大型、可灌流、可包埋細胞的多孔隙水膠支架之研究

Abstract

儘管1990年代以來組織工程領域大量的研究投入，目前能應用在臨床上的產品仍僅限於少數厚度較薄或代謝活性較低的組織，例如皮膚、氣管、大血管、和軟骨等。臨床上大部份的組織替代品必需是三維的形態，而且要能有足夠的大小。組織工程要達成這樣的需求，必需同時能克服許多的困難，包括提供充分的灌流與質傳(mass transfer)、均勻有效的細胞撒播(seeding)等。更進一步還要於體外建立完整穩定的微循環網絡(microvasculature)、最終製備出可施行血管接合手術的皮瓣(flap)。目前常用的三維細胞培養方法有細胞聚合球體(aggregates)、水膠包埋(in situ forming hydrogels)、預製多孔型支架(preformed porous scaffolds)等，前兩者一旦尺寸加大便難以維持足夠的灌流與質傳；而後者則是會有細胞分佈不均勻、細胞流失、細胞承受過強剪力等問題。除此之外，目前仍未有報告能夠於體外培養出與組織整合的微循環網絡，其中一個主要問題是未能提供穩定的完整結構與灌流，同時又有適合內皮細胞貼附生長的空間。本研究的目的是結合水膠包埋與預製多孔型支架的優點，研製出大尺寸的多孔型水膠支架，可在培養的環境中均勻地包埋細胞、保護細胞，又能有充足的孔隙可供灌流。並且希望能在此穩定灌流的水膠支架當中除了培養目標組織之外還能建立內皮細胞覆蓋的微循環結構，為製備帶血管皮瓣的目標奠立基礎。 本研究可分為三個主要階段：第一階段先確立支架的製程；第二階段測試各種細胞在支架裡的生長特性；第三階段以骨組織植體培養的模型研究水膠型三維灌流系統對骨組織與微循環網絡形成的效應。在第一階段的研究中，我們以明膠(gelatin)為基質，利用轉麩醯胺脢(transglutaminase)交聯來製成可包埋活細胞而且含有巨觀孔隙的水膠支架。不同的配方與製程製造出的水膠支架評估其操作性、孔隙率、孔洞聯通性、滲透率、以掃描式電子顯微鏡觀察其構造等。找到適合的支架製程後，進一步以各種不同類型的細胞測試靜態培養與灌流培養狀態的細胞存活與細胞增殖特性，並以螢光顯微鏡與共軛焦顯微鏡觀察其細胞形態與分佈等特性。在骨組織植體的研究中測試不同的細胞組合、灑播方式、製備流程、與生長補給(growth supplement)，以共軛焦顯微鏡與定性定量檢測來評估於體外灌流系統培養的植體，找出適合形成完整內皮細胞覆蓋的條件與環境，並評估俱有原始微循環網絡的大型三維骨組織植體是否適合長期的體外灌流培養。 我們成功地研製出大尺寸的多孔型水膠支架，可均勻地包埋細胞，又有充足的孔隙可供灌流。我們的方法製備的支架大小與形狀本質上沒有限制。在長達六週的灌流實驗當中，支架的完整性維持良好。經測試多種細胞株與primary cells (NIH 3T3, HuH-7, AML12, hADSC, HUVEC, human chondrocyte, mouse hepatocyte)，除mouse hepatocyte外，均能在調整條件後得到良好的存活率與增殖能力。此外，不同類型的細胞或細胞組合在培養過程中有不同的型態與重新排列(rearrangement)，顯示本方法與材料除了細胞適應性良好，也能提供細胞所需的環境線索，細胞培養在此類支架中，將可能擁有相當的重塑潛力。在骨組織植體的模型中，灌流培養的環境相對於靜態培養與二維培養可達到顯著較好的骨質形成與礦化。以二階段順序灑播hADSC與HUVEC可成功地使內皮細胞完整覆蓋植體孔隙，同時又不會干擾到水膠中骨組織的誘導與生成。 我們的研究成果證明了製備包埋細胞的多孔隙水膠支架的可行性。更因為它具備了結構完整性與良好的灌流特性，能夠達成組織工程所需的足夠大小與三維培養，同時也能於體外形成內皮細胞完整覆蓋的微循環網絡，使我們得以再朝組織工程的夢想再邁向前一步。未來的研究方向除了組織工程方面需要再針對各種不同的組織設計最適合的材料組成與培養環境、進一步於動物體內驗證其優點、設法結合微循環網絡與輸入輸出血管，其他包括體外細胞增殖、體外維生系統、藥物測試平台…等，也都是可能獲得進展的應用。

Despite the large volume of research in tissue engineering since 1990s, only a few products are clinically applicable up to now. These products are mainly thin or metabolically inactive tissues such as skin, trachea, large vessels, and cartilage. For most tissues, functional constructs should be three-dimensional (3D) and have clinically relevant sizes. Creation of such constructs has been limited by many challenges, such as insufficient perfusion and mass transfer in the interior regions of large and avascular constructs, inhomogeneous and inefficient cell seeding, unable to establish stable microvasculature in vitro, and not to mention the difficulty to fabricate a well organized flap with preformed artery and vein for surgical anastomosis. Commonly used methods of 3D cell culture are cell aggregates, in situ forming hydrogels, and preformed porous scaffolds. The former two methods have difficulty in providing sufficient perfusion and mass transfer when the size increased. On the other hand, preformed porous scaffolds have problems in cell seeding and distribution; during perfusion, less adhesive cells may be lost, and cells are exposed to non-physiological shear forces. Besides, one of the major problems in establishing microvasculature integrated with engineered tissue in vitro is lack of integral and stable scaffold suitable for perfusion and additional attachment of endothelial cells. The purposes of this study were, by combining the advantages of in situ forming hydrogels and preformed porous scaffolds, to establish the optimal regimen and process of fabricating large porous hydrogel scaffolds, which are able to encapsulate viable cells in situ, while at the same time can provide plentiful porosity for adequate perfusion. Furthermore, as the groundwork for vascularized flap, we want to demonstrate the ability to establish primitive microvasculature covered with endothelial cells in this stably perfused scaffold. This study consisted of three parts. Part one was to establish the process of fabricating the scaffold. In the part two, the growth characteristics of different types of cells in the scaffold were investigated. Using bone tissue graft engineering as a model, we assessed the effects of 3D hydrogel perfusion system on the formation of bone tissue and microvasculature in the part three. To achieve the goals of cell-laden hydrogel with macro-porosity, we designed different fabrication methods using gelatin as the matrix and transglutaminase as the cross-linking agent. Scaffolds of different regimens and fabrication processes were assessed for gross handling properties, porosity, pore interconnectivity, permeability, and the structures were observed by scanning electron microscopy. Suitable scaffolds were further tested for cell viability and proliferation in static culture and perfusion culture conditions. Epifluorescence microscopy and confocal microscopy were used to evaluate the cell morphology and distribution with different types of cells. In the study of bone tissue engineering, we tested different combinations of cells, methods of seeding, processes of fabrication, and growth supplements to figure out conditions suitable for viable bone tissue formation with integral endothelial covering. The feasibility of long term in vitro perfusion culture of the large 3D bone tissue graft was evaluated. In this study, we successfully fabricated large, macro-porous hydrogel scaffolds, which could encapsulate viable cells evenly, and provide plentiful and interconnected macroporosity for perfusion. This method could be scaled up, and the scaffolds maintained their structures and integrity after six weeks of perfusion. Cell lines and primary cells were tested for compatibility with the scaffold, including NIH 3T3, HuH-7, AML12, hADSC, HUVEC, human chondrocyte, mouse hepatocyte. All the cell types except for mouse hepatocyte demonstrated good survival and proliferation after adjustments of the culture conditions. Moreover, different types or combinations of cells exhibited different morphology and rearrangement, suggesting that the materials used in this method could provide substantial environmental cues and remodeling potential for the cells and engineered tissues. In the bone tissue graft engineering model, perfused constructs showed better bone tissue formation and mineralization than statically cultured ones. Two-staged seeding of hADSC and HUVEC could successfully achieve integral endothelial covering on the pore surface without compromise of osteogenic induction and bone tissue formation in the hydrogel. The results demonstrated the feasiblility in fabricating cell-laden macro-porous hydrogel scaffolds. The constructs were integral in structure, and suitable for perfusion. They could meet the size and 3D requirements of tissue engineering, and primitive microvasculature with integral endothelial covering could be established in vitro. To move forward toward successful tissue engineering, studies involving specialized modification of the materials and culture environment for specific types of tissues should be instituted. The advantages of in vitro established primitive microvasculature should be examined by in vivo studies. Incorporating engineered microvasculature with afferent and efferent vessels remains a challenging task. Other possible applications of this method may include in vitro proliferation of cells, extracorporeal life support, and drug test system.