| BackgroundBone defects,especially the treatment of critical bone defects,are a major clinical problem.One of the important methods in treating critical bone defects is use of bone graft replacement technology to implant bone graft substitute materials.Therefore,the bone defect is repaired.Although new technologies and new materials are emerging in an endless stream,there is still a certain deficiency in the treatment of critical bone defects.Our laboratory originally synthesized strontium-contaning ?-hemihydrate calcium sulfate(Sr-?-CSH).On the basis of maintaining well bone conductivity and biocompatibility of calcium sulfate,it also has osteoinductivity,but it still has defects in its degradation rate.To solve this problem,In the later stage,our laboratory conducted a research,The Sr-?-CSH was combined with nano-hydroxyapatite(nHA),and it was verified by experiments that the mixture still have a good biocompatibility,the degradation rate was moderate,and it was also osteoinductive,but its bone repair ability on critical bone defects still can't fully satisfied us.Aspirin,a synthetic compound introduced for treating humans more than 100 years,is a very popular antipyretic,anti-inflammatory and analgesic drug which is the most active component of the non-steroidal anti-inflammatory medication.Recently,researchers have found other roles of aspirin in disease.Aspirin has a certain impact on bone metabolism and bone health,aspirin can promote osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs),inhibit the adipogenic differentiation of BMSCs,furthermore,it can activate the activity of osteoblasts and inhibit the activity of osteoclasts,resulting in an increase in bone formation.The aim of this study is to attempt to incorporate aspirin into Sr-a-CSH/nHA composites in order to achieve a superior bone graf material for treating critical bone defects.Methods1.The physical characterization and in vitro degradation of aspirin-loaded Sr-a-CSH/n-HA composites.Firstly,5%Sr-?-CSH was prepared in the same way as before(using coprecipitation and hydrothermal method).Secondly,preparing different concentrations of aspirin solution(50vg/ml?200vg/ml?800vg/ml?3200vg/ml)with distilled water,and then mixed 5%Sr-a-CSH and n-HA in a fixed mass ratio,to get the Sr-?-CSH/n-HA.Thirdly,mixed the mixture with different concentrations of aspirin solution according to a fixed liquid-solid ratio,then molding,fixing,and drying to obtain cylindrical composite samples.Physical properties and characterizations was determined by X-ray diffraction analysis(XRD),Fourier transform infrared spectroscopy(FT-IR),compressive strength test and electron microscopy(SEM)2.The effects of aspirin-loaded Sr-a-CSH/nHA composites on proliferation,differentiation and migration of BMSCs regarding SD rats.The Sr-a-CSH/n-HA composite extracts containing different concentrations of aspirin were prepared according to international standards,and the BMSCs were cultured with the extract solutions.The effects of the extracts solutions on the proliferation of the cells were detected by CCK-8 method.The effect of the extract solutions on cell migration ability was examined by the scratch method.The effects of extract solutions of Sr-?-CSH/n-HA composite on osteogcenic related genes(ALP.BSP?RUNX2)expression was determined using real-time quantitative PCR(qRT-PCR),and the effects of extract solutions on cell osteogenic mineralization were detected by alizarin red stainin(AR).3.The effects of aspirin-loaded Sr-?-CSH/n-HA composite on critical bone defects.The bone defect model we used was a critical bone defect model of the middle tibia of SD rats.Sr-?-CSH/n-HA composite and aspirin-loaded Sr-?-CSH/n-HA were used respectively.The effects of aspirin-loaded Sr-?-CSH/n-HA composite on the repair of critical bone defects was evaluated by imaging(X-ray,Micro CT)and histomorphometric staining suchas HE staining,goldner trichrome staining,immunohistochemical staining(OCN,RUNX2,OST)and TRAP staining.Results1.XRD and FT-IR analysis showed no significant changes in the basic crystal form of the main components(CSH,nHA and Sr ions)on aspirin-loaded Sr-?-CSH/n-HA composite.The results of SEM observation showed that the aspirin-loaded Sr-?-CSH/n-HA composite was porous,the surface of the material was rough,and the crystals were tightly bounded.The compressive strength of composites can reach the level of normal cancellous bone compressive strength even aspirin was loaded,and as the concentration of aspirin increased,the compressive properties of the material did not decreased significantly.2.The aspirin-loaded Sr-?-CSH/n-HA composite has no cytotoxicity to BMSCs,furthermore it has a significantly increased proliferation,migration and osteogenic differentiation of BMSCs compared to CSH/n-HA composites which did not contain aspirin.3.X ray and Micro CT showed that each composite implanted bone showed a certain degree of bone repair,but the bone repair of the aspirin loaded Sr-?-CSH/n-HA group was significantly better than the Sr-?-CSH/n-HA group.The CT data of BV/TV,Tb.Th,Tb.SP.,and Tb.N in the experimental groups at 4 and 12 weeks after surgery showed that the aspirin-containing bone material group(50vg/ml,200vg/ml,800vg/ml)BV/TV,Tb.Th,Tb.N were significantly higher than the simple material group and positively correlated with the concentration of aspirin,while Tb.SP.was significantly lower than the simple material group and negatively correlated with the concentration of aspirin.It is statistically significant.The 800vg/ml aspirin Sr-CSH/n-HA group was the best in the repair of bone defects among all groups.Tissue section staining results:HE:showed that the bone morphology of composite containing aspirin was better than none aspirin group,and immunohistochemical staining showed that the expression of bone-related genes(OCN,OST,RUNX2)in the composite containing aspirin was significantly increased compared with none aspirin group.The Osteoclast-associated gene(TRAP)expression was significantly down-regulated and was dose-dependent.Conclusions1.The aspirin loaded Sr-?-CSH/n-HA composites still have good porosity,and the compressive strength is not significantly decreased compared with Sr-?-CSH/n-HA composites,and there is no significant difference in in vitro degradation rate among groups.Aspirin loaded Sr-?-CSH/n-HA can release a certain concentration of aspirin during the degradation process,which has a certain promoting effect on osteogenic induction.2.Aspirin-loaded Sr-?-CSH/n-HA composite material has no cytotoxicity,furthermore,it can promote the proliferation,migration and osteogenic differentiation of BMSCs more effectively compared with Sr-?-CSH/n-HA,what is more,Aspirin-loaded Sr-a-CSH/n-HA has a significantly increased up-regulation of BMSCs osteogenesis-related gene expression compared to Sr-?-CSH/n-HA,indicating that aspirin loaded Sr-?-CSH/n-HA has a better bone induction compared with Sr-a-CSH/n-3.aspirin-loaded Sr-a-CSH/n-HA composite has a more overall repair effect in the bone remodeling process in vivo,in addition,the osteogenesis effect of aspirin-loaded Sr-a-CSH/n-HA composite is significantly better than that of the Sr-a-CSH/n-HA composite.Tissue section morphology staining indicated an increased expression of osteogenic markers and decreased expression of osteoclast markers. |
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