| Summary: | 碩士 === 國立臺灣大學 === 化學工程學研究所 === 97 === Suitable cell sources with good expansion technique, as well as excellent biomaterials are important for tissue engineering applications. In this study, we aimed at the application of bone tissue engineering and the feasibility of in vitro expansion of bone marrow mesenchymal stem cell line (BMSC) and stem cells from human exfoliated deciduous teeth (SHED), and the development of biomaterials with osteoinductive characteristics.
First of all, chitosan-based biomaterials were prepared namely, C (chitosan), CG (chitosan/gelatin), C-A (chitosan/ascorbic acid), CG-A (chitosan /gelatin/ascorbic acid)). By using inverted microscopy and measuring the DNA quantity, we found that gelatin could improve cell adhesion, and thus improve the proliferation rate.
In this study, we also used real-time PCR and flow cytometry to detect the gene and protein expression of osteocalcin (OCN). Adding ascorbic acid and gelatin to chitosan biomaterials had little effect on the gene expression of OCN in BMSC. When BMSC exposed to osteogenic stimulus (OS), the gene expression of OCN in BMSC cultured on CG-A would decrease. When there was no OS, the expression of OCN in SHED cultured on C-A approximated that cultured in osteogenic medium. When exposed to OS, BMSC cultured on TCPS were 50 % OCN-positive, on C 1.8 %, on CG 9.8 %, on CG-A 11.0 %; SHED cultured on TCPS were 7.6 % OCN-positive, on CG 10.0 %. When there was no OM, the OCN-positive BMSC cultured on CG-A was 20.5 %.
In addition to the expression of OCN, we also measure the activity of alkaline phosphatase (ALP). We found that the activity of ALP peaking at the 3rd day after osteogenic differentiation, while SHED didn’t have this intendency.
In addition to the gene and protein expression, we also measured the function of cultured cells by using Alizarin red S staining and the content of calcium of BMSC or SHED. When BMSC cultured on CG-A under OS, the content of calcium increased to 1.56 folds of BMSC cultured on TCPS. For the case of SHED, the increment was up to 50 folds. Even if there was NS, SHED cultured on C-A still had similar amounts of calcium content.
We demonstrated that the adsorption of ascorbic acid onto biomaterials had effect similar to the presence of ascorbic acid in medium. When there was NS, the OCN-positive BMSC cultured on CG-A was 20.5 %. These biomaterials (C-A and CG-A) enhanced the activity of ALP, and increased the content of calcium. Therefore, these biomaterials could be good candidates for bone-related tissue engineering applications.
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