利用聚二甲基矽氧烷微陣列薄膜形成具誘導毛囊新生能力的真皮乳頭微組織並應用於毛囊重建工程上

Abstract

對嚴重掉髮及患有雄性禿的患者而言，利用組織工程的方式使毛囊再生來治療毛囊缺失可望成為未來一個重要的醫療方法。在毛囊重建工程中，毛囊內的間 葉細胞群，包含真皮乳頭及真皮鞘細胞被指出具有分化表皮角質細胞以形成完整的毛囊結構，但這項能力只存在當真皮乳頭或真皮鞘細胞聚集成多細胞球(微組織)的情況下才可在無毛的皮膚上新生毛囊。 而本研究的目的在於發展一個方式可以大量形成真皮乳頭微組織，使用的聚 二甲基矽氧烷(PDMS)製造的微陣列孔洞薄膜配合真皮乳頭細胞自我聚集的特性，最後希望可以有效率的進行毛囊重建的工作，亦即，先大量擴展真皮乳頭細胞的數量；將真皮乳頭細胞培養成具有誘導能力的微組織；將微組織移植到無毛的皮膚上進行毛囊重建工作。為了達到此目的，我們使用高生物相容性的 PDMS 做為材料製造微陣列孔洞薄膜，薄膜上含有 200μm 到 800μm 不同大小的微孔洞，由於真皮乳頭細胞對 PDMS 的貼附性不佳，因此會掉落到微孔洞內後自行產生聚集的細胞團。我們發現當細胞密度為 2.1*105/cm2 時，在大小為 200μm 以及 300μm 的微陣列孔洞薄膜中(含 100 個微孔洞)可以形成規則排列且大量的均勻球狀微組織。 而為了確認這些為組織誘導毛囊新生的能力，首先我們對為組織進行真皮乳頭細胞特殊細胞標記，包含 α-SMA 及 NCAM 的免疫螢光染色，發現其確實仍保有這些特殊蛋白。之後在一般被認為與真皮乳頭誘導能力有關的鹼性磷酸酶上，我們的微組織也同樣有大量表現。最後，利用真皮乳頭微組織混合新生鼠的表皮細胞植入裸鼠背部，也發現有新生的毛髮結構。 因此藉由本實驗提出的方式，我們確實可以在高細胞密度下利用 PDMS 微孔洞薄膜來形成大量的真皮乳頭微組織，而近一步應用在毛囊工程上及研究毛囊細胞間的交互作用上。

For severe cases of hair loss or alopecia, regeneration of hair follicles (HFs) by tissue engineering is a promising future treatment. To regenerate HFs in hairless skin, the unique HF mesenchymal cells, including dermal papilla (DP) cells and dermal sheath (DS) cells, have been demonstrated to be able to transdifferentiate epidermal keratinocytes into HF structures when the in vitro expanded DP cells are transplanted to the dermis as multicellular aggregates. The aim of this study was to develop a strategy for mass generation DP microtissues from dissociated DP cells by patterned Polydimethylsiloxane (PDMS) membranes and self-assembly of DP cells. After all, we hope we can have an efficient approach for HF regeneration: expansion of DP cells; cultivation of DP cells into inductive microtissues; transplantation of DP microtissues to the hairless skin. For this purpose, we used biocompatible material, PDMS, to fabricate patterned membranes containing many pores with size from 200μm to 800μm. Because DP cells are poorly adherent to the PDMS surface, they tend to aggregate in the pore of the membrane. Therefore, we could generate uniform patterned spheroid DP microtissues within 3 days when the cell density was 2.1*105cells/cm2 on the membrane containing 100 pores with size between 200μm to 300μm. To investigate the possibility of HF reformation induced by DP microtissues, at first, we examined the preservation of the phenotype of DP cells in microtissues. The expression of two markers of DP cells, α-smooth muscle actin (α-SMA) and neural cell adhesion molecule (NCAM), is well preserved in microtissues. Second, cultured DP microtissues also expressed alkaline phosphotase (AP) which has been regarded as an indicator for hair inductivity. Third, typical HF structures formed when mixing DP microtissues with epithelial cells of C57B/6 neonatal mice. Hence we can generate a large-scale DP microtissues by using patterned PDMS membranes at a higher seeding density. This system is of potential to be applied to HF engineering and the investigation of cellular interaction in the pattered follicular cells.