微細纖維狀仿生薄膜及立體生物性支架之製作及其做為全層皮膚應用之探討

Fabrication and application of biomimic fine-fibrous membranes and three-dimensional biodegradable scaffold in skin tissue engineering

葉佐儀(TSO-I YEH)

指導教授 ： 曾厚

臺北醫學大學/醫學院/醫學科學研究所/碩士(2008年)

摘要

組織工程中「人工全層皮膚」的發展被認為可替代傷口敷料之治療方式，以創造無疤痕組織的皮膚再生。本研究將人工皮膚分成表皮層及真皮層進行模擬。研究主要利用電氣紡絲技術，以生物可降解性高分子PLLA、PBSA為原料，製備粗細差異近十倍之纖維狀薄膜，經表面被覆玻尿酸(Hyaluronic acid)、膠原蛋白(Collagen)及聚葡萄胺糖(Chitosan)等不同物質後，首先進行毒性測試，再進行HaCaT、人類角質細胞(Keratinocyte)貼附及增生試驗；Keratinocyte並進行多層化試驗。由毒性測試結果確認經電紡技術製備之纖維薄膜可有效提供細胞貼附與生長；再由HaCaT及Keratinocyte結果顯示10w/v % PLLA對於細胞之貼附及增生均具較佳；於薄膜表面被覆HA、Collagen則可有效增加細胞貼附及增生效果，其表現甚至優於商品化之細胞培養皿；同時，結果亦顯示：播植細胞前將纖維薄膜浸潤於細胞培養液中，對於未被覆之纖維薄膜於細胞生長表現可大幅提升至與被覆HA或Collagen相近。本研究同時利用Paraffin及PLLA製備多孔質結構物作為真皮層基質之用；製備之轉速與Paraffin spheres的大小成反比關係；而PVA溶液濃度對Paraffin spheres粒徑的影響則不明顯，但會造成不規則狀之Paraffin spheres。大粒徑之Paraffin spheres所形成之多孔成型物愈脆弱。多孔質結構物經細胞毒性測試亦確認可適於細胞生長。總結，本研究結果可推論10w/v% PLLA為較相似於細胞間質之結構，故細胞生長結果最佳；而多孔質結構體推論亦將提供細胞生長甚至產生血管化之良好環境。

關鍵字

人工皮膚 ； 電氣紡絲 ； 多孔質結構物

並列摘要

It’s well known that in the living system, the extracellular matrix (ECM) plays an important role in controlling cell behavior. The ECM is composed of a basement membrane (BM) and complex cross-linked network of proteins and glycosaminoglycans(GAGs), and those non-woven fibrous architecture are already reach nano-sized. It is therefore important to know how to fabricate an artificial nano-sized fibrous structure for use in organ repair and tissue regeneration.
In present study, the major goal is to mimic the epidermal and dermal-like structure as a artificial skin. Biodegradable polymer Poly L-lactic acid (PLLA) and Poly butylenes succinate-co-adipate(PBSA) were used to fabricated various three-dimensional fibrous scaffolds via electrospinning process. Culture HaCaT and epithelial cell (keratinocyte) to determine whether the fibrous scaffolds are perfect for cell growth. The results show that 10w/v% PLLA and coated with HA、collagen respectively on the surface performed the better environment for cells growth. The second section was to create a dermal-like structure. A novel processing technique has been developed to create three-dimensional biodegradable polymer scaffolds with well-controlled interconnected spherical pores. Paraffin spheres were fabricated with a dispersion method, and were bonded together through a heat treatment to form a three-dimensional assembly in a mold. The inter-pore connectivity could be controlled by varying the heat treatment time of the paraffin spheres and the rotation rate , and mechanical properties could be controlled by varying the porosity of the scaffolds. Then the PLLA solution was casted onto the scaffold to form the framework. With an interconnected macroporous structure that promotes cell seeding throughout the interstices of the 3D porous scaffold, these novel matrices may be an excellent scaffold for vascularization in tissue engineering.

並列關鍵字

artificial skin ； electrospinning ； Keratinocyte ； 3D porous scaffold

參考文獻

1. D.N. Herndon, R.E. Barrow, R.L. Rutan, T.C. Rutan, M.H. Desai, S. Abston, A comparison of conservative versus early excision. Therapies in severely burned patients. Ann Surg, 1989. 209(5): p. 547-52; discussion 552-3.

2. J.K. Rose, D.N. Herndon, Advances in the treatment of burn patients. Burns, 1997. 23 Suppl 1: p. S19-26.

3. V. Falanga, Chronic wounds: pathophysiologic and experimental considerations. J Invest Dermatol, 1993. 100(5): p. 721-5.

4. T. Phillips, B. Stanton, A. Provan, R. Lew, A study of the impact of leg ulcers on quality of life: financial, social, and psychologic implications. J Am Acad Dermatol, 1994. 31(1): p. 49-53.

5. F.V. Bittencourt, A.A. Marghoob, A.W. Kopf, K.L. Koenig, R.S. Bart, Large congenital melanocytic nevi and the risk for development of malignant melanoma and neurocutaneous melanocytosis. Pediatrics, 2000. 106(4): p. 736-41.