Construction Of A Novel Individualization Tissue Engineering Bone And Its Osteogenesis In Vivo And In Vitro Experimental Study

OBJECTIVE: To prepare polycaprolactone(PCL)scaffolds by three-dimensional printing technology by combining platelet-rich plasma and bone morphogenetic protein BMP2 in order to construct individualized composite tissue engineering bone with fine bone repair ability.METHODS: PCL scaffolds with different porosity were constructed by 3D printing technology and their mechanical properties and porosity were tested.Platelet-rich plasma(PRP)was obtained by Landesberg secondary centrifugation.The characterization of PRP was detected by platelet count,electron microscopy and HE section staining.PCL/PRP composite tissue engineered bone and PCL/PRP/BMP2 composite tissue engineered bone were prepared by freeze-drying method,and their related characteristics were observed.Bone marrow mesenchymal stem cells(BMSCs)were obtained,isolated and cultured by whole bone marrow adherence method from New Zealand white rabbits,and their growth status was observed regularly.The BMSCs were identified by osteogenesis,adipogenesis,chondrocyte "three-line" differentiation,Western Blot and immunofluorescence methods.The osteogenic differentiation ability of PRP/BMP2 was observed at the cell level in vitro.Different groups were set up: blank control group,PRP group,PRP/BMP2 group and osteogenic medium group.The osteogenic differentiation ability of experimental group was observed by alkaline phosphatase staining and alizarin red S staining.And the expression of ALP and OCN was detected by PCR to evaluate the ability of promoting osteogenic differentiation in the experimental group.The osteogenic differentiation and proliferation ability of tissue engineered bone was observed in vitro.Groups were set up: PCL scaffolds(ordinary medium group),PCL/PRP composite scaffolds,PCL/PRP/BMP2 composite scaffolds and PCL scaffolds(osteogenic medium group).The proliferation of BMSCs were measured in the former three groups and the activity of alkaline phosphatase(ALP)were measured in each group.A 15 mm long bone defect in the middle radius of New Zealand white rabbits was established.48 rabbits were divided into 4 groups(6×2 rabbits in each group).Groups were set up: blank control group,simple PCL stent group,PCL/PRP composite tissue engineering bone group and PCL/PRP/BMP2 composite tissue engineering bone group.At the 4th and 12 th weeks,tissue specimens were obtained for gross observation and X-ray observation of bone defect regeneration;Tissue sections were stained with hematoxylineosin(HE)and Mason trichrome staining to further observe the bone regeneration effect of composite tissue engineered bone at histological level.RESULTS: PCL scaffolds with appropriate porosity and mechanical properties can be obtained by 3D printing technology;ideal platelet-rich plasma can be obtained by Landesberg secondary centrifugation method;PCL/PRP-/BMP2 composite tissue-engineered bone can be prepared by freeze-drying method.Rabbit BMSCs obtained by whole bone marrow adherence method were spindle-shaped and adherent cells,which were stable in the process of culture.The BMSCs could differentiate into osteoblasts,adipogenic cells and chondroblasts under the conditions of in vitro induction,and had the characteristics of multifunctional stem cells.The testing of the surface markers showed that the BMSCs express CD29 and CD44,but do not express CD34 and CD14.Observation of PRP/BMP2 promoting osteogenic differentiation in vitro showed that alkaline phosphatase staining and alizarin red S staining were the highest in PRP/BMP2group;the expression levels of osteogenic related genes OCN and ALP mRNA were significantly higher than those in blank control group,PRP group and osteogenic medium group;the activity of alkaline phosphatase protein(ALP)in PRP/BMP2 composite tissue engineered bone group was significantly higher than that in blank control group,PRP group and osteogenic medium group.The results of general observation and X-ray examination in animal experiments showed that the remodeling bone in bone defect area in PCL/PRP/BMP2 group was significantly better than blank control group ?PCL group and PCL/PRP group at 4 and 12weeks;HE staining and Masson staining further showed that the remodeling bone tissue in PCL/PRP/BMP2 group was significantly more than that in PCL group and PCL/PRP group at 4 and 12 weeks,while in blank control group,the broken ends were closed and a small amount of bone tissue grew in the bone defect area,which could not achieve self-repair.Conclusion: The individualized composite tissue engineered bone can be prepared by3 D printing technology combined with platelet-rich plasma and bone morphogenetic protein 2.This kind of tissue engineered bone has good bone regeneration ability and is expected to become an effective method for individualized bone defect treatment.