Experimental Study On Repair Of Mandibular Defects With Tissue Engineering Bone Constructed By The Co-culture System Composed Of Adipose-Derived Stromal Cells And Vascular Endothelial Cells

Objective:In order to further solved the problems that the slow revascularization of tissue engineering bone and the retardation of new bone growth, it need implant not only the stem cell that have differentiation potential to osteoblasts, but also VECs to promote angiogenesis.VECs have the ability of enhancing ADSCs to ossify and new blood vessel can provide nutrition for the ossification of the stem cell.The co-culture seed cells of tissue engineering are increasingly acknowledged that it is the promising seed cells to be used in tissue engineering.The purpose of this study was to analyze the influence on ossification and proliferation of ADSCs affected by VECs in the system of co-culture in vitro or vivo;To determine the capacity to repair bone defects of the co-cultured ADSCs and VECs as seed cells in bone bioengineering.Methods:(1)To isolate mononuclear cells from umbilical cord blood,they were cultured in conditioned medium.The immunofluorescent staining were used to observe expressions and distributions of some special surface marker,such as CD34,CD133,Flk-1 and vWF on adherent cells after 3 weeks and 6 weeks.(2)To isolate and culture rat adipose mesenchymal stem cells(ADSCs) cultured by four types of blood serum culture-medium,the immunofluorescence method were used to observe expressions and distributions of some special surface marker,such as CD34,CD133 and Flk-1 on adherent cells;They are induced by osteoinductive culture medium, alkaline phosphatase(ALP) and Von Kossa staining were done on the 14th day to test the characteristic of ossification.(3) To co-culture VECs and ADSCs by the proportion of VECs,3:1,1:3,1:1 and ADSCs,morphological changes were observed by inverted microscope:The adhesive and proliferation conditions of bone marrow stromal stem cells were evaluated by MTT;Alkaline phosphatase(ALP) and Von Kossa staining were done respectively on the 7^th day and 14^th day,ALP and osteocalcin(OC) were detected respectively on the 4^th day,7^th day and 14^th day.(4) The PDPB materials were made with porcine spine bone.It was modified by fibronectin to made partially deproteinised biologic bone(PDPBB),the surface information of PDPBB was observed with a scanning electron microscope;The PDPBB materials were inoculated separately with VECs,ADSCs and the co-culture cells according to the ratio of 1:1,the absorbance was tested by MTT automated for assessment the adhesive and proliferation conditions of three different cell on PDPBB, the most proper occasion was analyzed and it was observed with a scanning electron microscope.(5)VECs,ADSCs and co-cultured cells marked by BrdU were compounded separately with the PDPBBs in vitro,the tissue engineering bones were transplanted into both sides of femoral muscles bags in SD rats;The CD3,CD4,CD8 factors were detected with flow cytometry to analyze the CD4/CD8 of T lymphocytes subgroup in peripheral blood;It was to compare the new bone mass with hard tissue slices;The immunofluorescence staining of Brdu antibody was separately carried on in order to observate the proliferation and differentiation of the seeds implanted cells in vivo.(6)Take 60 healthful SD rats,randomly divided into 5 groups(A group:PDPBB +vascular endothelial cells;B group;PDPBB+adipose-derived stromal cells;C Group:PDPBB +1:1 co-cultured cells group;D group:simple PDPBB group;E groups:blank control group),to make 0.5×0.4cm critical bone defect in mandibular body used bone rongeur,respectively,every tissue engineering bone group were grafted into the mandibular defect models in rats;E group was blank control group; To execute X-ray examination and histological examination by hard tissue biopsy and analysis the amount of osteoblasts.Result:(1) The results of the immunofluorescent staining were positive on CD34,CD133,Flk-1 and vWF after 3 weeks,but their results were only positive on CD34,Flk-1 and vWF after 6 weeks,the part of adherent cells were positive on CD133.(2)It is thus clear that the cell shape of new-born calf serum(NBCS) group is single in culturing ADSCs,presenting fusiform;the results of the immunofluorescent staining were negatively on CD34,CD133 and Flk-1;The ALP staining of majority cells and red calcium nodus were positive after osteogenic induction;But that of fetal bovine serum(FBS) was complex,we can find that a great quantity polyhedral cells offer nest growth and took on a cobblestone morphology,the fusiform cells is existing near here;the results of polyhedral cells were positive on CD34 and Flk-1,the part of polyhedral cells were positive on CD133,it is negatively on the fusiform cells;A great quantity polyhedral cell was died after osteogenic induction,forming blank;The ALP staining of majority cells and red calcium nodus were positive in some fusiform cells (3)There were many synaptics connection in the 1:1 and 3:1 group and part of the cell form cell clusters on the 14^th day in vitro,no cell clusters observed in other groups;According to growth curvature,the absorbance of every group increased gradually,it reached the peak in ADSCs,3:1,1:3 and 1:1 ratio group on 12^th day and the highest was the 1:1 group,but the time was 10^th day in the VECs group,the differences had statistical significance.(4)In vitro,ALP staining of the ADSCs,3:1 and VECs group were negative on 7^th day,but part of the cell were positive in the 1:3 and 1:1 group;Von Kossa staining of every group was negative on 7^th day;on the 14^th day, ALP staining was also negative in the ADSCs and VECs group and it was positive in the 3:1,1:3 and 1:1 group,Von Kossa staining was positive in the little cell of the 3:1,1:1 and 3:1 group and other groups were negative.The ALP value of every group increased gradually,it was highest in the 1;1 group on the 4^th,7^th and 14^th day,there were no changes on the ADSCs and VECs group;multiple comparison of the value showed remarkable increase between the 1:1 group and other groups(P＜0.01);The OC value of every group increased gradually,it was highest in the 1:3 group on the 4^th day,but the OC value of the 1:1 group was highest on the 7^th and 14^th day,multiple comparison of the value showed remarkable increase between the 1:1 group and other groups(P≦0.01).(5)The PDPB materials made with porcine spine bone had homogeneous pore distribution and formed with a large number hydroxyapatite nets, there were many granular protein crystallization in the surface of PDPBB materials by fibronectin.The growth curve of cell on the PDPB had a 'S' shape,but that of on the PDPBB had no 'S' shape.The proliferation of every groups reach tip on tenth day, there was significantly different between every group(P＜0.01).The absorbance of every group increased gradually and it reach tip on 10^th day,the highest was the co-culture cells group according to the ratio of 1:1,it have significantly different when every group was compared each other(P＜0.01);On 10th day,a great deal of cells adhere on the PDPBB materials in the co-culture cells group and showed a nestlike distribution.(6)In ectopic osteogenesis,the granulation tissue had grew into the pore space of the tissue engineering bone after two weeks;The four weeks we could see a large number of multinucleated osteoclasts located in the surround of the scaffold materials and evolved into its internal,destructed,absorpted the scaffold materials; Homogeneous,non-cell bonelike material began to appear in the edge of scaffold materials;Many vasa vasorums took shape in the pore space of the tissue engineering bone;After 12 weeks the scaffold materials began to absorb,edge blur and easy break;the bonelike material began to appear in the edge of scaffold materials in every group,the largest was the co-culture group and the less was the PDPBB group;there are ossification centers in the surrounding of matertials in part of slices,we could see no considerable lymphocyte infiltration in each group.(2)New bone formation of the co-culture cell group was the largest,the less was the PDPBB group,it is statistical significance that the differences between other groups and the co-culture cell group (P＜0.01).(7)According to the CD4/CD8 curve of T lymphocytes subgroup in peripheral blood we could see:It gradually increased during 1-8 weeks in the PDPBB group,reached the highest point after 8 weeks and then gradually reduced,there was statistical significance campared with the control group(P＜0.05);In the 1st week it was the highest and decreased gradually between the 1st week and the 3rd week in the VECs Group,its shape almost changed into a straight line in the consecutive nine weeks;The shape of the ADSCs group,the co-cultured cell group and control group was almost a straight-line,it was no statistically significant compared with control group(P＞0.05);(8)Brdu immunofluorescent staining shows that the implanted cells of each group were scattering distribution,the cells gradually proliferated and formed "nestlike",the implanted vascular endothelial cells in VECs group and the co-cultured cells group formed large quantity new vessels,but it was no positive cells in the control group.(9)The first two weeks of repairing bone defects,a large number of fibrous tissue enveloped the implanted tissue-engineered bone in each group;bone defects had been closed by fibrous tissue in the control group;In the fourth week implanted materials could not be moved in each group,the muscle and fibrous tissue markedly thickened around PDPBB composited vascular endothelial cells,link up tissue engineering bone with mandible,blood supply of this group was richer than other groups;In the eighth weeks,the gap between mandible bone and the tissue engineering bone constructed by PDPBB composited the co-cultured cells has disappeared,the surface of some materials have been corticalization;In the twelfth week,the tissue engineering bone have been connected with mandibular bone in each group.In the simple PDPBB Group,the adipose-derived stromal cells group and the vascular endothelial cells group,part of the materialhave been absorbed,but, generally the materials have retained the original form;The shape of the co-cultured cells group was similar to normal mandible,the surface of some materials have been corticalization;the bone defect of the control group have not been repaired,the surface of bone defects has been closed and formed a semi-circular bone defect area. (10)In X-ray examination,the gap between the tissue engineering bone of the vascular endothelial cell group and mandibular defects was wide in the second week,new bone callus formation around bone defects;in 4th-8th week the callus gradually increased, in the twelfth week,we could see that the gap between scaffold material and mandibular defects gradually become vague,but the general form of scaffold materials still exist.The callus around the bone defects of the adipose-derived stromal cells group begin to generate in the second week,in the twelfth week,the density of tissue-engineered bone was similar to normal bone;The callus of the co-cultured cells group gradually increased from the 4th week to the 12th week,the eighth week obtained osseous fusion.,the gap have disappeared;in the 12th week,the bone density was significantly increased,the shape of the tissue engineering bone has basically returned to normal;Scaffold materials density of the simple PDPBB group reduced significantly compared with the normal bone in the twelfth week;The bone defect area gradually decreased in the control group in the eighth week and the twelfth week, the surface of bone defects has been closed and formed a semi-circular or triangle bone defect shape.(11) In histoiogic diagnosis,new bone formation of the co-cultured cell group was the largest,the vascular endothelial cells group come second,the simple PDPBB group was the lowest,it is statistical significance that the differences between other groups and the co-culture cell group(P＜0.01);there was statistical significance campared the VECs group and the ADSCs group(P＜0.05);the differences between the VECs group and the simple PDPBB group was statistical significance(P＜0.01).Conclusion:(1)The mononuclear cells from rat umbilical cord blood were induced into endothelial progenitor cells after 3 weeks in vitro.the part of adherent cells differentiated into mature vascular endothelial cells.(2)It can obtained comparatively pure ADSCs that apply nutrient medium contains NBCS;The cells used the medium contains FBS is abound in vascular endothelial cells.(3)The co-culture cell can stimulate proliferation each other and ADSCs are ossified by VECs in the system of co-culture VECs and ADSCs in vitro,it can reach the best result when the proportion is 1:1.(4)Fibronectin could improve the adhesive and proliferation of cells on the PDPB materials.(5)The adhesive and proliferation of the co-culture cells was better than the single kind ceils and the most proper occasion to transplanted is the tenth day.(6)New bone formation of the co-culture cell group was the largest,vascular endothelial cells can enhance the osteogenic potential of the tissue engineering;Each tissue engineering bones adhered Cells can not caused severe immune rejection in vivo.(7)Implanted cells of tissue engineered bone can considerable proliferate in vivo and participate in the formation of new tissues.(8)New bone formation of the co-culture cell group was the largest,vascular endothelial cells can enhance the osteogenic potential of the tissue engineering.