摘要
組織工程為近幾年來新興的生醫技術，其主要結合醫學應用與工程材料知識，研發能修復人體病變組織與器官的零件，以克服器官捐贈來源短缺的需求。理想的組織工程人工細胞外間質，必須盡量模擬真實生物體內的細胞外間質組成，以提高材料對細胞的相容性。同時必須具備一多孔性的結構，以提供足夠的空間讓宿主的血管與細胞遷入，以進行修復的動作。另外隨著植入生物體內時間的增加，人工細胞外間質必須可以被生物體內的酵素或水分所分解，而逐漸由新生細胞所分泌的細胞外間質所取代，以達到組織再生與修復的目的。
天然生物組織材料如牛、馬或豬等的結締組織，擁有完整的細胞外間質，包括了結構性纖維和親水性物質等，不需再接枝其他成份來模擬生物體的細胞外間質環境以提高材料對細胞的相容性。另外，去細胞後的生物組織能夠形成一3-D多孔性結構，而且仍保有生物組織細胞外間質的大部分組成，因此應該可以被當做組織工程人工細胞外間質使用。另外，在我們先前的研究結果裡，已證實以genipin交聯處理的生物組織有相當好的生物相容性。
因此本論文主要的目的為，應用組織工程的技術，評估以天然交聯劑genipin交聯去細胞的牛心包膜，做為組織工程人工細胞外間質的可行性。論文裡，評估了不同孔洞結構以及經不同交聯程度處理的去細胞牛心胞膜，在生物體內的再生與修復模式。同時我們也以genipin交聯處理去細胞的牛心包膜，做為修補心血管的人工補綴片，以評估發展組織工程人工補綴片的可行性。另外，我們也評估了人參萃取物ginsenoside Rg1(Rg1)當做新型促進血管新生因子的可行性，以解決在組織工程應用裡血管新生不足的問題。最後我們也探討了，利用去細胞牛心包膜內醣胺素鏈上的磺酸基(sulfate group)或羧基(carboxylic acid group)來與bFGF結合，以增加裝載bFGF的量並提高bFGF在生物體內的穩定性以避免蛋白質失去活性。
在第一部份的實驗裡，我們以Triton X-100、protease inhibitor、DNase及RNase等試劑去除牛心包膜內的細胞後。分別利用定性與定量分析的方法探討去細胞前後，牛心包膜內細胞外間質組成的變化。同時，我們也比較去細胞前後以及經genipin交聯後變性溫度與機械性質的差別。另外，我們也利用了醋酸處理與酵素處理的步驟，來增加去細胞牛心包膜的孔洞大小及孔隙度。實驗裡，我們也探討了不同孔洞大小及孔隙度的去細胞牛心包膜，在植入生物體內後血管新生的情形。實驗結果證實，牛心包膜在去細胞的過程中，除了洗去了部份醣胺素與水溶性蛋白外，原來的膠原蛋白以及大部分的醣胺素，仍然保存於其天然細胞外間質的結構內。另外，牛心包膜在去細胞之前與之後的變性溫度與機械性質，並沒有明顯的差異。動物實驗結果顯示，增加去細胞牛心包膜的孔洞大小及孔隙度，可以加快宿主血管滲入試片內的速度。
在第二部份的實驗裡，我們以genipin做為生物組織材料的交聯劑，並利用組織工程的技術來評估不同交聯程度的去細胞牛心胞膜，在植入老鼠背部後的再生與修復能力。實驗裡，分別在3天、1個月、3個月、6個月與1年後取樣，針對植入後試片的免疫反應、細胞遷入種類與滲入數目(infiltrated cell number)、組織修復時新生的細胞外間質組成、鈣化程度以及機械性質等加以評估。實驗結果顯示，不同交聯程度的去細胞牛心包膜會影響其在生物體內被分解的速率，而造成不同的組織再生型式。
在第三部份的實驗裡，我們以genipin交聯處理去細胞的牛心包膜(AGP補綴片)，做為修補狗肺動脈的人工補綴片，以評估發展組織工程人工補綴片的可行性。實驗的對照組為glutaraldehyde交聯處理去細胞的牛心包膜(AGA補綴片)與genipin交聯處理牛心包膜(GP補綴片)。在實驗的設計上，我們分別在1週、1個月與6個月後分別取樣，針對植入後的人工補綴片的免疫反應、細胞遷入種類與組織修復時新生的細胞外間質組成、生化性質、鈣化程度、血栓程度與沾黏程度等加以評估。實驗結果顯示，植入狗體肺主動脈1個月後，在AGA與AGP補綴片內明顯有組織再生的情形。包括宿主的纖維母細胞、肌纖維母細胞與內皮細胞都可以遷徙與增殖在補綴片內或上。相反的，宿主的細胞無法滲進GP補綴片內。另外，由於genipin的細胞毒性遠低於glutaraldehyde，因此在AGP補綴片內組織再生的情形比AGA補綴片來得明顯。然而在植入6個月後，在AGA與AGP補綴片內以及AGA、GP與AGP補綴片的內膜增生處都發生了軟骨與骨化生的病理現象。
在第四部份的實驗裡，我們以genipin來交聯經醋酸澎潤與酵素處理後的去細胞牛心包膜，當做人工細胞外間質。然後把Rg1以gelatin包覆在人工細胞外間質內後，植入於老鼠的背部，探討了包覆有Rg1的人工細胞外間質，在生物體內對血管新生以及組織再生與修復的影響。實驗裡，我們以包覆bFGF以及未包覆任何生長因子的人工細胞外間質當做對照組。然後分別在1週與1個月後取樣，針對植入生物體後人工細胞外間質內的血管新生數目、免疫反應、細胞遷入種類與滲入數目、組織修復時新生的細胞外間質組成、生化性質以及變性溫度等加以評估。實驗結果顯示，Rg1為一種有效促進血管新生的新型生長因子，且可加速組織再生及修復的速率。
在第五部份的實驗裡，我們利用去細胞牛心包膜內醣胺素(GAGs)鏈上的磺酸基(sulfate group)或羧基(carboxylic acid group)來與bFGF結合，以增加裝載bFGF的量並提高bFGF在生物體內的穩定性以避免蛋白質失去活性。實驗設計上，我們利用hyaluronidase來分解去除牛心包膜內的GAGs，以製備含有不同GAGs含量的去細胞牛心包膜。實驗裡，我們測試了含有不同GAGs含量的去細胞牛心包膜，可以裝載bFGF的量以及其體外的釋放行為，另外我們也把裝載bFGF的測試試片，植入於老鼠的背部。然後於1週後取樣，針對植入後測試試片內的血管新生數目、免疫反應、細胞遷入種類等加以評估。實驗結果顯示，未被hyaluronidase分解的去細胞牛心包膜比被hyaluronidase分解的去細胞牛心包膜，擁有較多的GAGs。因此除了可以裝載較多的bFGF，還可以有效的調控並持續釋放去細胞牛心包膜內的bFGF，並且在植入生物體內後，能夠持續有效的促進血管新生。

Abstract
A cell extraction process was employed to remove the cellular components from bovine pericardia, leaving a framework of largely insoluble collagen, elastin, and tightly bound glycosaminoglycans (GAGs). The acellular tissues then were fixed with a naturally occurring crosslinking agent (genipin) as a tissue-engineering extracellular matrix (ECM). Effects of the ECM porous structure, GAGs content and its degradation rate as well as incorporation with a novel angiogenic agent (ginsenoside Rg1) on the tissue regeneration patterns in the acellular ECM were investigated subcutaneously in a rat model. Applications of the acellular ECM as a patch in repairing defects in the pulmonary artery in a canine model were evaluated.
In the first study, a cell extraction process was used to remove cellular components from bovine pericardia. Varying pore sizes and porosities of the acellular tissues were then created using acetic acid and collagenase. These tissue samples were fixed with genipin. The study was to investigate the ultrastructures of these acellular tissues and their biochemical and mechanical properties. Additionally, the effect of porous structures of acellular tissues on their in vivo angiogenesis was investigated subcutaneously in a rat model.
After cell extraction, electron microscopy indicated that all cellular constituents were removed from the tissue. The acellular tissues formed distinct patterns in pore size, porosity following treatment with acetic acid and collagenase. Biochemical analyses confirmed that these acellular tissues with distinct porous structures consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans. The thermal stability, and mechanical properties of the bovine pericardial tissue remained unaltered after cell extraction. The porous structures of the implanted samples seem to determine whether successful microvessel-ingrowth takes place. The acetic-acid-treated and collagenase-treated tissues, due to their high pore size and porosity, showed a large number of microvessels infiltrating into the interstices of the implanted samples. In contrast, a low density of microvessels was observed infiltrating into the acellular tissue and penetration of microvessels into the cellular tissue was never encountered.
In the second study, the acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completed degraded before the infiltrated cells began to secrete their own extracellular matrix.
In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.
In the third study, the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart. At 1-month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde-fixed acellular patch was significantly thicker than its genipin-fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipin-fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehyde-fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart.
At 6-month postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk following implantation or a lack of angiogenesis in the regenerated tissue observed at 1-month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia.
In the fourth study, effects of ginsenoside Rg1 (Rg1), a natural compound isolated from Panax ginseng, on angiogenesis and tissue regeneration in a genipin-fixed acellular tissue (ECM) in vivo were investigated. Basic fibroblast growth factor (bFGF) was used as a control. The results obtained at 1-week postoperatively showed that the extent of angiogenesis in the ECM was significantly enhanced by bFGF or Rg1. At 1-month postoperatively, vascularzied neo-connective tissues were found to fill the pores within the ECMs loaded with bFGF or Rg1. There was a significant increase in the neo-capillary density from 1 week to 1 month for the ECM loaded with Rg1, while that observed in the ECM loaded with bFGF stayed approximately the same because of the limitations of protein stability. These results suggested that Rg1 may be a new class of angiogenic agent and may be loaded in the ECM for accelerating tissue regeneration.
In the fifth study, the distinct varying GAGs content of genipin-fixed acellular tissue (ECM) loaded with bFGF, and its release from the ECM was analyzed. The tissue response to the ECM loaded with bFGF was evaluated by H&E after subcutaneous implantaton in rats. It was found that attachment of GAGs to ECM increased the bFGF binding capacity double and resulted in a more gradual and sustained release of bFGF in vitro. The in vivo results obtained at 1-week postoperatively showed that the extent of angiogenesis in the attachment of GAGs to ECM was significantly enhanced by bFGF.

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