Tunable Osteogenic And Osteoinductive Capabilities Of Hydroxyapatite Through Regulating Its Crystallinity

Hydroxyapatite?HA?is a biocompatible and osteoconductive bone substitute,but clinical applications of which are still limited by the poor biodegradable and osteogenic capabilities caused by its high crystallinity after high-temperature sintering.This study aimed to illuminate the effects of crystallinity on the physicochemical and biological properties of HA through comparatively investigating the physicochemical properties of HA powders,the in vitro cytocompatibility and osteogenic differentiation ability of HA powders,and the in vivo osteogenesis and angiogenesis of porous HA microsphere scaffolds.Hydroxyapatite precursors were synthesized by a chemical precipitation method.Microwave and calcination methods were further utilized to treat the precursors for the production of HA powders with gradient crystallinities?0%-100%?,respectively.With the increasing crystallinity of HA,BSA adsorption capacity of HA powders gradually weakened.As the crystallinity increased beyond 60%,protein adsorption amount of HA powders achieved an equilibrium,regardless of different initial protein concentrations.HA powders with different crystallinities all revealed good cell affinity as co-cultured with mouse bone marrow mesenchymal stem cells?mBMSCs?which adhered and spread well.HA powders with crystallinity above 60%remarkedly promoted cellular viable and proliferation abilities,whilst the powders with crystallinities of 0%and 45%significantly up-regulated the ALP activity and mRNA expression of osteogenesis-related genes?such as ALP,Runx2 and OCN?of mBMSCs.Agarose/HA composite scaffolds were prepared through mixing agarose with HA powders using a mechanical agitation and lyophilization method.Compared to pure agarose scaffold,agarose/HA scaffolds had greater surface roughness,more micropores and higher mechanical strength.The scaffolds containing HA with lower crystallinity?<45%?had stronger BSA absorption ability owing to their higher porosity compared with other scaffolds.Rabbit bone marrow mesenchymal stem cells?rBMSCs?exhibited higher viable and proliferation abilities on the composite scaffolds with HA crystallinity over 60%,whilst had greater spreading areas and ALP activity on the scaffolds with low-crystallinity HA?<45%?.Results illustrated that lower doses of Ca²⁺released from high-crystallinity HA?>60%?could benefit cell viability and proliferation activity;low-crystallinity HA?<45%?had better protein adsorption capacities and could generate more favorable Ca²⁺-containing microenvironments for the notably enhanced osteogenic differentiation of BMSCs.HA microspheres with different crystallinities were prepared using an extrusion-spheronization method scaffold and bonded with poly?lactic-co-glycolic acid??PLGA?to prepare porous HA microsphere scaffolds.The microsphere scaffolds had three-dimensional interconnected macroporous structure with porosity of 35%-41%and compressive strength of 6-8 MPa.After implantation in rabbits dorsal muscles for 2 weeks,all the microsphere scaffolds hardly biodegraded;at week 4,slight degradation occurred in the microsphere scaffolds with HA crystallinities of 0%,45%and 60%.Histological and immunohistochemical results showed that a large number of inflammatory cells accumulated in the scaffolds after 2 weeks and disappeared at week 4.Among them,the microsphere scaffold with HA crystallinity of 45%was completely encapsulated with fibrous tissues and notably induced the ingrowth of large numbers of large-diameter new blood vessels,indicating an excellent angiogenesis capability and a great osteoinductivity potential of HA-based bone substitutes for clinical applications.