| Part I:The study of preparation and biological characteristics of nHA/PA66composites tendon-bone interface bolt for ACL reconstructionObjective:To research and prepare a novel nHA/PA66composites tendon-bone interface bolt, and examine the structure and biological characteristics, explore the possibility to be a tissue engineering scaffold.Method:Scaffold preparation:Phase transfer method were used to prepare nHA/PA66composites. Structure observe:observe the composites appearance in general and scanning electron microscopy examine the incisal surface. Biocompatibility test:Many methods were applied to detect the biological properties of nHA/PA66composite, such as:bone marrow mesenchymal stem cells dual culture with nHA/PA66composite in vitro, systemic toxicity, intradermal stimulation and muscle implantation test etc.Results:Scanning electron microscopy showed the pore size of nHA/PA66composite materials meet the design; nHA/PA66composites did not affect the proliferation and differentiation of osteoblasts which mineralized initiation of the third generation of BMSCs;acute toxicity and intradermal stimulation were not found in experimental animals; nHA/PA66composites implanted into the muscle did not cause inflammation,and degraded gradually.Until16weeks,the composites had basically completely absorbed and replaced by bone.Conclusion:In our study, the preparation off nHA/PA66composite is simple, and the material parameters are easy to control, the composite has a three-dimensional porous structure, pore size range200μm from500μm; there is a good biocompatibility combined nHA/PA66composite with BMSCs, and favorable for adsorption and proliferation of BMSCs, It has a good osteoinductive activity in the process of bone repair; the results of systemic toxicity, intradermal stimulation and muscle implantation test show that nHA/PA66composites has good biocompatibility and osteogenic properties in vivo, nHA/PA66composites is an ideal bone tissue engineering scaffold material. Chapter II Preparation and detection of platelet-rich plasmaObjective:In this study, two-step centrifugation was used to prepare platelet-rich plasma, and detected platelet-derived growth factor. The purpose is to obtain comforming PRP and provide theoretical suppot in universal clinical application for the future.Methods:PRP preparation:10ml autologous whole blood was obtained from ear central artery in the rabbit under sterile condition, and it was sent to centrifuge treatment. The blood was divided into2layers after the first centrifugation, drawing the upper plasma layer to another centrifuge tube, and the plasma is divided into2layers after the second centrifugation, the underlying layer is PRP; Platelet count and PDGF bioactivity test were performed in whole blood and PRP.Results:10ml autologous whole blood can be prepared for1.5ml PRP; platelet concentration of whole blood is393.4x109/l; platelet concentration of the whole blood is1751.3×109/l. Platelet concentration of PRP platelets is4.46times that of whole blood; PDGF concentration measured in whole blood is34.21ng/ml; PDGF concentration measured in whole blood is142.75ng/ml. PDGF concentration in PRP is4.17times that in whole blood.Conclusion:PRP contained growth factor which contribute to tendon-bone healing after ACL reconstruction; two-step centrifugal is a simple and efficient method for preparation of PRP. Chapter Ⅲ:PRP/nHA/PA66composites on Tendon-bone healing after ACL reconstruction with autologous semitendinosus tendon in rabbitsObjective:The purpose of our study is to observe the effect on tendon-bone healing after ACL reconstruction with PRP/nHA/PA66 composites and supply theoretical foundation for applying tissue engineering technology in clinic for the future.Methods:Sixty healthy adult New Zealand white rabbits,20rabbits were randomised divided into A, B, C three groups. Tibial tunnel entrance of A group without any intervening treatment;5mmx2mmx2mm nHA/PA66composites implanted to tendon loop which tied at the distal tendon graft with suture in B group;5mmx2mmx2mm PRP/nHA/PA66composites implanted to tendon loop in C group. After2W,4W,8W.16W,5rabbits sacrificed respectively for gross observation, CT scanning and3-D reconstruction, biomechanical test and histological observation. All the properties are compred with the normal joint tissue respectively.Results:1. Biomechanical test:The maximum load was no significant difference among A,B and C group in2W(P>0.05), The variation tendency of maximum load is shown C>B> A in4W and8W(P <0.05); The variation tendency of maximum load is shown C> A and B> A in16W(P<0.05),but there is no significant difference between C and B groups (P>0.05).2.CT scanning and3-D reconstruction:The CT values was no significant difference among A,B and C group in2W(P>0.05);The variation tendency of CT values of EA(Extra-Articular, EA) were shown C>B>A in4W and8W(P<0.05);The variation tendency of CT values of EA is shown C> A and B> A in16W(P<0.05), no significant difference between C and B group(P>0.05)3. Histological observation:Apperance and maturation of chondrocyte, tidemark and insertion constructure in C group is eralier and better than in B and A group.Conclusion:Growth factors which found in PRP can enhance tendon-bone healing after ACL reconstruction; PRP/nHA/PA66composite have good biocompatibility and osteogenic activity; The detection of PDGF and BMP-2by immunohistochemistry staining is an effective method to detect bone formation. PRP/nHA/PA66composite materials can promote tendon-bone healing after ACL reconstruction, and may be is a new scaffold for ligament tissue engineering.
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