The Research Of Sensory Nerve To Promote The Massive Bone Defects

Background:The treatment of long bone defects is a difficult problem of traditional orthopedic.Adopt the method of autologous bone graft while the effect is good,but because of the limitation of bone mass,most of the patients.And allogeneic bone graft,due to the immune response,treatment effect is poor.With the development of material science,the use of artificial bone graft,and the method of osteogenesis effect is poor,clinical application has not been made good progress.In recent years,with the emergence of tissue engineering bone,for the treatment of long bone defect provides an ideal way.However,while compared with the general artificial bone graft,tissue engineering bone osteogenesis effect is relatively good,but still not reach the expected level.In recent years to improve the effect of tissue engineering bone ossification of study is very hot,the main direction of concentration in the blood supply and nerve control two aspects.Good blood supply can provides oxygen and nutrients for bone tissue engineering,as well as some osteogenesis contributing factor,so the vascularization contributes to the growth and regeneration of the bone,it has been widely recognized in the industry.But innervation to the mechanism of action of tissue engineering bone ossification is not very clear,it needs us to further research.Studies have found that sensory nerve can promote tissue engineering bone ossification,the osteogenesis effects similar to those of vascularization,and motor nerve has no effect.The results of this study show that sensory nerve and motor nerve for tissue engineering bone osteogenesis effect is not the same.Of this study is based on the findings,by comparing the different of sensory nerve and motor nerve,and to explore the sensory nerve,promote the mechanism of action of long bone defect,and provide new methods for the treatment of long bone defect.Objective:Explore the sensory nerve,promote the mechanism of action of long bone defect repair in rats,provide new method for the treatment of long bone defect.Methods:The first part: the sensory nerve long bone defects in rats model of production and osteogenesis effect observationUsing adult SD rats as experimental animals,apply the GFP + in bone tissue engineering material of BMSCs as seed cells,and then make simple bone defect model respectively the blank group,the bone tissue engineering material implanted into control group and the group of sensory nerve transplantation animal model.In 1 week,4 weeks,8 weeks respectively,taken CT,X-ray,and Micro-observe osteogenesis effect.Fixed,decalcified,after the completion of the section,HE stain and immunohistochemical confocal images,detection of histological observation.The second part: Saphenous nerve and muscle branch of sciatic nerve of rats were extracted surgically as sensory and motor nerve tissue,respectively.The nerve homogenate(10 mg/ml)was prepared as per 10 mg tissue with 1 ml osteoblast inducing conditional media,and 10 times diluted after filtration purification.Cultivation GFP + rat pups BMSCs in vitro was trained to P3 generation after adding six different concentration gradient of sensory and motor nerve homogenate.Cell proliferation quantity detection and alkaline phosphatase staining were used to assess the proliferation and differentiation of BMSCs post 14 days.The third part: trained sensory neurons under influence of bone marrow stromal stem cellsMixing trained group: SD rat newborn rat DRG,mixed with GFP + BMSCs trained,the status of cell growth and change.Co-culture group in isolation: SD rat newborn rat DRG,according to 1 x103 cells after digestion/Transwell Chambers,the proportion of vaccinated sensory neurons,six were given,and the six BMSCs trained in the orifice plate,as the experimental group;In the control group of six orifice vaccination BMSCs cells,only a hollow Transwell little room.Cultivate fluorescence microscope cell growth in the process of change,after 10 d is used to detect the number of CCK-8 cell proliferation,and immunohistochemical staining,cell images taken by laser confocal microscopy.The fourth part: iTRAQ quantitative analysis of protein screening of sensory nerve and motor nerve differences.Take the sciatic nerve muscle branch of the 300 kunming mice as a motor nerve,saphenous nerve as a sensory nerve,each divided into two groups,a total of four groups,do iTRAQ quantitative analysis,screening differences of the two proteins.Results:The first part: the sensory nerve transplantation of osteogenesis effect is better than that of blank group,the quantity of seed cell proliferation and survival are significantly greater than the control group.The second part: According to the results of CCK-8,sensory nerve homogenate of BMSCs proliferation promoting effect was significantly higher than motor nerve homogenate,and concentration of sensory nerve homogenate in 1 mg/ml appeared to be most effective.A high concentration of motor nerve homogenate inhibited the proliferation of BMSCs significantly.The sensory and motor nerve homogenate could not promote BMSCs differentiation to osteogenesis as shown from ALP staining.The concentration of 0.01 ~ 1 mg/ml of sensory nerve homogenate can accelerate the proliferation of BMSCs.The concentration of 1 ~ 10 mg/ml of motor nerve homogenate can inhibit the proliferation of BMSCs.The third part: spectrum experiment spectra of the communist party of China 314047,through the Mascot after analyzing software,match to the spectra of quantity is 33559 pieces,including the Unique spectra of 27693 copies,number identification to 2613 proteins,9445 peptides,including 8655 Unique peptides.Co-culture group.The fourth part: mixed BMSCs feel to neuronal migration and enrichment phenomenon.Isolation trained group,experimental group cells than the control group obviously,and the difference was statistically significant(P < 0.05).Direction differentiation ability of experimental group is less than the control group.ConclusionThe sensory nerve has to promote the effect of tissue engineering bone spicules.Its mechanism may be that sensory nerve can promote BMSCs proliferation,and suppress the osteogenetic differentiation.And the effect factors of exists in the difference of sensory nerve and motor protein.