The Effect Of The Local Denervation Of Sympathetic On Vascular Wall On The Bone Tissue Engineering Osteogenesis In Vivo

In orthopaedic clinical work, critical-sized bone defect and bone nonunion,caused by many factors, is still a worldwide problem in orthopaedic surgeons. At present autologous bone graft, allograft and artificial bone filling, etc, are commonly used in clinical treatment. But, these methods have a lot of defects such as limited donor sites,immunogenicity and lack of biological activity,which limit the clinical application of these methods. The emergence of tissue engineering bone can solve this problem. And the vascularization and neuralization of bone tissue engineering are critical to make sure the clinical transformation of tissue engineering bone. So it is urgent to study the mechanism of vascularization and neuralization in the repairing process of tissue engineering bone and to find the best way to achievevascularization and neuralization of tissue engineering bone.Previous studies found that vascular bundle and sensory nerve bundle implanted in bone tissue engineering has the same effect on its neuralization. However, the vascular bundle used in the experiments contains a lot of sympathetic nerveswhich distribute in vascular adventitial. Whether it is the vascular itself which promotes the neutralization or it is the function of sympathetic nerveon the vascular adventitia? How do blood vessels and nerves play a role in the process of promoting tissue engineering bone? Do they have effectson each other? These scientific problems is still unknown. Therefore,the first part of this study trys to establish a sympathetic denervation model of local vascular wall, then local sympathetic denervation vascular and normal blood vessels were implanted in tissue engineering bone. Then we observe the effects of these two factors on bone formation,vascularization and neuralization of tissue engineering bone. And clarify the roles of blood vessels and nerves and their interaction in the process of bone tissue engineering repairing large segmental bone defects.Part 1 A study on constructing a rabbit model of local sympathetic denervation of femoral artery by microsurgery method Objective:To study the feasibility of microsurgical technique to denervate sympathetic of femoral artery in rabbit, providing a reliable animal experimental model for further study of the mechanism of neuralization in bone tissue engineering.Methods:21 New Zealand white rabbits were randomly divided into 4 groups: A the control group(n=3), B the 4 weeks group(n=6), C the 8 weeks group(n=6) and the 12 weeks group(n=6). Bilateral femoral arteries of the 21 rabbits were exposed. Adventitia of femoral arteries in 3 test groups were removed for about 2cm by microsurgical technique,whereas adventitia of the control group remained intact without any treatment. The arteries samples were collected at 4 weeks,8 weeks and 12 weeks after treatment. The structure of vascular were indicated by hematoxylin-eosin(HE) staining, and the distribution and volume of the sympathetic fibers were evidenced by glyoxylic acid8 staining and the expression of tyrosine hydroxylase(TH),the marked protein ofsympathetic.Results:The adventitia of 3 test groups were invisible or lost most of it while the control group remained intact shown by HE staining. For glyoxylic acid staining, the fluorescence intensity value 0f the control group, 4 weeks group, 8 weeks and 12 weeks was0.08124±0.00260,0.02920±0.00206,0.02661±0.00233,0.03094±0.00211,n=6respectively. The distribution and fluorescence intensity of sympathetic nerve were both significantly reduced in test groups compared to the control group(P<0.05). And no statistical difference was found among the 3 test groups(P>0.05). Semi-quantitative analysis of the expression of TH was 0.8626±0.03519 ? 0.3631±0.03019 ?0.3964±0.02239?0.3487±0.02356 respectively,n=6, which showed the same tendency as glyoxylic acid staining test.Conclusion:Microsurgical technique is promising as an ideal method for the local denervation of sympathetic nerve from artery system as it can significantly reduce sympathetic fibers on adventitia without regeneration during the experimental period.Part 2 Construction of tissue engineering bone in vitro Objective:To culture the seed cells BMSC on the TCP porous biological ceramic scaffold materials, so that the seed cells BMSC can survive and grow on the scaffold material, and ensure the experiment in vivo can be carried out smoothly.Methods:Extract fetal rabbit primary BMSC and cultured to P3.Inoculate the BMSC on the beta TCP porous bioceramic scaffold material at the density of 10^7/ml beta TCP porous bioceramic scaffold material, the material is a cuboid. The height of it is 15 mm and the bottom of the surface is a 4mm X4 mm square. In order to eliminate the gas in the material,we use the negative pressure suction method so that the cell suspension can fullyget into the internal pore structure, and then put the composite into culture. After 5 days of culture, the samples were fixed with glutaraldehyde and then they were observed by scanning electron microscopy to confirm the adhesion and growth condition in the material of BMSC.Results:The growth of BMSC taken from fetal rabbits was good, and the primary cells adhered to the dish bottom on a monolayer, presented a long spindle shape or a spiral shape. After 5 days of culture, BMSC were attached to the surface of TCP material, and the growth of the seed cells were in good condition according to the the result of SEM. A large number of BMSC were attached to the inner surface of the TCP which can be observered after incision, and the cells were also in good condition.Conclusion:BMSC can attach to the TCP porous ceramic scaffold materials, and the growth state is good, so it can be used to repair large segmental bone defects in rabbit bone defect.Part 3 the effect of the local denervation of sympathetic on vascular wall on the bone tissue engineering osteogenesis in vivo Objective:To explore the effect of the local denervation of sympathetic on vascular wall on the bone tissue engineering osteogenesis in vivo.Methods:10 rabbits were randomly divided into 3 groups. group A contains 2 rabbits, group B4 and group C 4. Rabbits in group A were implanted in tissue engineering bone alone,group B: tissue engineering bone and vascular bundle with its adventitia removed and group C : tissue engineering bone and common vascular bundle. After implantating tissue engineering bone into the rabbit radial segmental bone defects for 12 weeks, gross observation, X-ray, micro CT, tetracycline and calcein fluorescent test and VG staining were carried out to detecte the osteogenic effect. Compare their different effects on tissue engineering bone osteogenesis.10Result:Gross observation In group B(transplanting vascular without adventitia) and group C(transplanting common vascular),the broken ends of the bone defects healed. The scaffold materials of bone tissue engineering were not visible on the surface of defect; while bone tissue engineering scaffold material in the control group(transplanting tissue engineering bone alone) was still clearly visible on the surface. The healing of bone defect was poor.X-ray Compared with the control group, the X-ray examination of the vascular transplantation group and adventitia removed group showed that material shadow had become vague and even recanalization of bone marrow cavity could be observed, but no visible differences were found between these two groups; while in the control group,?-TCP material shadow is still clear and can easily identify the profile of the material.Micro CT three-dimensional reconstruction the area of bone region in group B(transplanting vascular without adventitia) and group C(transplanting common vascular)was much larger than that of the control group; no statistical difference was found between group B and group C. The residues of material area in group B and group C was obviously smaller than that of the control group(p<0.05). Similarly, no statistical difference was found between group B and group C(p>0.05).Tetracycline and calcein fluorescence double labeling experiments: from three groups of mineral deposition rate MAR(mineral deposition rate) comparison chart it can be seen that the rate of group B(transplanting vascular without adventitia) and group C(transplanting common vascular) is higher than that of group A(control group)(P < 0.05),but no statistical difference was found between group B and group C(p>0.05).VG staining From VG staining of the photos it can be seen that the area of red bone-forming region in group B and group C is much larger than that of group A. And no visible difference was found between group B and group C.Conclusion:Transplanting common vascular bundle and vascular bundle without adventitia for 12 weeks can both improve tissue engineering bone osteogenesis significantly in vivocompared to tissue engineering bone alone. And there is no statistical difference between transplanting common vascular bundle and vascular bundle without adventitia after transplanting for 12 weeks.