以天然高分子為支架製成之組織工程替代物通常有機械強度不足的問題，機械力的刺激被認為在組織的成長與重塑中扮演重要的角色。本研究以十字形含纖維母細胞之第一型膠原蛋白凝膠為學習模型，以客製可進行動態拉伸之生物反應器營造以下不同的力學環境:自由懸浮培養、等雙軸固定拉伸、定應變之等雙軸往復式拉伸 (CES-7%; CES-20%) 和逐步漸增應變之等雙軸往復拉伸(ICES)。在六天後，我們以客製之雙軸機械測試裝置，定量膠原蛋白凝膠的雙軸機械性質，以非線性光學顯微鏡之二倍頻與雙光子吸收分別觀察膠原蛋白纖維及細胞，以免疫組織化學染色觀察其細胞增生及平滑肌動蛋白的表現，及以RT-PCR定量第一型膠原蛋白、第三型膠原蛋白、纖維連接蛋白、平滑肌動蛋白、週期素D1和轉型生長因子基因的mRNA表現。我們發現產出的凝膠之剛性與其培養之力學環境相關，高低排序依序為: ICES> CES-20%>CES-7% >等雙軸固定拉伸。在非線性光學顯微鏡觀察到膠原蛋白二倍頻的影像中ICES表現最為強烈的纖維微結構。於細胞的型態和分化上，動態拉伸(CES-7% ; CES-20%和ICES)細胞增生的表現量較自由懸浮培養和等雙軸固定拉伸培養來的不明顯，而於α-SMA的表現則為等雙軸固定拉伸高度表現，而定應變等雙軸往復拉伸CES-7%則無明顯表現，但於較大應變CES-20%和逐步漸增應變之等雙軸往復拉伸(ICES)則又再次表現α-SMA。綜合以上凝膠之剛性與胞外間質的微結構及細胞的表現型有關，逐步漸增應變之等雙軸往復拉伸培養環境對於重塑胞外間質、調節細胞型態和TGF-β1的表現較為劇烈。

Insufficient mechanical strength is a main problem of tissue engineered grafts fabricated from natural polymers. Mechanical stimuli have been proven as an important role in turnover of artificial tissues and matrix reorganization. In this study, we used fibroblast-seeded collagen gels as a model to study the mechono-biological responses under defined cyclic biaxial mechanical stretching. The fibroblast-seeded collagen gels were cultured under five different mechanical environments: free-floating, static equibiaxial stretching, constant strain amplitude cyclic equibiaxial stretching (CES-7% and CES-20%) and stepwise incremental strain amplitude cyclic equibiaxial stretching(ICES). We used a custom-made biaxial mechanical testing device to examine the mechanical properties of the gels. Nonlinear optical microscopy was used to observe the collagen fibers and cell in the gel. Cell proliferation and expression of smooth muscle α actin were quantified by immunohistochemistry. Furthermore, RT-PCR was used to quantify gene expression of collagen type I, collagen type III, fibronectin, α-SMA, cyclin D1 and TGF-β1. Results showed that the stiffness of the gels were dependent on the culture mechanical environments: ICES> CES-20%> CES-7% > equibiaxial strain 10%. The image from nonlinear optical microscope illustrate that the gels cultured under ICES had the most intense fibrous microstructure. No significance difference was observed in cell proliferation among gels cultured under CES-7%, CES-20% and static groups, while the expression of α-SMA showed opposite trends. In summary, the stiffness of the gels appeared to correlate the microstructure of the gels as well as the phenotype of the cells. Gels cultured under ICES regulate phenotype of fibroblast, remodeling of matrix, and turnover of cell.

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