Experimental Research On Construction Of Tissue Engineering Blood Vessel Scaffold With Cells From Bone Marrow Mononuclear Cells

Part I Study the Culture Condition and Biological Characteristics of Endothelial Progenitor Cells from Rat Marrow in VitroObjective To elucidate a simple method of isolating endothelial progenitor cells ( EPCs) from rat marrow monocytes (MNCS) and observe the endothelial cell specific expression profile during proliferation and differentiation in vitro.Methods MNCS were isolated from rat marrow using ficoll density gradient centrifugation, induced, proliferated and differentiated in EGM-2 culture medium(the experiment group) and common medium (the control group) respectively. The EPCs were identified by immunofluorescent staining (CD34,VWF dipl-mark and FLK1) at 4,7,10,14,21 d, respectively. The proliferation of EPCs was evaluated by MTT, and endothelial cell specific markers (VWF,FLK1) were determined with western blotting and Real-time PCR at 1,4,7,10,14,21 d, respectively, and compare with the control group. Results During culturing process, the cells became spindle shaped and displayed endothelium-like cobble-stone morphology with outgrowth at 4 d and 7 d, but cells of the control group were worse in the condition and the morphous than the experiment group .Result of MTT indicated that the number of EPCs increased to peak at 10 d (P<0.05). Percentage of the cells that were positive for CD34/VWF was 90% at 7 d, and that of the control group extremely low, Percentage of positive was no increase. Expression for VWF at 1 d was not found with western blotting, however,gradually increased at 4 d and reached to the top during 10 d to 14 d. The results with Real-time PCR indicated that the expression of VWF and FLK1 mRNA within non-induced cells was not found, gradually increased at 4 d and reached to top at 14 d, the low-expression at 1 d was significantly different from the other ones (P<0.01).Conclusion These data demonstrated that EPCs could be isolated from rat marrow monocytes (MNCS) and cultured in vitro. The EPCs could be used as endothelial seeding cells in vascular tissue engineering.Part II Study the culture and bionomics of smooth muscle progenitorcells from rat marrow in VitroObjective To elucidate a simple method of isolating smooth muscle progenitor cells ( SPCs) from rat marrow monocytes (MNCS) and observe the smooth muscle progenitor cells specific expression profile during proliferation and differentiation in vitro.Methods MNCS isolated by ficoll density gradient centrifugation from rat marrow,induced and proliferated and differentiated then identified by immunofluorescent staining (α-SMA,CD14) at 4, 7, 10, 14, 21 d, respectively. and smooth muscle cells specific markers (α-SMA, CD14) were determined with western blotting and Real-time PCR at 1, 4, 7,10, 14, 21 d, respectively.Results During culturing, cells start adherence and became spindle shaped with outgrowth at 4 d and 7 d , and present typical"peak""valley"at 14 d (3 generation). dipl- positive forα-SMA,CD14 at 4 d. Expression forα-SMA at 1 d not found with western blotting ,but it gradually enhanced at 4 d and reached to the top from 10 d to 14 d ,and still to remain high-level after 21 d .The results with Real-time PCR indicated that the expression ofα-SMA mRNA within noninduced cells was not found ,but after being induced it gradually enhanced at 4 d and got to top at 14 d, and still to remain high-level after 21 d , low-expression at 1 d was significantly different from the other ones(P<0.01).Conclusion These data demonstrated that SPC could be isolated and cultured from rat marrow monocytes (MNCS), and during culturing Smooth muscle cell-specific marker expressions, such asα-SMA and CD14 potently enhanced from 1 d to 21 d. SPCs can be used as smooth muscle seeding cells for constructing vascular tissue engineering.Part III Study the growth effect on endothelial progenitor cells with extracellular matrix scaffoldObjective To explore the characteristics of endothelial progenitor cells (EPCs) on pressed and unpressed extracellular matrix (ECM) scaffolds and to find a novel bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels.Methods EPCs were induced from mesenchymal stem cells isolated from rat bone marrow and cultured in EGM-2 culture medium. The scaffold was comprised from three major ECM proteins (i.e. fibrinogen, fibronectin and laminin) which can be easily molded into mat-like form and gelled by the addition of fresh plasma. Then, pressure was applied to make a pressed scaffold. EPCs were seeded on both pressed and unpressed scaffolds. the surface structure of the scaffold and growth state of EPCs on the scaffold were observed and analysed by electron microscope. The characteristics of those EPCs on different kinds of scaffolds were studied with EPC-specific VWF by immunofluorescent, western blotting and a real-time PCR technique at DIV3 (days in vitro), DIV7, DIV10, DIV14, DIV21,respectively. In addition, the hydromechanical characteristics of four kinds of scaffolds were detected by a rheometer.Results The ratios of cell adhesion at 1 h,3 h,5 h after seeded on pressed scaffold and unpressed scaffolds were 25.3%,60.4%,90.5% and 10.5%, 30.4%,66.3% respectively. Cell numbers on the two scaffold types at DIV1, DIV3, DIV7, DIV10, DIV14, DIV21 increased in evidence. pressed scaffolds has got a larger cell number(p<0.001)at DIV1, DIV3, DIV7,but no significant difference was found after DIV10. Furthermore, cell shapes of EPCs on pressed scaffolds were more mature and more similar to endothelial cell. A level cell surface on pressed scaffolds was achieved. However, on unpressed scaffolds, cell shapes were irregular and gaps between cells at the top of the scaffold were rather distant. Western blotting assays revealed: EPCs on pressed scaffolds expressed more protein VWF at DIV3,vDIV7, vDIV10, but no significant difference after DIV14. Real-time PCR results showed: EPCs on the two different groups of scaffolds all expressed VWF gene, The quantity of their expression in the two groups were all enhanced at DIV7(p<0.05), peaked at DIV14(p<0.01)and still maintained at a high level at DIV21. The quantity of VWF gene expression in the pressed group was much higher than that in the unpressed group at DIV3, DIV7, DIV10, but there was no significant difference after DIV14. Hydromechanical result show torque of pressed and growthed-cell scaffold was obviously lower than other scaffold (p<0.01).Conclusions Pressed ECM scaffolds can promote adhesion, proliferation and differentiation on EPCs. Pressed scaffolds can be used as the matrix for EPC and fabricated into a novel synthetic tissue bio-engineered vascular scaffold. Part IV Study the growth effect on smooth muscle progenitor cells with extracellular matrix scaffoldObjective To explore the characteristics of smooth muscle progenitor cells (SPCs) on the extracellular matrix (ECM) scaffolds and to find a novel bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels.Methods second generation SPCs from mesenchymal stem cells isolated and cultured and induced from rat bone marrow were growthed on the scaffold(the experiment group), that comprises three major ECM proteins (i.e. fibrinogen, fibronectin and laminin) which can be easily molded into mat-like form and gelled by the addition of fresh plasma.and cells were the control group on the cover glass. the surface structure of the scaffold and growth state of SPCs on the scaffold were observed and analysed by scanning electron microscope (SEM)and transmission electron microscope (TEM);The characteristics of those SPCs on the experiment group and the control group were studied with SPC-specificα-SMA by immunofluorescent, ,western blotting and by an real-timePCR technique at DIV3 (days in vitro), DIV7, DIV10, DIV14, DIV21.Results The ratios of cell adhesion at 1 h, 3 h, 5 h after seeded on scaffold and control group were 26.40%,61.40%,91.50% and 11.10%,30.55%,66.90% respectively. Cell numbers on the experiment group and the control group at DIV1, DIV3, DIV7, DIV10, DIV14, DIV21 increased in evidence. the experiment group has got a larger cell number(p<0.001)at DIV1, DIV3, DIV7,but no significant difference after DIV10. Furthermore, cell shapes of SPCs on scaffolds were more level, more compact and have major ecphymas as typical smooth muscle cell by SEM. Detected by TEM , SPCs of the control group were monolayer ,on the other hand ,ones of the experiment group were multiplayer which was 3D stereochemical structure and got close to natural vascular. However, cell shapes of the control group were irregular and gaps between cells at the top of the scaffold were rather distant. Western blotting assays revealed: SPCs on ECM scaffolds expressed more proteinα-SMA at DIV3, DIV7, DIV10 ,but no significant difference after DIV14. Real-time PCR results showed: SPCs on the two different groups all expressedα-SMA gene, The quantity of their expression in the two groups were all enhanced at DIV7(p<0.05 ), peaked at DIV14(p<0.01) and still maintained at a high level at DIV21. The quantity ofα-SMA gene expression in the experiment group was much higher than that in the control group at DIV3,DIV7,DIV10(p<0.05), but there was no significant difference after DIV14. that of the control group significant decrease after DIV21.conclusions scaffolds can promote adhesion, proliferation and differentiation on SPCs. ECM scaffolds can be used as the matrix for SPCs and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.Part V Experimental Research on Construction of Tissue engineering Blood Vessel scaffold with cells from smooth muscl progenitor cells and endothelial progenitor cellsObjective To explore the characteristics of smooth muscle progenitor cells (SPCs) and endothelial progenitor cells ( EPCs) on the extracellular matrix (ECM) scaffolds and to find a novel seeds cells and bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels.Methods induced, proliferated and differentiated in different culture medium from rat bone mononuclear cells (BMCs). SPCs and EPCs of the second generation from mononuclear cells isolated and cultured and induced from rat bone marrow were growthed on the scaffold(the experiment group), that comprises three major ECM proteins (i.e. fibrinogen, fibronectin and laminin) which can be easily molded into mat-like form and gelled by the addition of fresh plasma. and cells were the control group on the cover glass. the surface structure of the scaffold and growth state of SPCs and EPCs on the scaffold were observed and analysed by scanning electron microscope (SEM)and transmission electron microscope (TEM).Results BMCs were induced in EGM-2 culture medium, and BMCs were induced in M-199 culture medium with bFGF. second filial generation SPCs that induced were planted on the ECM-scaffold, SPCs under inverted/ fluorescent microscope present typical"peak""valley"at 4 d , and second filial generation EPCs were planted on the scaffold with the SPCs after 4 d, A level cell surface on scaffolds was achieved under SEM After10 d. And multiplayer SMCs were found on the surface of monolayer EPCs under TEM, which had three diamensions stereochemical structure with endomembrane and tunica media and got close to natural blood vessel. The growth of the control group cells with monolayer construction was weaker .Conclusions BMCs will be most ideal cells source for Tissue engineering Blood Vessel. ECM scaffolds can promote adhesion, proliferation and differentiation on SPCs and EPCs.ECM scaffolds can be used as the matrix for SPCs and EPCs and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.