| Background & ObjectivesHow to promote the healing of bone defect has been an important issue in the research field of bone repair. At present, the focus on the healing of bone defect is how to promote and strengthen the creeping substitution in bone and formation of new bone by using a variety of growth factors. Platelet-rich plasma (PRP) is platelet concentrates got by concentrating autologous whole blood. The platelet concentration in PRP is at least 4 times more than that in the whole blood. PRP contains high concentrations of various growth factors, including ADP, ATP, Angiopoietin-2, Tissue-Activating Peptide-Ⅲ, Platelet-derived growth factor (PDGF), Transforming growth factor-P (TGF-(3), Vascular endo-thelial growth factor (VEGF), Insulinlike growth factor (IGF), Epidermal growth factor (EGF) and etc. These factors have the functions of accelerating the healing and regeneration of injury tissue. The preparation of PRP is cheap and simple. Autologous blood has neither toxicity nor immunogenicity. The growth factors released by PRP are in accordance with the ratio of factors from autologous blood. Therefore it is regarded as an ideal source of growth factors for clinical treatment of bone defect. Bone morphogenetic protein (BMP) can induce undifferentiated mesenchymal cells differentiating into Osteoblast and enhance the growth and proliferation of bone cell. It can also enhance the synthesis and calcification of collagen so as to form new bone and promote ectopic bone formation or bone formation in situ. The joint effect of several growth factors released by activated PRP can obviously promote the healing and remodeling on bone defect. We make use of histological observation and computer image analysis to observe the influence of PRP and BMP on bone defect, providing a theroy basis for clinical applications in bone defect and repair.Materials and Methods1 Animal grouping and model preparation 48 healthy New Zealand white rabbits were randomly divided into A, B, C and D group. Every group contained 12 New Zealand white rabbits. The radial bone defects of rabbits in group A were implanted with the compound tissue engineering bone (PRP+FG+bBMP). B and C group was respectively implanted with compound materials (PRP+FG) and (bBMP+FG). According to the size of bone defects. we implanted about 2.0 ml these compound materials. After that, we sutured the incision and injected penicillin 400000u/d for 3 days.2 The preparation of PRP The whole process of PRP preparation was strictly sterile.0.03g/L sodium pentobarbital was injected into the ear vein of rabbits for anesthesia. Then we extracted 5ml autologous blood from the rabbit central ear artery and put it into 10ml centrifuge tubes according to the Landeaserg PRP extraction method.3 Construction of tissue engineered bone 48mg bBMP was immersed into distilled water and then put it into 4℃refrigerator overnight. Then bBMP was ground into powder and was mixed with PRP and FG. We put coagulant (1ml mixture of 10% calcium chloride and 1000u thrombin) into container which contained this complex. According to 1ml:200u PRP/bBMP, we made the mixture of PRP, bBMP and FG into gel, which was implanted into the radial defects in rabbits. The production and application of the PRP compound gel should be finished within 40 minutes.4 General observation Animal diet, mental state, wound healing, changes of implants volume and the extent in which the implants had the tightness with the surrounding tissue and changes in implant surface and whether it had inflammatory response, etc.5 Histological observation We took the central tissue of bone defects after 4,8 and 12 weeks and then observed its bone formation through HE slice.6 X-ray observation After 4,8 and 12 weeks, the two fore limbs of rabbits were X-rayed to observe the repair on radius'defect.7 Computer image analysis Two HE sections were selected from each specimen. Four visions were taken from each section. Then we used QWIN image analysis systems to compare area percentage of new bone formation among the three groups.8 Statistical Methods The statistical software SSPS16.0 for windows was exploited to analyis data. All the data were expressed by (x±s). Deviation and difference were compared using the one way ANOVA and LSD test between each group in every time point. The significant difference was judged by P<0.05.Results1 General situation The rabbit's consciousness, diet, action, hair color, urine and stool was normal. There was no swelling, bleeding and purulent secretion on the incision of rabbits. The wound healed within six days.2 Histological observation 4 weeks later, In group A, we could see there was affluent neovascularization on bone defects and more fibrous callus formation. Osteoblasts and chondrocytes were active in proliferation. In group B and C, we could see fibrous tissue structure on bone defects in which there was various amount of osteoblasts and chondrocytes. In group D, we could see fibrous tissue structure on bone defects surrounded by small amount of inflammatory cells and osteoblasts. The osteogenesis in group A was better than that in group B and C.8 weeks later, In group A, abundant bone matrix and new bone formation on radius'defects and the number of cartilage cells decreased. The new-formed bone interweaved on radius'defects of rabbits in group B and C. The Bone matrix and calcium deposition in group B and C was not as good as that in group A.12 weeks later, in group A, mature bone structure was mainly lamellar bone and small amount of scattered woven bone. The bone marrow cavity is completely re-passed. In group B, There was more woven bone, osteoblasts, osteoclasts and a small amount of myeloid tissue could be seen. In group C, Bone maturation was not good and we could see blood cells in the marrow cavity. In group D, we could see that the radius'defect was still filled with fibrous tissue and small amount of bone matrix and new bone.3 X-ray observation Group A:4 weeks after operation, we could see that blurred shadow was patchy in distribution on the radius'defects. The two ends of fracture was clear and had highter density.8 weeks after operation, we could see continuous high-density shadow and callus formation on the radius'defects, which formed a bridge between the two ends.12 weeks after operation, we could see a uniform shadow of high density on the radius'defects. The bone marrow cavity was re-passed, with the cortex bone shell of long bone continued. Group B:4 weeks later, we could see continuous flocculent blurred shadow on the radius'defects.8 weeks later, we could see continuous high-density shadow on the radius'defects and the callus formation in group B was less than that in group A.12 weeks later, the radius'defects in rabbits was nearly repaired and the bone marrow cavity was mostly re-passed. Group C:4 weeks later, we could see that there was low-density callus formation on the radius'defects and the two ends of fracture.8 and 12 weeks after operation, we could see that the radius'defects were filled partly with new bone and the bone marrow cavity was partly re-passed. Group D:4 weeks after surgery, there was no obvious new bone formation and the two ends of bone defects were very clear.8 and 12 weeks later, Marrow cavity was partly closed. The bone defects didn't be repaired.4 Computer image analysis Over time, new bone was gradually on the increase and the compound bio-material was gradually decomposed.4,8 and 12 weeks after surgery, area ratio of newly-formed bone on the surface area of radius defects in group A was significantly higher than that in group B, C and D. Area ratio of newly-formed bone on the radius in group B was significantly higher than that on the radius in group C from 8 to 12 weeks after surgery.Conclusions1 Both PRP and BMP have an effect on improving the generation and repair of radius'defects in rabbits, but the former is better than the latter in the respects of forming new bone.2 The tissue engineering material containing PRP and bBMP have a better effect on the bone generation and maturity of radius'defects in rabbits than tissue engineering material only containing PRP and bBMP.3 The tissue engineering material containing PRP, FG and bBMP has a high value of clinical application owing to its simple preparation, obvious osteogenic effects and non-immunogenicity,. |
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