Research On The Osteogenesis Effect Of Tissue Engineered Bone Fast Constructed By Seletive Cell Retention Technology In The Goat Spine Fusion Model

Background : Bone defects induced by various kinds of causes are commonly seen in clinic. As a gold standard, autogenous bone transplantation can not satisfy the requirement for bone defects therapy because of limited bone mass and operation complications in donor site. Tissue engineered bone (TEB) constructed by bone marrow stem cells (BMSCs) and biomaterial in treating bone defects in animal studies had obtained certain effects, but the shortcomings were: BMSCs in little resource, long cycle of culture and amplification in vitro, low adhesion rate of cell to scaffold and high specification, thus limited the application in bone defect therapy. Autologous red bone marrow percutaneous injection had obtained some success in treating clinical bone nonunion and bone defect, but the method of direct injection, had the defect of partly stem cell running off. Selective cell retention (SCR) is a technique of bone marrow stem cell enrichment, the mechanism is seletive retenting stem cells and facilitating ossify factors when bone marrow pass through the adequate mesh and favourable surface adhesiveness of scaffolds. The TEB constructed by SCR were replanted immediately for treating bone defect, could obtained the same effect as autogenous bone transplantation. But due to intellectual property rights conservation and enter restriction, enrichment technique and correlated manufactures harded to applicate in our country. To generalize SCR technology in our country,our research corps had roughly sdudied the performance and the osteogenesis effect of PLL-DBM in athymic mouse subcutane,and found that the osteogenesis effect of PLL-DBM enriched bone marrow stem cell was same as that of TEB.objective : (1) To study the osteogenesis effects of tissue engineered bone fast constructed by SCR in goat spine fusion model; (2) To explore the mechanism of tissue engineered bone fast constructed by SCR;(3) To manufacture enrichment device and optimize its function parameter.Methods:⑴Demineralized bone matrix decorated with poly-L-lysine (PLL-DBM) as a new kind of bone marrow stem cells enrichment scaffold, its microstructure such asporosity and pore size were determined by scanning electron microscope, its content of calcium and phosphonium were detected by spectrum analysis,the combination condition of PLL with DBM and the content of major immunogenicity amino acids were determined by component analysis of amino acids,and detected its biomechanic property by biomechanic test.⑵Fast constructed tissue engineered bone by enriching bone marrow stem cells with Seletive Cell Retention Technology ,the osteogenic effects of the following four groups grafts were evaluated in goat lumbar intertransverse fusion model: groupⅠA : TEB fabricated by PLL-DBM enriched bone marrow; groupⅠB:autogenous iliac bone; groupⅡC : DBM soaked with bone marrow; groupⅡD:blank DBM. At 8,16 weeks after operation, goats were sacrificed respectively, and fused segments were sampled for X-ray, three-dimensional CT and CT value, histologic analysis and biomechanical test were done to compare and evaluate their osteogenic ability.⑶New enrichment scaffold were handled by SCR, the enriching effects of PLL-DBM to fibroblast colony forming unit ( CFU-F) were observed before and after enrichment to determine the enriching effects of PLL-DBM for bone marrow stem cells, contents of PDGF and IGF-I in bone marrow supernatant were detected by ELISA method before and after enrichment.⑷TEB were constructed quickly after bone marrow enrichment by SCR,than removed the cells in TEB by freeze-drying keeping 36h to become freeze-TEB(FTEB). the PLL-DBM combinated with bone marrow by SCR (SCR group), the freeze-TEB (FTEB group), and DBM (DBM group) were implantated into subcutaneous area of nude mouse, drow the materials at 4, 8 days to observe cells recruitment ;drow the materials at 4, 8, 12 weeks, image density, histological changes of bone graft area and ossify effects were determined by X ray, CT scanning and HE staining.⑸designed and manufactured enriching device ,and detected the enriching effects of PLL-DBM for NCs to determine the optimization function parameter including circulation times and flow rate of bone marrow.Results:⑴Steady ivory converage were formed at exterior and interior surface by PLL. The porosity of PLL-DBM is (70±6)%, the pore size is (472.5l±7.02)μm. there are many micropore about 100μm size among pores, a great quantity of pore cross-connected each other in the interior pore, and formed smaller, more steady mesh structure in the natural pore and spider-web pattern; spectrum analysis showed that DBM and PLL-DBM had no conspicuous wave crest of calcium and phosphonium; component analysis of amino acids showed that PLL-DBM and DBM had contained more collagen amino acids (GlY, Arg,Lys),but few major immunogenicity amino acids(Trp,TYr,CYs),and PLL-DBM had conspicuous Lys wave crest compared to DBM ; biomechanic property showed that,when scaffold had compressed by 60%,the maximum load and bending strength of DBM significantly superior to that of PLL-DBM(P=0.022, P=0.012),but the elastic modulus had no significantly difference(P=0.225).⑵X ray showed that ,at 8 weeks after operation, the fusion range ofⅠA was significantly less than those ofⅠB , only the segment around the inner side of vertebra fused largerly ,but significantly superior to that ofⅡC , there basically were no fusion inⅡD ; at 16 weeks after operation, the fusion range ofⅠA andⅠB were basically similar, all fused completely, and the bone density was basically same as the density of transverse processes; however, the fusion range ofⅡC was significantly less than those ofⅠA andⅠB, only the segment around the inner side of vertebra fused partly inⅡC; there basically were no fusion inⅡD , only there were island-like osteogenic chips between the transverse processes . At 8 weeks,the score of X ray ofⅠA was 7.17±1.17, significantly less to 9.00±1.10 ofⅠB(n=6,P<0.05),but significantly superior to that ofⅡC andⅡD, by 16 weeks after operation, the score of X ray ofⅠA andⅠB had no significantly difference(n=6,P>0.05), but significantly superior to that ofⅡC andⅡD . At 16 weeks after operation, CT value ofⅠA andⅠB group showed no significant difference (P> 0.05), significantly superior to that ofⅡC (n=6,P <0.01), but CT value ofⅡC was significantly superior to that ofⅡD . Histological score was same as the result of X ray .At 16 weeks, biomechanical test on the fusion segments showed that the maximum load and bending strength inⅠA group had no significant difference with those inⅠB group (n=6,P> 0.05); those inⅠA group were higher than those inⅡC Group (n=6,P <0.01,P <0.05), and those inⅠB group were higher than those inⅡC Group (n=6,P <0.01), which was significantly higher than those inⅡD Group (n=6,P <0.01).⑶The concentration enrichment multiple rate of PLL-DBM for CFU-F was 5.68±0.49, but that of DBM was only 2.23±0.27,the difference between PLL-DBM and DBM was significant(n=4,P<0.01), concentration enrichment multiple rate of PDGF was 13.624±1.251,concentration enrichment multiple rate of IGF-I was 36.311±5.562 in the enrichment scaffold. (4) The results of boneformation into subcutaneous area of nude mouse show that much of CNs were recruited into the scaffold of SCRgroup and FTEBgroup at 4th day,by 8th day much more CNs were recruited into the scaffold,but few CNs into the he scaffold of DBM group.X ray showed that ,at 4 weeks,there was no significant bone developing all groups, the density of each composite implantation increase gradually followed the time, and had significant difference at each time point when compared with single DBM; the density in SCR group was similar to that in FTEB group, but was superior to DBM group. At 12 weeks , CT value of SCRgroup was (687.67±18.55)HU, CT value of FTEBgroup was (674.33±12.21)HU,and that of DBM group was (57.88±5.47)HU. HE staining showed that the osteogenesis effect of FTEBgroup was similar to that of SCRgroup,but was superior to DBM group. PLL-DBM scaffold degradated gradually as time passed, bone trabecula and small vessels formed gradually into the scaffold;rigid bone tissue were seen at 12weeks, and medullary tissue and vessels were seen in some region of implantation site in SCRgroup and FTEBgroup,the scaffold of DBM group was degradated basicly. (5)For the enriching device designed and manufactured ourselves, the optimization combination function parameter was circulation times 4 times/ flow rate of bone marrow (100ml/3min),no good enriching effect in more circulation times and slower flow rate of bone marrow.Conclusion:⑴Allochthonous goat's PLL-DBM possess three dimensional space as autogenous bone and PLL for cell adhesion, and have favourable cellular compatibility, low immunogenicity, histocompatibility and biodegradability, can promote the adhesion and proliferation of MSCs, is an ideal enrichment scaffold for bone marrow stem cells.⑵TEB constructed fast by SCR have favourable bone formation, and its osteogenesis effects in goat intertransverse fusion model is similar to that of autogeneic iliac bone.⑶Enrichment scaffold can increase the concentration enrichment multiple rate of CFU-F significantly, and increase the contents of PDGF and IGF-I in TEB.(4) The growth factors enriched in enrichment scaffold can recruit the cells and the growth factors around the region of implantation site,which maybe the main role of the mechanism of high ossify activity of TEB constructed fast by SCR.(5)The enriching device designed and manufactured ourselves is easy to hand and control,and has complete function.The optimization combination function parameter was circulation times 4 times/ flow rate of bone marrow (100ml/3min).