Fabrication And Enhanced Osteo-differentiation Ability In Vitro Of N-HA/Resveratrol/Chitosan Composite Microspheres

Objective(s):With the development of minimally invasive techniques for bone repair,injectable microsphere scaffolds have also made great progress in the field of bone repair.In order to improve the anti-inflammatory and osteogenesis effects,we employed resveratrol(Res)with anti-inflammatory,antioxidant and osteogenic effects,n-HA(nano-hydroxyapatite)with bone conductivity and osteoinductivity,and Chitosan(CS)with good biocompatibility,non-toxic and antibacterial function to prepare n-HA/Res/CS composite microspheres.Vanillin was used as a crosslinking agent to crosslink and cure the composite microspheres for purpose of stable microspheres.Methods:We prepared nano-hydroxyapatite/resveratrol/chitosan composite microspheres(n-HA/Res/CS)with different contents of Res by emulsion chemical cross-linking method.The physical and chemical properties were characterized by FT-IR,XRD,SEM,laser particle size analysis,swelling rate analysis,HPLC,fluorescent staining,etc..Live/dead staining,CCK-8,RT-qPCR,immunofluorescent staining analysis and alizarin red staining were used to assess cell viability,proliferation,anti-inflammatory,and osteogenic differentiation.Results:The main steps of Res-loaded microspheres prepared by emulsification cross-linking are briefly described,including emulsification rate,phase volume ratio,composition of dispersed phase,and cross-linking method.The results showed that the content of Res in n-HA/CS matrix is 0.75?9.07%.The encapsulation rate of Res in composite microspheres is 13.42?39.43%.Morphological observation showed that microspheres with a diameter size ranged from 30.75 to 110.87 um with moderate swelling ability.In vitro degradation showed that vanillin-crosslinked n-HA/Res/CS composite microspheres could degrade and release Res at a controlled release rate,which also confirmed the feasibility of the composite microspheres adapting to drug release in a physiological environment in bone tissue engineering,the biocompatibility experiment of mesenchymal stem cells(MSCs)seeded on the composite microspheres showed good biocompatibility.MSCs seeded on composite microspheres,and used RT-qPCR and alizarin red staining assay to detect the mRNA level of osteogenic-related genes Runx2,ALP,Col-1 and OCN and the amount of mineralization in the osteogenic medium,the results also showed that the composite microspheres promote bone differentiation and extracellular matrix calcification.After seeding RAW264.7 onto the composite microspheres,the results from RT-qPCR and immunofluorescence staining showed that the Res-loaded composite microspheres had anti-inflammatory capacity and could reduce mRNA expression of pro-inflammatory cytokines TNF-?,IL-1? and iNOS RAW264.7 cells.Conclusion(s):We successfully prepared n-HA/Res/CS composite microspheres with different Res content.The optimized CM-30 showed a better drug sustained-release profile,which significantly improved the anti-inflammatory and osteogenic capbility,and can provide a certain theoretical basis to use microsphere drug loading to treat bone related diseases for the future bone tissue engineering.