Experimental Study On Bone Repairing Segment Tibia Defects Of Swine With Allogeneic Tissue Engineering Bone

Caused by severe trauma, tumor resection and so on, large segment bone defects were always tough problem perplexing orthopedics doctor, treated mainly with autogenous bone transplantation, allogeneic or xenogenic bone grafts, biomaterial replacement and other approaches. Autogenous bone grafting is the gold standard and the most effective methods for treating osseous defects, but autologous bone grafts often don`t satisfy the amount of bone tissue needed for reconstruction and would bring patients new wound, the other ways have also no satisfied effect. In recent years tissue engineering bone brings new hope for bone defect repairing, it applied the principles of engineering and the life sciences to develop biological functional artifical bone, which involves three main elements: seed cell, scaffold and tissue bone construction technique. Mesenchyma stem cells (MSCs) are the most commonly used and ideal seeds cell. According to the gene origin of MSCs there has the classification of autologous and allogenic tissue engineering bone, and the research currently focus on autologous tissue engineering bone which has manifestated good ability of bone formation and has repaired bone defect in animal experiment. In the clinical trial it also has experimented successfully and obtained better effect in repairing bone defect. But it has some shortage such as no pre-construction, long waiting time of common 3~4 weeks and so on, which limit the clinical application and the industrial production and that would be overcomed by tissue engineering bone constructed with allogenic MSCs which would be able to satisfy the clinical application aim of "with take with use".The immunogenicity and ossification ability of allogenic tissue engineering bone are the key of applicating in the clinic.Its immunogenicity mainly concerns with the seed cell. Reseach had demonstrated MSCs only expresses MHC-I and don`t express MHC-II, Fas/FasL, CD80, CD86, CD40 and CD40L. In vitro experiment: the mixed lymphocyte culture (MLC) demonstrated that MSCs did`t cause the allogenic lymphocyte to be activated and proliferate; In vivo experiment: allogenic MSCs can exist for long time and don`t be distinguished and eliminate. Therefore it suggest MSCs has low or even no immunogenicity. it was also manifested MSCs has the immunosuppression function and may induce the immunity tolerance. Even after osteogenic induction the immunogenicity of MSCs didn`t become more strengthen.there are few reports about allogenic tissue engineering bone. Research on it`s immunogenicity were rare. It (De Kok et al) confirmed no immunity reject response through examination of the circulating antibody and inspected bone defect better healing in the dog experiment applicating allogenic tissue engineering bone to repair dog tooth socket bone defect without immunoinhibitor. But Hiroyuki et al constructed tissue engineering bone with allogeneic MSCs to repair bone defect of rat and dicovered that in the experiment group treated with immunoinhibitor bone defect had been repaired, but not using immunoinhibitor the contrast group hadn`t repaired bone defect, it demonstrated that the ossification ability of allogenic tissue engineering bone has closely correlation with its immunogenicity. At present only small animals such as rat been studied on allogenic tissue engineering bone, there has no same conclusion for immunogenicity through peripheral humoral immunity inspection and the contrast study of treatment with immunoinhibitor after transplantation. it is still confused whether reject response exist and how is the degree of reject after transplantation of allogenic tissue engineering bone, Therefore this experiment plans to study the immunogenicity and ossification ability of allogenic tissue engineering bone in big animal. Swine were similar to human in organ and immunity system and were seleced as experiment animal. The Guizhou mini-pig and the Guangxi Pama mini-pig were two different kinds Inbred strain animals selected respectively as the donater and the acceptor in experiment, establishing mid-segment 2cm defect model of mini-pig shinbone, discussing the separation and purification of small pig marrow MSCs, constructing tissue engineering bone with osteoinducted MSCs from donater seeding in scaffold in vitro. According to its transplant characteristic, to monitor reject response after transplantation by flow cytometry, ELISA, RT-PCR and so on, observing the repair of defect through X- line inspection, tissue special staining, biological mechanics test .The main results and conclusions are as follows:1. The result of CDC presented strong masculine between the two strains of the Guizhou mini-pig and the Guangxi Pama mini-pig and showed negative inside the strain; The SI was more than 3 by mixing in 3H-TdR after MLC between the two strains,which far smaller than 3 in the strain. It meaned the two kinds of strains do not match and inside the strain has good identity, also manifested the possibility of reject would be great after transplantation between the two strain mini-pigs. It concluded that the Guizhou mini-pig and the Guangxi Pama mini-pig are able to satisfy experimental require.2. Fixed with plate, the mid-segment 2cm defect model were setted up, which were not able to naturely heal after 12 weeks through X-line inspection, observation of the roughly specimen and tissue dyed witn HE. Therefore the segment tibia 2cm bone defect model of mini-pig is fit for the goals of experiment.3. Comparing with Ficoll, Percoll associating with adherence may obtain MSCs with good cell vigor, purity and ossification activity from mini-pig bone marrow, it demonstrated Percoll is more suitable to separate and purify MSCs from mini-pig marrow than Ficoll, To identify matched the attribute of MSCs.4. The immunogenicity of Mini-pig MSCs is low and that has no significant changes in different time after osteogenic induction. Porousβ-TCP was selected as scaffold to construct tissue bone, The rate seeding in scaffold of MSCs was 63±14% by precipitation repeatedly, Under electron microscope seed cell adhered and extended full in the scaffold surface and hole.β-TCP didn`t influence the biological performance of seed cell. The masculine MSCs is brown by BrdU marking, which achieved good mark in 10 umol/L and 48 h hatching.5. The experiment mini-pig were randomly divided into A, B, C and D group, representing respectively allogenic tissue engineering bone group, autogenous tissue engineering bone group, pure TCP groupand blank defect group. Postoperative immunology monitoration results are as follows:CD4+, CD8+ and CD4+CD8+ double positive T cell of peripheral blood each group didn`t have significant changes in different Postoperative time, It was no significant changes that A group compared with other groups in the same time; The early time lymphocyte transformation experiment displayed the SI of each experimental group was more small than 2, between groups no significant deviation; In postoperative one MLC of A group the donater osteoinducted MSCs hadn`t made accepter lymphocyte proliferate,the cytotoxicity of T lymphocyte was low in A group; By ELISA the early peripheral serum IL-2 level has gradually elevated in each experimental group and that of A, B and C group having elevated obviously in 3 days(P<0.05) and there was no remarkable deviation A group compared to B and C group, starting to drop when 1 week; as well as the early IL-10 level of each experiment group mildly elevated, but not remarkable, gradually recovered normally after 2 weeks, no significant difference between groups; Through RT-PCR the mRNA expression of IL-2 and IL-10 in the early time were up-regulated in A, B and C group,but it was no significant difference between D and C group in the same time; The early times engineering bone tissue dyeing with HE were seen a few inflammatory cells infiltrating in A, B, C group after operation,as well as rare lymphocyte and monocyte infiltrating. By the immunohistoche mistry techniques, it also found the CD4+, CD8+ lymphocyte exsist in early time and there was no obviously deviation in C and D group. There hadn`t some deposition of IgG and C3 in C and D group; And in A group and B group little of IgG compound deposited in the crevice of scaffold, and C3 depositing hardly; The tissue has many newborn bone cell marking BrdU positive in A group after 8 weeks; Conclusion: there had mild inflammation in early time and reject response were not monitored after transplantation of allogenic tissue engineering bone, allogeneic MSCs exsisted always and formed the newbone. It suggested that the immunogenicity of allogenic tissue engineering bone is no or low.6. Mini-pigs with 2 cm shinebone defect were divided into A (allogenous group), B (autogenic group) and C group (pure TCP group) according to the treatment methods, then the osteogenesis and the repairing ability of each group were detected. The result is as follows: the X-line inspection demonstrated plate screw fixed good, scaffold degraded and replaced gradually by newbone and defect bridged; The same time X grading of A group was obviously higher than C group (P<0.05), no remarkably distinguishes from B group. The mRNA expression ofⅠtype collagen and osteocalcin increased gradually, significant stronger than C group (P<0.05) and obvious no difference contrast to B group. Appling HE dyeing to check the artificial bone tissue, it found thatβ-TCP degraded, meanwhile new bone and blood vessel formed gradually, finally replaced by mature bone tissue in postoperative 16 weeks. The histology grade of A group of was obviously higher than that of C group(P<0.05), and no remarkable denivation compare with B group.As well as the sirius red dyeing of tissue deteced that in A group I collagen producted greatly, obvious more than C group (P<0.05),and there was no differences between A and B group. In postoperative 16 weeks the vascularizing level of bone tissue in A and B group is good, difference in C group of. At the same time inspecting the bone density of new bone, it found that the bone density of A and B group are close to the normal,low of C group. there was statistics significance A group comparing with C group.Biological mechanics tests suggested that A group surpassed C group. Conclusion:allogenic tissue engineering bone had a good ossification ability and were able to repair the critical bone defect. on the other hand it would infer that it has no or low immunogenicity which do not influence ossification activity.CONCLUSIONThe monitoring hadn`t displaied exsistence of rejection After transplantation of allogenic tissue engineering bone ,which had successfully repaired the bone defect and has same curative effect compareing autogenous tissue engineering bone. It suggested allogenic tissue engineering bone has low or no immunogenicity and good osteogenesis activity. This study produced an experiment biasis for allogenic tissue engineering bone entering into clinical application.