- 學位論文
Human dental pulp stem cells expressing TGF{221}-3 transgene for cartilage-like tissue engineering
Human dental pulp stem cells expressing TGF{221}-3 transgene for cartilage-like tissue engineering
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
並列摘要
A major challenge facing the tissue engineering discipline is cartilage tissue repair and engineering, because of the highly specialized structure and limited repair capacity that cartilage possesses. Dental pulp stem cells (DPSCs) were identified about a decade ago as a potential candidate for cell based therapy and tissue engineering applications. The present study aimed to utilize gene therapy with isolated DPSCs to induce chondrogenic transgene expression and chondrogenic lineage differentiation, with the ultimate goal of engineering cartilage tissue-like constructs. We isolated DPSCs from human teeth extracted for orthodontic treatment. We further enriched the isolated population using immunomagnetic bead selection, which increased stem cell markers: Stro-1 and CD146, compared to unselected population. The DPSCs showed the ability to differentiate into the chondrogenic lineage when induced with recombinant hTGFβ-3 and when transduced with hTGFβ-3 transgene. We successfully constructed the recombinant adeno-associated viral vector encoding the human TGFβ-3, and determined the best multiplicity of infection for DPSCs. The transduced DPSCs highly expressed hTGFβ-3 for up to 60 days. Expression of chondrogenic markers; Collagen IIa1, Sox9, and aggrecan was verified by immunohistochemistry and mRNA. We successfully fabricated an electrospun nano-fiber scaffold upon which morphology, proliferation and viability of the DPSCs were examined. DPSCs attached and proliferated on nano-fiber scaffolds demonstrating better viability compared to micro-fiber scaffolds. Transduced cells expressed hTGFβ-3 protein up to 48 days. Cells seeded on nanofiber scaffolds showed higher expression levels compared to micro-fiber scaffolds or culture plate. Scaffolds seeded with DPSCs were implanted in nude mice. Immunohistochemistry for TGFβ-3 DPSCs constructs (n=5/group) showed cartilage-like matrix formation with glucoseaminoglycans as shown by Alcian blue. Immunostaining showed positivity for Collagen IIa1, Sox9 and aggrecan. Semi-thin sections of the transduced DPSCs constructs examined by transmission electron microscopy (TEM) showed chondrocytic cellular and intra-cellular features, as well as extracellular matrix formation (n=2/group). In vivo constructs with the TGFβ-3 DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSCs constructs (n=5/group). Western blot analysis confirmed this expression pattern on the protein level (n=3/group). Engineered constructs mechanical properties were examined and compared to patellar bovine cartilage to assess functionality (n=5/group). TGFβ-3 transduced DPSCs constructs showed a higher equilibrium elastic modulus compared to nontransduced constructs. Micro-fiber scaffolds constructs showed a higher elastic modulus (0.11 MPa, 18% of bovine cartilage), compared to nano-fiber constructs modulus (0.032 MPa, 6% of bovine cartilage). Nano-fiber based constructs showed a similar Poisson‘s ration to bovine cartilage, while that of micro-fiber scaffolds was lower. As an alternative gene delivery method, electroporation parameters for DPSCs transfection were optimized, and compared to commonly used chemical transfection methods. TGFβ-3 transfected DPSCs showed a significantly higher relative TGFβ-3 mRNA and protein expression compared to non transfected control and to eGFP transfected DPSCs. Transfected DPSCs showed increased relative expression of chondrogenic markers; Collagen II, Sox9 and aggrecan, compared to non transfected DPSCs. Successful chondrogenic differentiation of DPSCs gene therapy with TGFβ-3 transgene, and seeding them on PLLA/PGA scaffolds makes it a potential candidate for cartilage tissue engineering and cell based therapy.