於乙烯-乙烯醇共聚物上高效率製造可注射之毛囊狀真皮乳突-角質細胞混合性微組織應用於毛囊再生

Abstract

先前研究中指出，將毛囊之真皮乳突細胞培養於乙烯-乙烯醇共聚物poly(ethylene-co-vinyl alcohol) (EVAL)，能使真皮乳突細胞自我聚集成立體微組織，並保有誘導毛囊新生之能力。而毛囊的生成時必須要有表皮細胞參與，因此植入的位置要非常精準地接近表皮才能成功誘導出毛囊，但這於臨床使用上是具有困難度的。 目前在毛囊再生的研究中，大多需以胚胎鼠或新生鼠做為細胞來源，才能誘導毛囊再生。但胚胎或新生細胞來源非常不易，且具有倫理道德上的爭議。而禿髮大多發生於成年人，目前利用成體細胞誘導毛囊生成是極困難的。因此本實驗構想為以成鼠的真皮乳突細胞與腳掌之角質細胞做為細胞來源，將此兩種細胞同時培養於EVAL，希望此兩種細胞能於EVAL上形成大量混合性微組織，並具有毛囊再生之能力，提供一個利用自體細胞生成大量毛囊狀微組織之方式，運用於毛囊再生。 由實驗結果中發現，真皮乳突細胞於EVAL上能形成許多球形微組織，角質細胞於EVAL不貼附，呈懸浮狀態。而將真皮乳突細胞與角質細胞共同培養於EVAL上也能形成許多混合性球形微組織。 進而針對混合性球形微組織做分析，其結構為真皮乳突細胞分佈於微組織中心而角質細胞分佈於外圍，形成類似毛囊構造 (folliculoid)。在培養後第一天與第三天，微組織內之細胞存活率仍保有九成，顯示細胞形成微組織後，依然具有其活性。在基因表現上，角質細胞朝向表皮分化的基因表現會下降 (keratin 1, loricrin)，朝毛囊分化的基因表現會上升 (keratin 6)。功能上，這些微組織在植入動物體後能生成新的毛囊與髮幹構造。 我們進一步探討細胞於材料上貼附與移行之關係，推測微組織形成之機轉。真皮乳突細胞於EVAL具有中度的貼附率且有較高的移動速率。而角質細胞不易貼附於EVAL，但易貼附於真皮乳突細胞。藉由真皮乳突細胞於EVAL上形成微組織之同時，能帶動貼附於其上的角質細胞。可能由於真皮乳突細胞間黏著性大於角質細胞，因此真皮乳突細胞在移動形成微組織之同時會往細胞團中心聚集，角質細胞則被動地被推向微組織的外圍，最後會因微組織外圍的角質細胞不易貼附於EVAL，使原本貼附於EVAL的微組織離開基材表面，懸浮於培養液中。 利用此方式製造出的微組織，細胞間能直接互相接觸，提高細胞間交互作用，增加真皮乳突細胞將表皮的角質細胞誘導為毛囊中的角質細胞之機率，且能於短時間獲得大量微組織，因此更能實際運用於臨床研究上。

Dermal papilla cells can self-assemble into spheroidal microtissues on poly(ethylene-co-vinyl alcohol) (EVAL) membranes and these microtissues are able to induce hair follicle neogenesis in our previous study. However, epidermal cells are necessary for hair follicle neogenesis. Therefore, the dermal papilla microtissues should be precisely injected to the dermal-epidermal junction. However, it is extremely difficult in clinical practice. According to the study of hair follicle regeneration so far, it is necessary to use embryonic or newborn cells as the source for successfully making the hair follicle in vitro. But it is hard and has provoked a great deal of controversy. Furthermore, the alopecia usually appears in adults. Therefore, using adult cells is difficult to induce hair follicle. The purpose of this study is to generate functional dermal papilla-keratinocyte hybrid microtissues by substratum-facilitated cell self-assembly using adult rat cells for hair follicle regeneration. We found that dermal papilla cells alone can attach and form spheroidal microtissues on EVAL membrane, but keratinocytes alone fail to attach and become suspended. When dermal papilla cells and keratinocytes are simultaneously seeded, they are able to form hybrid microtissues on EVAL membrane. Structurally, the hybrid microtissues have dermal papilla cells aggregated in the core and keratinocytes sorted to the surface, forming a structure mimicking bulbs. Cell viability remains as high as 90% in the microtissues. In gene expression, keratin 1 and loricrin are down regulated while keratin 6 is up regulated. Functionally, after being transplanted to nude mice, the microtissues are able to regenerate new hairs. Analyzing behavior reveals that keratinocytes tend to attach to dermal papilla cells. Consequently, the keratinocytes are carried by the motile dermal papilla cells on EVAL membrane and are pushed to the outside of microtissues by dermal papilla cells during the self-aggregation. Finally, the microtissues spontaneously detach from EVAL membrane.