Constructing Tissue-engineering Cartilage By HUMSC Compound With C/P(LLA-CL)Scaffold In Vitro

Objective To explore the strategies of isolation and culture of human umbilical cord-derived mesenchymal stem cells, evaluated their biological characteristic and expected it to provide experiment foundation for the study of seed cell of tissue engineering.Methods Obtain the wharton's jelly of the fetus umbilical cord in sterile; the tissue were digested by different enzyme solutions. Then the digested cell suspensions were plated and cultured in DMEM/F12medium containing10%FBS; While needing a cell growth to80-90%fusion,1:3spread generation culture. Observed the morphologic changes of the cells by the inverted microscope; the cell cycle and the immunophenotypic characterization of adherent cells were analyzed by flow cytometry, The growth curve of the cells was made by MTT method. The differentiation potential of the cells were tested using standard differentiation conditions for osteoblasts and adipocytes.Results A few adherent cells could be observed only1-2hours after cultivation. After6～8hours numerous adherent cells can be observed as long or short spindle-shape or multangular shape; Logarithm growth period appeared in the2-3th day after cultivation when the cells were almost fibroblast-like cells; Cells exhibited spindle-shape fused more than90%in the4～5th day. Cell adherence and proliferation were speeded up after passaged with1:3ratio; The passage cells which most fibroblast-like shape began to adhere within2hours after sub-culture and proliferate rapidly. The cell cycles evaluation by flow cytometry showed the3rd passage of the cells had powerful proliferation potential. Meanwhile, The P3passage of the cells expressed CD13, CD29,CD44,CD105and do not express surface markers of hematopoetic cells(CD45,HLA-DR) and endothelial cells(CD31); The growth curve made by MTT showed the P3,P5,P7of the cells had powerful proliferation ability; Moreover, these cells could differentiate into osteoblasts and adipocytes under specified culture condition.Conclusion We have successful developed a simple and efficient method to isolation and culture of MSC derived from umbilical cord which can be used as a new source of seed cell for tissue engineering. Chapter2Comparison of potential differentiation into chondrocytes between human mesenchymal stem cells derived from umbilical cord and bone marrowObjective Both the mesenchymal stem cells derived from human umbilical cord(HUMSC) and bone marrow(BMSC) have the potency of multilineage differentiation; Observe the characteristics of differentiating into chondrocytes and detect the closed related gene expression about the HUMSC under certain induce fluid; To compare the difference in chondrocytes differentiation ability between the HUMSC and BMSC.Method Obtain the MSC derived from the wharton's jelly of the fetus umbilical cord which digested by different enzyme solutions; The third passage of BMSCs and HUMSC at a density of2×105/cm2were both divided into two groups. Cells in the induction group and the control group were respectively inducated in the chondycytes inductive medium and the DMEM medium; Observed the growth state and the morphologic changes of the cells by the inverted microscope; After two weeks the cells were harvested and stained by using toluidine blue and collagen Ⅱ antibody; RT-PCR was used to detect and compare the expression of collagen Ⅱ, aggrecan, Sox-9mRNA among each group.Result The fibroblast-like shape HUMSC gradually became triangle,polygon and roundness shape and could assemble themselves together by inverted microscope and HE stain. At14days following chondrogenesis induction both the experiment group show positive to toluidine blue stain and collagen Ⅱ stain; However the HUMSC induction group exhibited a significantely strong reaction to the stain; The HUMSC control group appear to weak positive to both stain but the BMSC control group were detected as negative reaction. The result of RT-PCR revealed that the HUMSC little expressed Sox-9,CoL2aland aggrecan mRNA without induction; the gene express were significantly reinforced under chondrogenesisin duction (P<0.05); A negative result and weak positive expresstion were detected respectively in the BMSC control group and induction group; The HUMSC induction group display stronger positive result in gene expresstion compare to that of the BMSC induction group(P<0.05).Conclusion Both the HUMSC and BMSC can differentiate into chondrocyte under chondycytes-inductive medium; However, the former has the higher chondrogenesis property than the latter; So the HUMSC should be selected in preference as seed cells in cartilage tissue engineering. Chapter3Study of properties and biocompatibility of a novel scaffold using P/LLA-CL composited with chitosanObjective To fabricate a new novel scaffold by P(LLA-CL) mixed with chitosan and to explore the properties and biocompatibility of the scaffold for investgating its possibility application in the cartilage tissue engineering. Method The porous CVP(LLA-CL) scaffold was prepared and served for the scanning electron micfoscope (SEM)observation; Then the pore diameter,the porosity and water absorption rate analysis of the materials were investigated; After induced by chondrogenesis conditioned medium, the P3HUMSC were seeded into the experiment scaffold. The scaffold without chitosan was used as control group. Cell proliferation and differentiation were analyzed using inverted microscope. HE stain was used to observe the cells distribution in each scaffold; The adhesion ratio of the3rd passage was detected by cell counted in4,8,12,24h; At the1th,3th,5th,7th day of culture, the MTT was used to analyse the influence which scaffold on the cell proliferation; At one and two weeks culture, The SEM was used to observe and analyse the growth state of cells on the scaffold; The histocompatibility of the scaffold was tested by the nude mouse subcutaneous embedded experimentResult The scaffold was three-dimensional porous cylindrical structure and looked like sponge under SEM observation; the scaffold had good pore interconnectivity with pore diameter(183.56±16.78)um,(91.56±1.27)%porosity and (218.66%±1.55)%water absorption rate; The cells suspension was absorbed quicker in C/P(LLA-CL) than that of P(LLA-CL); HE stain showed abundant cells scattered in pores of the C/P(LLA-CL) scaffold in contrast to the inner cell density in the control scaffold; MTT result revealed that the two scaffolds not only had no adverse effect on the cell proliferation but also the C/P(LLA-CL) scaffold can promote cell proliferation; The cells adhesion ratio were observed gradually increase both in the two scaffolds and the experimental group had higher adhesion ratio than the control one at the same point (P<0.05).Two weeks after induction the SEM micrographs indicated that cells covered the C/P(LLA-CL) scaffolds uniformly with much matrix secretion;yet cells in the control scaffolds secreted less matrix; In the subcutaneous embedded experiment the C/P(LLA-CL) scaffold can effectively reduce the inflammatory reaction which revealed the scaffold has good histocompatibility.Conclusion The porous C/P(LLA-CL) scaffold has good pore diameter and porosity, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering. Chapter4Construction of tissue engineering cartilage by combined HUMSC with C/P(LLA-CL) scaffold in vitroObjective To explore the possibility of construction tissue engineering cartilage by HUMSC compound with C/P(LLA-CL) porous scaffold in vitro. Methods The P3HUMSC were seeded on C/P(LLA-CL)scaffolds which be pre-treatment; Chondrogenic inductor or general media were respectively used according to different group; After3weeks, the compounds were harvested and HE,toluidine blue stain and collagen Ⅱ stain were applied. At7th,14th,21th day of culture the RT-PCR was used to detect the expression of Sox-9,CoL2a1and aggrecan mRNA; Western blot was applied to detect the expression of collagen Ⅱ and aggrecan protein.Results The surface of the cells-scoffold complex in the experimental group was gradually became smooth and lenitive; The cartilage-like tissue formation can be observed after3weeks induced; However the size of the complex were diminished in the control group; At3weeks, the HE stain demonstrated that cartilage-like tissue been formed in the induced group which was not observed in the control group; a strong positive staining for toluidine blue stain was investigated in induced cells and extracelluar matrix, so were for collagen Ⅱ immunohistochemistry. But in control samples, the outcome was weak positive for toluidine blue and collagen Ⅱ staining; The RT-PCR revealed that the gene expressions of mRNA for Col2al, Sox-9and aggrecan gradually up-regulation at7th,14th,21th day after induced cultured. The level of the mRNA expression in the experimental group were significant higher than the control group at the same time point (P<0.05); The result of westem-blot were concordant with RT-PCR which indicated that the productions of Col2al protein and aggrecan protein also increased by degrees in the experiment group and significant difference was found compare with the control group(P>0.05).Conclusion HUMSC combined with C/P(LLA-CL) porous scaffolds induced in vitro can successfully construct tissue-engineering cartilage which may be helpful for the study to repair the injuried cartilage.