利用疏水性PDMS形成具3D結構的真皮乳頭微組織並結合去細胞之豬皮進行體外培養應用於毛囊重建之研究

Abstract

禿髮，對現代人而言是令人困擾的症狀，不管男性或女性都飽受所苦。然而在現今各式各樣的治療方法裡，例如光治療、藥物治療或植髮等，卻有治療效果不穩定以及影響健康的疑慮。因此，隨著組織工程的發展及近年來於毛囊再生的研究，對未來禿髮的改善提供了可行的方法。毛髮自然的發育過程是透過表皮與間葉細胞一系列的交互作用產生。而位於毛髮底部的真皮乳頭細胞便是一種特化的間葉細胞，當毛囊細胞聚集形成立體球狀的構造時，配合與表皮細胞共培養能促使表皮與毛囊細胞的交互作用，即便毛囊細胞經過多代培養之後，仍能使其保有誘導毛髮新生的能力。 因此，本研究的目的在於透過培養真皮乳頭細胞以及表皮細胞形成立體微組織球態，觀察其是否能在非生物體內環境下促使毛髮新生。我們選用聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)作為材料，由於其本身的材料特性，包含具備良好的生物相容性，以及不利於細胞貼附的性質，將真皮乳頭細胞培養於此材料表面便能形成微球組織。加上表皮細胞於毛囊微組織外層包覆形成'core-shell'的形態，並透過免疫螢光染色進行真皮乳頭活性分析(α-SMA、NCAM)，確保其仍保有促毛囊新生的能力。另外，為了提供微球組織適當的生長環境，我們把經由凍融、酵素及界面活性劑等去細胞方式處理的豬皮，經過冷凍乾燥後使用雷射雕刻機於其上產生陣列孔洞，並將core-shell形態的表皮-毛囊細胞微球組織植入此去細胞豬皮中，植入固定時間後透過切片及染色觀察微球組織於其上之發育形態，以證明3D培養的真皮乳頭和表皮細胞配合適當的支架能模擬生物體內毛囊的生長環境，並具有潛力應用於未來的毛髮重建工程上。

Alopecia is a disturbing syndrome for modern people, it means that many men and women have suffered from it. . Although there are different methods such as phototherapy , drug treatments, and hair follicles implantations et al to solve the hair loss problem, none of them have stable and effective treatment results. Also, they may have some side effects which could affect patient's health. Due to the development of tissue engineering and study on hair regeneration over the few decades, the researchers open a new door to improve the unmet needs for hair loss. The natural hair developing process depends on a series of interactions between epidermal cells and the dermal papilla (DP)cell, located in the bottom of hair bulb, which is a specialized mesenchymal stem cell. When those DP cells are cultured in three-dimentional spheroid structure , followed by adding epidermal cells , the epidermal cells will cover the DP spheroid, forming core-shell micro-tissue. The ability to induce hair regeneration could be retained in vitro even the cells are cultured after a few passages. As a result, the aim of this study is to develop a method to form DP-epidermal microtissue easily, and to investigate whether these microtissues could induce hair regeneration in vitro. Polydimethylsiloxane (PDMS), used in this study, is a suitable material for culturing DP spheroids, because it is characterized by excellent biocompatibility, and low attachment for cell culture. After the epidermal cells are co-cultured with DP to form 'core-shell' structure, their hair inductive activity is confirmed by immunochemistry staining , such as α-SMA and NCAM. In addition, to provide the DP-epidermal microtissue with an appropriate developing environment, the porcine skin treated by a series of decellularization process was fabricated by laser engraving to produce microwell arrays on the surface. Then, the DP-epidermal microtissues are implanted into the acellular skin microwell arrays for pre-determined intervals ,and the cells morphology on the skin scaffold during cultivation was observed and validated by histological analysis to prove that 3D cells culture and a suitable growing environment could mimic the natural developing process in vivo and have potential for hair regeneration.