An Experimental Study On Variant Deproteinized Osteo-Articulation Combined With Calcium Phosphate Cement And RhVEGF+rhBMP-2 To Repair Osteoarticular Defect

Osteo-articular defect caused by external injury,infection and tumor is a great challenge faced with by orthopaedic surgeons. Osteo-articular transplantation with autos blood vessel inosculation has the best effect,but the resource of donor site is very limited,technical difficulty is great and the donor sites have many complications.So it is only adapted to repairing tiny section osteo-articular defect and important osteoarticular defect of hand.Other prosthesises, such as metals, polymers and ceramics,can be used to repair osteoarticular defect but they have some common issues:easily fracture, subsidence, fatigue fracture rebuilding requirement and so on. Osteo-articular allograft has never developed generally mainly because the immunological rejection issue is hard to overcome.The research on natural bio-derivating osteoarticular materials possessing biological activity is the important topic of the world tissue engineering realm at present.It is a revolution in the research realm of biomaterial that we can develop a bioprosthesis ,which can heal with acceptor and survive.It can also make many meromelia patients of osteoarticular defect return to society and benefit mankind.Recently,many researchs have discovered that deproteinized osteo-articulation (DPOA) is composed of inorganic bone mineralizer and bone collagen,whose immunogenicity is fairly low.It has the close-up pore- formatic reticularis system of natural bone tissue,which profits the input of new vessels and the immigration of mesenchymal cell,promotes the osteoblastic differentiation and the extracellular matrix synthesis,is easily gotten close and removed by the osteoclasts of acceptor,then induces bone formation.It is the effective frame adhered by osteogenesis cells.At the same time, although DPOA underwent a series of biological chemical treatment such as deproteinization and defatting, the researchs discovered the articular facet of the material was still smooth and glossy,and the material had the same shape structure with the osteoarticular of hosts, better mechanical strength and biomechanics property.It can be used as osteoarticular allograft.But the long and bulk DPOA is short of the capacity of bone induction,and the blood supply resource only origins from one extremity touching with hosts.Blood vessels grow into the material from proximate host bone,and the process is undoubtedly very slow.Thus it is a very necessary means to assist bone induction and vascularization growth factor to DPOA material.So we apply the most effective growth factor for bone induction and vascularization, vascular endothelial growth factor (VEGF) and bone morphogenetic protein(BMP-2).Moreover we make calcium phosphate cement(CPC) as the slow-release carrier for the above-mentioned growth factor,thereby achieve local persistent homo-density releasing of the growth factor,make the material"activate",heal with host bone during fairly short time ,become autologous tissue and repair osteo-articular defect at last. Etio-experiment unertakes research from 3 aspects,and the main means,results and conclusions as follow:1.The material repairing and reconstructing osteo-articular defect-the manufacture and correlation detection of DPOAWe made inferior segments of fresh adult mixtus dogs'femur to bio-derivate osteo-articular materials—DPOA by hydrogen peroxide- diethyl ether means.We certified that the protein and lipid component of DPOA made by the means had been basi-removed,which lowered or eliminated the immunogenicity ,through macro-style observation, scanning electron microscope observation, inverted phase contrast microscope observation and multi-item physical chemistry detections.Furthermore,the materials have the similar anatomy outline form and three dimensional pore-wire frame structure with primitive bone tissue.So it is an ideal osteoarticular allograft material.2.The experiment on CPC releasing rhVEGF and rhBMP-2 in vitro as carrierBecause of the features of CPC such as degradation,none heat production,we dissolved rhVEGF and rhBMP-2 with curing juice of CPC,then agitated it with CPC powder thoroughly and made CPC cure under ordinary temperature,which become CPC as the carrier of rhVEGF and rhBMP-2.We discovered the addition of above-mentioned growth factor did not influenced the curing and inverting into HA crystal of CPC,and there were not biological response between the growth factor and CPC,by means of X-ray diffraction analysis.We discovered rhVEGF had the similar releasing dynamics rule in vitro with rhBMP-2 through detection of high performance liquid chromatogram analysator.The growth factor explosingly released in about 24 hours,the released amount was about 13%;tacho-released from the first day to the seventh day, the released amount alined with time;the releasing speed became slow during the seven day and the eighth week.It certified the feasibility making CPC as the slow-release carrier of rhVEGF and rhBMP-2.3. An experimental study on variant deproteinized osteoarticulation combined with calcium phosphate cement and rhVEGF+rhBMP-2 to repair osteoarticular defect36 adult mixtus dogs were randomly divided into 3 groups:group A,group B,group C.Group A: CPC /rhVEGF/rhBMP-2/ DPOA transplanted group,CPC combined with rhVEGF+rhBMP-2 was well-distributedly coated on DPOA and partly plugged into the medullary cavity.Group B: rhVEGF/rhBMP-2/ DPOA transplanted group, rhVEGF+rhBMP-2 was directly dripped into DPOA.Group C:simple DPOA transplanted group.The correlation detections about bone formation, blood supply reconstruction, immunological rejection, vitodynamics test and so on,were made at post-operative corresponding time spots.We made X-ray observation,HE staining and immunohistochemistry analysis of collagen I, transmission electron microscope test, vas capillare analysis by ink perfusion, arterial perfusion blood flow observation of knee joint by ECT, vitodynamics test and T lymphocyte subpopulation analysis by flow cytometry,These tests displayed that the callus formation and union between the material and host bone in gtoup A were both better than those in group B and C,moreover there was the formation of chondrocyte clump at distal end of joint in group A;the vascularization and blood flow in group A were obviously more than those in group B and C;the early immunological rejection in group A was obviously lower than that in group B and C,but there was no significant difference in later immunological rejection in 3 groups.Summing up, CPC /rhVEGF/rhBMP-2/ DPOA transplantation utilized CPC as slow-release carrier of rhVEGF+rhBMP-2 to release rhVEGF and rhBMP-2 locally, persistently and effectively,thus induced the formation of bone and chondromatous,promoted the early re-vascularization of grafts,spelled affluent target cells and growth factor for bone union,created best microenvironment for bone formation,made grafts gradually"activate",achieved early bone union between grafts and host bone and made grafts become autologous tissue of accepters.It offered an effective therapeutic tool for repairation of osteoarticular defect.