Microvascularized Biological Bone Constructed With Autologous Pheriphral Blood Derived EPCs And BMSCs Seeding On PDPBB

[Objective and background] Bone defect is a disease with serious appearance and function damage. China is a country with a large population, In china, there are over3million patients with the bone defect of cranium, mandibula and limb caused by disease, athletic injury, trauma, traffic accident and natural calamity, and this number are increasing10%each year with population aging. Recently, as the explosion of the research of tissue engineering, engineering bone has already began to applied to reconstruct the bone defect. But the failure of engineering bone transplantation which induced the necrosis and absorption happened frequently for insufficient angiogenesis and rejection, and the sphacelus may harm the organism in vivo. These shortcomings severely hold back the clinic research and application of engineering bone. To construct an engineering bone with good histocompatibility, low rejection and good vascularization is a keypoint for bone defect repairment with engineering bone application in clinic. In this research, an co-culture system constructed with autoallergic peripheral blood derived endothelial progenitor cells (EPCs) and autoallergic bone marrow stem cells were seeded onto partially deproteinised biologic bone, to construct an autoallergic biological engineering bone. The efficiency of BMSCs on the angiogenesis of peripheral blood EPCs, and the EPCs on the osteogenesis of BMSCs were dynamic monitored both in vitro and in vivo. This research will provide a new method of constructing a new autoallergic engineering bone with no rejection and good vascularization,[Methods](1) Endothelial progenitor cells (EPCs) were isolated from rabbit peripheral blood and cultured in Endothelial cell medium (EGM). The expression of CD34, CD133, vWF on the third generation adherent cells were detected with immunofluorescent staining;(2) Bone marrow stem cells(BMSCs) were isolated from rabbit bone marrow blood and cultured in medium added1%fetal bovine serum. The expression of CD29, CD34, CD90on the third generation adherent cells were detected with immunofluorescent staining;(3) EPCs and BMSCs were co-cultured with the ratio of EPCs,2:1,1:2,1:1and BMSCs, the morphology change of cells were observed with inverted microscope, and the proliferation of cells in each group were evaluated by Wst-1;(4) Alkaline phosphatase (ALP) and Von Kossa staining of the cells in each group were performed on day3,7and14, the content of ALP and osteocalcin(OC) of the cells in each group were detected on day3,7and14with ALP Kit or OC Kit;(5) The total RNA of autologous co-cultured cells with best ratio, pure BMSCs and EPCs groups were extracted, and the expression of VEGF, Osteonectin, Osteopotin and Collagen Type I were detected with Real-time Fluorescent Quantitation polymerase chain reaction on day3,7and14;(6) The partially deproteinised biologic bone (PDPB) were prepared with fresh porcine spine bone and formed partially deproteinised biologic bone(PDPBB) with fibronectin modified, the cavity of PDPBB was observed with scanning electron microscope; The EPCs, BMSCs and the co-culture cells with the ratio1:2were seeded separately onto PDPBB materials, the growth and proliferation of cells on the PDPBB in each group were detected with Wst-1and scanning electron microscope, the most proper occasion for transplanting the biological bone back into rabbit was analyzed;(7)12health Newzealand white rabbit which has extracted peripheral blood and bone marrow few months ago were underwent the surgery of biological bone transplanted back into their body (PDPBB+peripheral blood EPCs; PDPBB+BMSCs; PDPBB+autologous co-culture system with best ratio with best ratio). The engineering bone were respectively transplanted into the muscle of Newzealand white rabbit (autologous co-culture system transplanted back into donor animals); the microvascularizaion were detected with Immunohistochemistry staining as CD34, CD105and ZO-1.[Result](1) Higher purity EPCs could be isolated with Ficoll. The CD34, CD133and vWF of peripheral blood derived endothelial progenitor cells were positive with immunofluorescent staining on week3weeks;(2) Higher purity BMSCs could be isolated with the Ficoll. The CD29、CD34and CD90of peripheral blood derived endothelial progenitor cells were positive with immunofluorescent staining on week3weeks;(3) In vitro, there were many synaptics connection between cells in the autoallergic co-culture system group and some cells formed clusters on the day14. The absorbance of each group increased gradually and reached the peak on day12, and the absorbance of1:2group was highest, and there are statistical significant difference when compared with each group (P<0.01);(4) In vitro, ALP staining of the EPCs and BMSCs group were negative on day3, but some cells in autologous co-cultured group were positive; EPCs group were still negative on day7, some positive cells were observed in BMSCs group and more positive cells were observed in positive in the co-culture group. The ALP content of each group increased gradually and highest in the co-culture group on the day3,7and14, there are statisticly significant different between each group (P<0.01); (5) Von Kossa staining of the EPCs and BMSCs group were negative on day3, but some cells in autologous co-cultured group were positive; EPCs group were still negative on day7, some positive cells were observed in BMSCs group and more positive cells were observed in positive in the co-culture group. The OC content of each group increased gradually and highest in the co-culture group on the day3,7and14, there are statisticly significant different between each group (P<0.01);(6) The mRNA expression of VEGF, Osteonectin, Osteopotin and Collagen Type Ⅰ were gradually increased when detected on day3,7and14with Real-time Fluorescent Quantitation PCR. A few OsteonectinN Osteopotin and Collagen Type Ⅰ and lower mRNA were expressed by pure BMSCs and EPCs. The expression of VEGF, Osteonectin, Osteopotin and Collagen Type Ⅰ were highest in autologous in co-culture system, with significant statistic difference when compared with other groups (P<0.01);(7) The electron microscope detection:There were a large number of hydroxyapatite nets on the PDPB materials made with porcine spine bone, a lot of granular protein crystallizations were observed on the surface of PDPBB prepared with fibronectin. The growth and proliferation of cells in autologous co-culture system on PDPBB were good, and formed cell clusters. A lot of collagens were created by autoallergic co-culture system. The adhesion of pure bone marrow stem cells and EPCs on PDPBB were not as good as cells in autologous co-culture system;(8) The proliferation of each group on PDPBB were detected with Wst-1. The absorbance of eacg groups gradually increased and reach the peak on day12, the highest was the autologous co-culture cells group with the ratio1:2, which have significantly different when compared with each other groups (P<0.01);(9) The microvascularization of the engineering biological bone in vivo were observed with immunohistochemistry, and CD105、CD34、及ZO-1were detected respectly. Granulation tissues were grew into the cavity of engineering bone on week2, and the positive cells of autologous co-culture system were gradually increasing on week2,4and8, and formed some microvascular. There were some positive cells and microvascular were observed in group EPCs, fewer positive cells were observed in group BMSCs and on microvascular structures were observed in this group. The expression of CD105、CD34、及ZO-1were highest in co-cultured group with significant different compared with other groups (P<0.01);[Conclusion](1) EPCs isolated from peripheral blood with Ficoll density gradient centrifugation were highly purified and no induction needed;(2) BMSCs isolated and purified with Ficoll density gradient centrifugation were highly purified and vivid;(3) In vitro, cells in co-culture system promote the proliferation of each other. The osteogenesis of BMSCs could be promoted by peripheral blood derived EPCs with the premium ratio1:2;(4) The microvascularization of EPCs can be promoted by BMSCs with the premium ratio1:2;(5) The proliferation and adhesion of cells on scaffold were promoted by eachother. The capability of cells growth and proliferation of autologous co-culture system were best than pure BMSCs and pure EPCs groups;(6) In vivo, the microvascularization of autologous co-cultured cells were best and formed lumen of vessels. EPCs could improved the blood supply for engineering bone with good angiogenesis and microcirculation capability;(7) The biological engineering bone constructed with peripheral blood derived EPCs, BMSCs and PDPBB are biological material with good microvasculaerization, histocompability, and suitable for clinic experiment and application.