HA/β-TCP/CS/PRF Temperature-sensitive Hydrogel Experimental Study On The Repair Of Rabbit Calvarial Defects

Objective: platelet-rich fibrin(PRF)and a new injectable scaffold materialhe that early work to build-hydroxyapatite(HA)/ tertiary calcium phosphate(β-TCP)/Chitosan(CS)temperature-sensitive hydrogel tissue engineering scaffolds,named it HTCP composite gel.And through cytology and animal experiments verify the osteogenesis effect.Methods: 1、HTCP hydrogel: 10 ml CS temperature-sensitive gel,1ml PRF suspension with a diameter of 0.25 mm of HA and β-TCP(a ratio of 3:7)mixture,37 ℃ incubator 15 min,the mixture solidifies into a solid,become HA/β-TCP/CS/PRF gel(HTCP).The use of PH indicator to determine PH value.Observed by the method of inclined tube HTCP temperature-sensitive hydrogel gelling time.2、HTCP hydrogel of rabbit bone marrow mesenchymal stem cells(BMSCs): the influence of the third generation BMSCs to 105 / cm2 cells concentration vaccination in 24 hole culture plate,adherent cells,the experimental group with conventional osteogenesis induced liquid add ing new HTCP gel preparation,the control group with conventional osteogenesis induced liquid.Osteogenesis induced by 8 to14 days,the status of two groups of cell growth,3 weeks on two groups of ALP staining,and determined by MTT colorimetric method.3、HTCP hydrogel repair Rabbit skull defec t experiment research: in the 24 New Zealand big eared white rabbit cranial parietal boneon the preparation of 4 of 6 mm diameter bone defect area.According to the different implant materials can be divided into four defect zone HTCP hydrogel group,HTC hydrogel and PRF group,and blank control group.Observations from 4,8,12 weeks.Line of experimental specimens gross observation,X-ray observation,CBCT observation,HE sta ining and BMP-2 immunohistochemical staining.The preparation of injectable materials of bone tissue engineering,put it in the body of minimally invasive,painless osteogenesis.The preparation of the tissue engineering bone material was validated by cell and animal studies.Thus to better guide clinical work.Methods: The HTCP hydrogel was prepared to observe the filling of the bone marrow stem cells.The cells proliferate in HTCP hydrogels and use ALP staining and MTT staining to verify the biological phase of the material Capacitance and osteo inductive ability.24 New Zealand big-eared white rabbit cranial parietal bone on the preparation of 4 of 6 mm diameter bone defect area.Defects in random into HTCP hydrogel as experimental group,HTC hydrogel,PRF group,as the control gro up,the other set up blank control group.Surving animals were observed for 4,8,12 weeks,killing animals to get samples.To observe the osteogenetic effect by general observation,X-ray,Micro CT,HEstaining and Immumohistochemical staining.Result: 1、HTCPWhite opaque liquid gel,HA and β-TCP powder particles suspended in solution.PH value of 6.5-7 or so have been measured,in 37 ℃ constant temperature box can be gelation let stan d for 15 minutes.2、determined by MTT colorimetry analysis of BMSCs in HTCP gel can survive and grow normally,no obvious cytotoxicity.ALP staining prompt HTCP hydroge l could promote osteoblast differentiation of BMSCs.3、animal experimental results:(l)gross observation showed that groups of bone defect area new bone formation increased gradually over time,at 4,8 weeks,the amount of new bone formation HTCP group were superior toThe control group,12 weeks,HTCP group with HTC did not see obvious difference,but is better than that of PRF group and the blank control group of bone defect repair.(2)imaging showed that each group defect photic zone area decreases gradually with the extension of time,and 4,8 weeks postoperatively,the experimental group photic zoneis smaller than the control group.At 12 weeks HTCP,HTC group,PRF group defect almo st no through the light and shadow,lamellar bone fusion together,with the surrounding bone density.(3)HE staining results,4 to 8 weeks after HTCP group,trabecular bone,fibr oblasts,etc.More than in the control group.HTC group and PRF group for guided bone Again Raw ability difference is not obvious,but the blank group of bone defect repair are promoting and guiding role.At 12 weeks,HTCP,HTC group,PRF group have a mature new bone and host bone fusion,is better than that of blank control group.(4)the BMP-2immunohistochemical observation: 4 weeks,groups of BMP-2 antibody staining were significantly positive,yellowing cytoplasm;8 weeks,HTCP group of BMP-2 dyeing strong positive,the rest of the group is positive,cytoplasm were dyed tan;At 12 weeks,the positive expression were reduced,no obvious difference.Conclusion: HTCP gel has goo d biocompatibility and can promote the differentiation of BMSCs development,early and mid in rabbit skull defect can obtain good osteogenetic effect,is better than the single use HTC or PRF.HTCP hydrogel as temperature-sensitive hydrogel tissue engineering bone frame materials can promote the healing of bone defect repair,has good clinical application potential.