A Novel Quercetin-Containing α-CSH/n-HA Composite:Preparation,Biological Properties And Osteogenic Potential In Bone Defects Repair

Background&ObjectiveBone defect is a difficult problem in clinical treatment.At present,it is urgent to invent new artificial bone replacement materials for bone defect repairing.Previous studies in our lab have shown that the composite material of alpha-calcium sulfate hemihydrate/nano-hydroxyapatite(a-CSH/n-HA)has good biocompatibility and bone conductivity,but poor bone induction performance.Therefore,this project innovatively combines Quercetin with a-CSH/n-HA to obtain a new type of artificial bone replacement material with good biosafety,moderate degradation rate and ideal bone induction performance.Materials&MethodsPart I Preparation and characterization of a novel Q-a-CSH/n-HA composite.The gradient concentration of Quercetin containing in a-CSH/n-HA was prepared by direct mixing method.X ray diffraction(XRD)was used to evaluate the composition of elements.Fourier transform infrared scanning(FTIR)was used to evaluate crystal structure.SEM(SEM)was used to observe the surface micro-structure.The influence of Quercetin on mechanical strength of materials was determined by pressure test.Determination of element composition were made by X-ray energy spectrum analysis(EDX);In vitro degradation assay and determination of Ca2+ and quercetin concentrations in degradation solution were also performed.Part II Potential of proliferation,migration and osteogenic differentiation of rat BMSCs cocultured with Q-a-CSH/n-HA composite.The extract of gradient concentration of Q-a-CSH/n-HA were prepared.Examination of Ca2+ and quercetin concentration in Q-a-CSH/n-HA extracts was also made.MTT assay exam the cytotoxicity of extracts;CCK-8 assay exam the effect of extract on BMSCs proliferation;Scratch assay evaluate the effect of gradient extracts on BMSCs migration;fluorescence quantitative PCR assay detected expression level of osteogenic differentiation genes of BMSCs affected by Q-a-CSH/n-HA extracts.Alizarin red and Von Kossa staining were used to detect the potential of BMSCs osteogenic mineralization.Part III Experimental study of a novel Q-a-CSH/n-HA composite used for the repairing of critical defect of tibia in rats.The critical tibial defect model of SD rats was established.50 male 8weeks old SD rats and 36 female lyear old SD rats were divided into 5 and 4 groups,and no graft,α-CSH/n-HA graft,0.5%Q-a-CSH/n-HA graft,1.5%Q-a-CSH/n-HA and 3%Q-α-CSH/n-HA graft were implanted respectively.X-ray examination and micro-CT were used to observe bone defect repair and new bone morphology.The new bone formation and material degradation were observed by the HE staining,Goldner staining and Safranin O staining.The number of RUN-X2/OSX/OCN positive cells was observed by immunofluorescence.The ability of colony formation was detected by CFU method.Ca2+ concentration in rats’ serum was also determined.Results1.XRD and FTIR showed that the diffraction and infrared spectra of Q-a-CSH/n-HA were similar to those of a-CSH/n-HA.EDX results showed that the composition of elements in each experimental group was basically similar,and the composition of material elements after degradation was richer.SEM showed that the addition of quercetin increased the number of microspheres and the porosity of the degraded material obviously.The mechanical strength of the material decreased with the addition of quercetin,but the lowest is 4.29±0.65 MPa of 3%Q-a-CSH/n-HA group was still higher than those of normal human cancellate bone.Quercetin had no significant effect on the degradation rate of the composite and the Ca2+ level in the degradation solution,but quercetin concentration in the degradation solution increased.2.With the increase of quercetin proportion in the material,Ca2+ concentration in the extraction liquid was indifference,but quercetin concentration increasing,proliferation rate and the migration ability of the rat BMSCs increase,expression of TNF alpha downregulated,but OSX,RUNX2,OCN,ALP,BMP-2,OPN,BSP,Smad2 and TGF-beta expression of BMSCs upregulated,osteogenesis differentiation and mineralization was also elevated,1.5%Q-a-CSH/n-HA group were the best,and pure a-CSH/n-HA group were the worst.And the extracts from all the research groups were not cytotoxic.3.X-ray and Micro-CT results showed that the rate of bone defect repair and formation of new bone in quercetin-containing materials group was significantly higher than those of the control group.The results of HE,Goldner and Safranin O staining showed that quercetin significantly increased the generation of new bone and promoted the degradation and absorption of material.Quercetin significantly increased the number of RUNX2/OSX/OCN positive cells revealed by immunofluorescence assay.These effects were best in young rats(1.5%)and older rats(3%),and worst in pure a-CSH/n-HA.The Ca2+ concentration in the young rats’ serum of the 1.5%group was significantly higher than that in the blank control group,the differences among other groups were not significant.The results of CFU method showed that the older rats in the 3%group and the younger rats in the 1.5%group were significantly higher than the control group respectively.Conclusions1.Quercetin had no significant negative effects on the crystal structure,chemical composition,degradation rate and Ca2+ release of the composites.As the quercetin concentration increased,the mechanical strength of the material decreased,while the surface microsphere structure,post-degradation porosity,and quercetin release increased.2.Q-α-CSH/n-HA has good biological safety.The increase of quercetin concentration in the composites directly influenced BMSCs,enhancing the ability of proliferation,migration,osteogenic differentiation and mineralization,and has an ideal potential of bone induction on BMSCs of rats.3.The animal model used in this study is real and effective.The new Q-α-CSH/n-HA composite material has more overall repair effects in the process of bone reconstruction in vivo,and the porous cavity formed after degradation is more conducive to the re-absorption of n-HA.The change of microenvironment in the modeling area and the release of quercetin and Ca2+ may synergize,and the anti-aging effect of quercetin and bone induction efficacy may also synergize,jointly promoting the proliferation,migration,recruitment and osteogenic differentiation of BMSCs,and ultimately promoting the repair and reconstruction of bone defects in rats’ tibia.