以脂源性幹細胞培養在具新穎性生物相容性孔洞型支架促進傷口修復

Adipose-Derived Stem Cells Cultured in Novel Biocompatible Porous Scaffold for Wound Repair

顏宏勳(Hung-Hsun Yen)

指導教授 ： 王惠民

高雄醫學大學/藥學院/香粧品學系碩士班/碩士(2016年)

摘要

本研究以模擬生物體及體外試驗探討3維多孔性支架對於傷口癒合之功效。此支架以天然且低生物抗原性之聚合物－膠原蛋白、玻尿酸與明膠做為原料，並以1-ethyl-3- (3-dimethylaminopropyl) carbodiimide交聯而成，分別調整玻尿酸含量：HA-L及HA-H。實驗將測試支架生物相容性，包含材料特性及其針對傷口修復的擬生物體與體外試驗。在機制中，化學交聯反應發生於玻尿酸的羧基，膠原蛋白和明膠的胺基上；傅立葉轉換紅外線光譜偵測出在支架上相同位置的交聯基團。在吸水比例上，HA-L及HA-H分別具有比自身重31倍與36倍吸水能力；而於生物降解性試驗證明，此支架能隨著時間被人體所分泌的酵素分解。從大鼠脂肪取得之脂肪幹細胞經由流式細胞儀鑑定其特異性指標( CD90、CD105與CD146：正表現；CD11b、CD31：負表現)，並植入支架培養。脂肪幹細胞能促進細胞增生，並分泌轉化生長因子(TGF-β)及血管內皮生長因子(VEGF)刺激血管的新生作用。實驗的第7天，細胞生長量分別於HA-L及HA-H支架比第一天的增加3.5和4.2倍；同時，TGF-β於HA-L及HA-H支架中的濃度分別為36 與39 pg/ml，VEGF的濃度分別為12 與 57 pg/ml，增加的VEGF濃度與支架中HA的比例有直接地關聯性。第14天及42天，脂肪幹細胞在支架上的分佈以螢光顯微鏡及掃描式電子顯微鏡觀察，發現細胞大量分布在支架中，並且細胞可分解支架。在脂肪幹細胞支架植入老鼠體內的試驗中，證明脂肪幹細胞能轉移至皮下組織，並且宿主的細胞亦能長入支架內。在組織切片下觀察，以正常皮膚與有植入支架的皮膚相比，並未發現有明顯的排斥反應發生。總和以上所述，細胞於兩種支架中，在第7與14天的生長量及血管新生因子分泌量，HA-H都優於HA-L支架，並且使用此材料結合脂肪幹細胞之培養能有效促進傷口修復。

關鍵字: 傷口修復、三維支架、脂肪幹細胞、轉化生長因子、血管內皮生長因子、活體

關鍵字

傷口修復 ； 三維支架 ； 脂肪幹細胞 ； 轉化生長因子 ； 血管內皮生長因子 ； 活體

並列摘要

This manuscript demonstrated ex vivo and in vitro experimental effects on wound healing using three-dimensional porous scaffolds. Scaffold were constructed by natural and low-reject polymers, collagen (Col), hyaluronic acid (HA), and gelatin (Gel). The scaffolds were separated into two types according to HA concentrations, HA-L and HA-H. Our scaffold was cross-linked with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), and this scaffold was then tested by biocompatibility assessments, included ex vivo and in vitro characteristics. In chemical reaction, cross-linking groups occurred between the carboxyl groups of HA and the amino groups of Col (Gel), and Fourier transform infrared spectroscopy (FTIR) also detected the consistent results in raw materials and scaffolds. The swelling ratio of HA-L and HA-H scaffolds was analyzed by water absorption capability and displayed 31 and 36 scaffold weight enlargement. In biodegradation examination, scaffolds were degraded by three enzymes: lysozyme, hyaluronidase and collagenase I enzymes in time. The adipose-derived stem cell (ASCs) from rat adipocytes were detected the specific cluster-of-differentiation marker (positive: CD90, CD105 and CD146; negative: CD11b and CD31) by flow cytometry. ASCs were co-cultured within these scaffolds that promoted cell proliferation and stimulated capillary angiogenesis, such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). Over the 7-day period, the cell growths of the scaffolds with HA-L and HA-H increased 3.5 and 4.2 folds more than on day 1. On day 7, the TGF-β concentrations of HA-L and HA-H were 36 and 39 pg/ml, and the VEGF concentrations were 12 and 57 pg/ml, respectively. The increase of VEGF concentration could be proven related to the scaffold HA amount. The morphology of ASCs in the scaffolds were inspected by fluorescence microscope and scanning electron microscope (SEM). In vivo histology examination, ASCs implanted scaffold transported into the subcutis, and the rat cells also proliferated into scaffolds. In histology section, the immune response rejection was no significant contrast between rat normal skin and ASCs scaffold. In the experimental data, the biocompatibility of HA-H scaffold was better than HA-L type, and therefore, ASCs HA-H scaffold improved wound repair efficiently.

Key words: wound healing; three-dimensional scaffolds; adipose-derived stem cells (ASCs); transforming growth factor-β (TGF-β); vascular endothelial growth factor (VEGF); in vivo

並列關鍵字

wound healing ； three-dimensional scaffolds ； adipose-derived stem cells (ASCs) ； transforming growth factor-β (TGF-β) ； vascular endothelial growth factor (VEGF) ； in vivo

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