以角質細胞及真皮纖維母細胞共養於雙層孔洞結構之明膠/葡萄胺聚醣基材作為皮膚組織工程之研究

Abstract

本研究的目的是希望以組織工程的方式發展出一個類皮膚的等價物（skin equivalent），來促進皮膚的修復與再生，藉以幫助臨床上大面積燒燙傷以及傷口難以癒合的病人。 首先需發展出一適合的皮膚支架(scaffold)，為了模擬皮膚細胞外基質(extracellular matrix)的成分，本研究選用明膠/軟骨硫素/透明質酸 (gelatin-chondroitin 6 sulfate-hyaluronic acid; G-C6S-HA)來當作基材。再利用不同冷凍速率之溫度下冷凍乾燥(freeze-drying)的方式形成具有雙層孔洞結構(bi-layer)的G-C6S-HA薄膜;並進一步利用碳二醯胺(carbodiimide)來交聯，以促進此G-C6S-HA薄膜的機械強度。 第一階段將先評估製備出的G-C6S-HA薄膜其孔洞型態(morphology)，物理化學性質(physical-chemical)，以及生物相容性(biocompatibility)，是否適合作為一皮膚再生的模型。 第二階段是利用此模型評估其在體外(in vitro)建構出皮膚等價物(skin equivalent)的可行性。培養過程中，首先利用本實驗室開發出的攪拌瓶反應器(spinner flask)，來種植皮膚真皮纖維母細胞(dermal fibroblast; FB)，以達到高種植效率(seeding efficiency)，以及細胞均勻分佈(homogeneous cell distribution)的優點;接著再將此種植有真皮纖維母細胞的真皮等價物(dermal equivalent)，接種上表皮的角化細胞(epidermal keratinocyte; K)，之後將此皮膚等價物在液面下(submerge)培養一段時間，再移到氣液介面下(air-liquid interface)讓皮膚表皮組織成熟分化(epidermalization)。經過三到四週的培養，表皮角化細胞已能從基底層(basal layer)開始往上分化出基底上層(suprabasal layer)和角質層(cornified squamous layer);真皮纖維母細胞同時也開始分泌出自己的細胞外基質來逐漸取代原來的支架位置，並發展出一類似真皮的結構。 第三階段將利用此模型在3D仿器官生長(organotypic culture)的模式下，探討皮膚基底層(basement membrane; BM)與細胞外基質(extracellular matrix; ECM)蛋白的生成與活性表現，並利用組織免疫化學法(immunohistochemistry)與即時定量聚合酵素連鎖反應(real time PCR)來加以定性並定量分析，探討其分泌量上的變化與受何種細胞所調控分泌。研究中發現BM and ECM蛋白的表現是受到表皮細胞與真皮纖維母細胞的精密調控(sophisticated interplay)，隨著培養的時間會有所變化，最後達到動態的平衡。 第四階段將種植有人類表皮角化細胞與真皮纖維母細胞(human K&FB)的G-C6S-HA皮膚替代物(skin substitute)，移植到免疫不全(severe combined immunodeficiency; SCID)的老鼠背部，評估其促進傷口修復(wound healing)的能力。結果顯示，在移植四週後，新長出來的皮膚組織具有完整的表皮與真皮構造，且沒有疤痕(scar)或組織沾黏(tissue adhesion)的情形產生，並擁有基底蛋白與分化蛋白的完整表現。 總結，利用冷凍乾燥與碳二醯胺交聯方式製備出的雙層孔洞結構之G-C6S-HA薄膜，具備有良好的型態，物理化學性質，以及生物相容性;在動物活體(in vivo)實驗上亦證明其具有促進傷口修復與良好的可吸收性(graft take)。未來此雙層孔洞結構之G-C6S-HA薄膜除了可當作支架，應用在組織工程上外;此人工皮膚替代物亦極具有潛力，應用在治療大面積燒燙傷與傷口難以癒合的病患身上，造福人群。

In the study, the gelatin-chondroitin 6 sulfate-hyaluronic acid (G-C6S-HA) membrane was designed and fabricated as the scaffold to mimic the skin extracellular matrix in composition for skin tissue engineering. The bi-layer G-C6S-HA membrane with different pore sizes on each layer was prepared using freeze-drying technique. The biological stability of the membrane was improved by 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide cross-linking process. The morphology, physical-chemical properties, and biocompatibility of the bi-layer G-C6S-HA membrane were evaluated to ensure bi-layer G-C6S-HA membrane applicable as a potential model for skin tissue engineering. To reconstruct the skin equivalent, dermal fibroblasts were dynamically inoculated by self-designed spinner flask for optimizing homogeneous cell distribution in the G-C6S-HA membrane. After seeding with epidermal keratinocytes, the skin equivalent was cultured in submerged condition followed by transferring to air-liquid interface for epidermalization. Three weeks later, the keratinocytes differentiated into an epidermis-like structure with suprabasal layers and cornified squamous layer. The lower part of the membrane has developed into a dermis-like structure with sparse cell distribution surrounding with newly synthesized extracellular matrix. Next, this reconstructed skin equivalent was adopted to investigate the phenotypic and molecular expression of basement membrane (BM) and extracellular matrix (ECM) proteins in vitro. The deposition of BM and ECM proteins secreted by keratinocytes and dermal fibroblasts was quantitatively characterized by real-time PCR and examined by immunohistochemistry. The dynamics of RNA expression and protein deposition indicated a gradual synthesis pattern and assembly of the different constituents in a sophisticated and tightly regulated epithelial-mesenchymal interplay. Lastly, G-C6S-HA membrane was seeded with keratinocytes and dermal fibroblasts; then grafted onto the dorsum of SCID mice to evaluate the effect on promoting wound healing process. Four weeks after transplantation, the regenerated skin exhibited a well-developed epidermis and dermis that expressed the differentiated markers and the specific basement membrane proteins. The bi-layer G-C6S-HA skin substitute not only had positive effect on promoting wound healing process, but also revealed high percentage of graft take rate. To sum up, it is suggested that this newly designed bi-layer G-C6S-HA membrane is valuable as a skin substitute and possess great potential in the treatment of clinical patients with extensive burns and chronic wounds in the future.