevent systemic bone loss by local subcutaneous drug delivery system.HA-based composite bone materials with the function of long-term controlled drug release may provide new strategies for the treatment of bone and joint diseases.