比較動態拉伸頻率對包覆於褐藻酸鈉水膠纖維中纖維母細胞生長與型態的影響

Effects of cyclic stretching on the growth and morphology of fibroblasts embedded in alginate hydrogel fibers

吳旻哲(Min-Che Wu)

指導教授 ： 林忻怡

國立臺北科技大學/工程學院/化學工程研究所/碩士(2014年)

https://doi.org/10.6841/NTUT.2014.00550

摘要

之前的研究發現，若將細胞包覆在褐藻酸鈉水膠之中，生長於褐藻酸鈉水膠纖維中的纖維母細胞，能夠有較快的細胞生長和較佳基因表現，但大多數的拉伸試驗都是將細胞種在材料表面，故本實驗將細胞包覆在5% RGD改質的褐藻酸鈉水膠中，並用快速原型系統製作成3D多孔隙支架，每天以15rpm及60rpm的頻率拉伸樣品1分鐘，而對照組則是靜態培養。於第1、4、7、14天，觀察細胞生長在不同環境下的細胞型態、增生、活性的差異。
在活細胞即時觀察影像系統下發現，實驗組及對照組的細胞會在纖維中移動。DNA定量結果顯示，第7天後實驗組的細胞比對照組生長的還要快。在早期（第1天及第4天）對照組的細胞活性明顯高於實驗組。隨著培養時間的增加（第7天及第14天），實驗組細胞的活性增加並超過了對照組的細胞。Live/Dead染色發現，細胞在1，4，7，14天大多是存活的。HE染色顯示纖維母細胞在拉伸後的14天內沒有明顯的細胞型態改變，但分散均勻，很少團聚。在第一型膠原蛋白免疫螢光染色觀察下，發現膠原蛋白圍繞著細胞，膠原蛋白也無增加的趨勢。

關鍵字

機械刺激 ； 褐藻酸鈉 ； 纖維母細胞 ； 快速成型

並列摘要

In this experiment, cell was mixed in 5% RGD-alginate and used rapid prototyping system to build the 3D porous scaffolds. The experimental group (E) was cyclically stretched for 1 min per day with a frequency of 15 rpm (E-15) and 60 rpm (E-60), respectively; the control group (C) was static culture. The cells were harvested on day 1, 4, 7, 14.
The cell proliferation results show that the cells embedded in alginate fibers grew overtime and that day 7 the cells subjected to stretching grew slightly faster compared to those under static culture. The viability of cells under static culture was higher than that of stretched cells in earlier days (day 1 and 4). As the culture time increased (day 7 and 14), the viability of stretched cells increased and surpassed that of static cells. We found that live/dead staining appeared the cells were mostly survival in day 1, 4, 7, 14. HE staining showed fibroblast stained cultures after 14 days of cyclic stretch have no apparent preferential orientation. Others have shown stretching of cells changes cell morphology. We did not observe this in our experiments. Immunofluorescence staining showed that type I collagen found around the cell and no collagen increased.

並列關鍵字

Mechanical stimulus ； Alginate ； Fibroblast ； Rapid prototyping

參考文獻

16.Sandra A L Moura, Luiza Dias C Lima, Sílvia Passos Andrade, Armando Da Silva-Cunha, Rodrigo L Órefice, Eliane Ayres, Gisele Rodrigues Da Silva. The role of macrophages in wound repair: a review. Plast Reconstr Surg 68 (1): 107-113, 1981.

1.Miron-Mendoza M, Koppaka V, Zhou C, Petroll WM. Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo. Experimental cell research 319: 2470–2480, 2013.

2.Mcgarry James G., Klein-Nulend Jenneke, Prendergast Patrick J. The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts. Biochemical and Biophysical Research Communications 330 (1): 341-348, 2005.

3.Thi MM., Kojima T., Cowin SC., et al. C. Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells. Am J Physiol Cell Physiol 284 (2): 389-403, . 2003.

4.Norvell SM., Ponik SM., Bowen DK., et al. Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules. J Appl Physiol 96 (3): 957-966, 2004.