| Mesenchymal stem cells (MSCs) are multipotent progenitors existing in adult tissues. They are able to differentiate into various lineages such as adipo-cytes,osteocytes,chondrocytes and other cell types both in vitro and in vivo.MSCs play an important role in the tissue renewal and repairing. Recent studies showed that MSCs have low immunogenicity and are capable of regulating immune respone. MSCs can be isolated from various tissues, which were first found in bone marrow. However, bone marrow-derived MSCs can only be obtained by invasive method which distribute sparsely. With the increased of ages, the numbers as well as expansion and differentiation potentials decrease accordingly. Therefore, it is quite necessary to search for new sources of MSCs for the application of regenerated medicine. It was demonstrated recently that placenta is rich for MSCs, human placenta-derived mesenchymal stem cells (PMSCs) can be differentiated into many tissue as bone marrow-derived MSCs under appropriate conditon. Regenerated Antheraea pernyi silk fibroin(A. pernyi SF) is a new type of biomaterials own by our country. It has the tripeptide sequence arginine-glycine-aspartic acid(Arg-Gly-Asp), which serves as a cell-adhesion site, besides the other similar chemistry structure to regenerated Bombyx mori silk fibroin(B. mori SF). Currently, there is no report about the study on the biocompatibilities of human PMSCs with regenerated A. pernyi SF.In this study, human PMSCs were isolated and expanded successfully in vitro.Moreover, the biocompatibilities of regenerated A. pernyi SF with human PMSCs were explored thoroughly, which can provide important experimental basis for the construction of "engineered bone" by using regenerated A. pernyi SF as a new biomaterial and human PMSCs as new stem cell candidate.Part I Isolation and Identification of Human Placenta-derived Mesenchymal Stem Cells in vitroObjective: To isolate and generate mesenchymal stem cells from human placenta and induce their osteogenic and adipogenic differentiation, which provides evidence for the application in tissue engineering and cell therapy.Methods: The placenta tissues were digested by collagenase IV and cultured with low-glucose DMEM supplemented with b-FGF. The adherent cells were collected and passaged. The phenotypes of the cultured cells were detected by flow cytometry. The osteogenic differentiation was induced withβ-glycerol phosphate disodium salt-pentahydrate, vitamin C and dexamethasone whereas the adipogenic differentiation was induced with 3-isobutyl-l-methylxanthine(IBMX), dexamthasone, indomethacin and insulin. The osteogenic differentiation was detected with alkaline phosphate assay and von kossa staining. The adipogenic differentiation was observed with Oil red O staining. Results: PMSCs were isolated and generated from human placenta successfully, which were fibroblast-like cells with high proliferation. Flow cytometry analysis showed that PMSCs expressed CD29, CD44, CD105, CD106 and CD166, but not CD34, CD45 and HLA-DR.The cells were highly positive for alkaline phosphate staining and also showed mineralization demonstrated with von kossa staining after three weeks culture induction of osteogenic differentiation. Futhermore, liquid vacuoles were detected by Oil red O staining after two weeks culture of the induction of adipogenic differentiation. Conclusion: Effective culture system was established to isolate and generate PMSCs with the osteogenic and adipogenic differentiation, which indicated that PMSCs could be served as a new mesenchymal cell source in tissue engineering.Part II The study on the biocompatibilities of regenerated Antheraea pernyi silk fibroin with human placenta-derived mesenchymal stem cellsObjective: To study the biocompatibilities of regenerated A. pernyi SF with human PMSCs, which provide the theoretical basis for the application of human PMSCs combined with regenerated A. pernyi SF in tissue engineering.Methods: Human PMSCs were prepared by the method described previously. Cells after 3 passages were used in the experiment. The films were prepared by coating tissue culture plastic with regenerated A. pernyi SF solution. The attachment of human PMSCs on the regenerated A. pernyi SF films was detected by cell counting and light microscope. MTT and cell counting were used to examine the viabilities and proliferation of human PMSCs on the regenerated A. pernyi SF films. The morphology of human PMSCs seeded on the regenerated A. pernyi SF films was observed by light microscope and HE staining. FCM was used to analyze the phenotype of human PMSCs seed on regenerated A. pernyi SF films.Results:The 1h adherent rate of human PMSCs on regenerated A. pernyi SF films was significantly higher than that of regenerated Bombyx mori silk fibroin(B. mori SF) films, collagen films and tissue culture plastics. MTT assay showed that the proliferation of human PMSCs on regenerated A. pernyi SF films was significantly higher than those of regerated B. mori SF films. HE staining indicated that the spreading of human PMSCs on regerated A. pernyi SF films was similar to that on collagen films and tissue culture plastics, and was better than that on regenerated B. mori SF films. Futhermore, FCM analyses showed that regenerated A. pernyi SF did not change the phenotype of human PMSCs.Conclusion:The regenerated A. pernyi SF can support the attachment, growth and phenotype maintenance of human PMSCs, which indicated that regenerated A. pernyi SF has good biocompatibilities with human PMSCs.In summary, effective culture system was estabilished successfully to isolated and expand human PMSCs in vitro. The differentiation potentials of human PMSCs were also identified under appropriate induction conditions. Futhermore, the biocompatibilities of regenerated A. pernyi SF with human PMSCs were investigated thoughly. The results indicated that the regenerated A. pernyi SF could support the attachment and expansion of human PMSCs effectively in vitro, and maintain the normal expression of various surface antigens. This study provides important evidence for the application of human PMSCs combined with regenerated A. pernyi SF in bone tissue engineering. |
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