| 摘要: | 臨床上生物組織常被用來當做製造人工心臟瓣膜、小口徑人工血管、人工補綴片及人工韌帶等材料的來源。這些生物組織材料的來源可以是牛、馬、豬或羊等哺乳類動物的心瓣膜、血管、心包膜及肌腱等等。然而這些生物組織材料在植入人體之前，都必須先經過交聯劑的交聯處理，交聯處理的目的主要是增加生物組織材料的穩定性，及降低其免疫排斥性與抵抗人體酵素的分解。目前被用來交聯生物組織的交聯劑都是由化學合成而來的，包括有：formaldehyde、glutaraldehyde、dialdehyde starch及epoxy compound等。而glutaraldehyde是目前臨床上使用最多的生物組織交聯劑。但是由於glutaraldehyde的毒性過高，而導致了經glutaraldehyde交聯處理過的生物組織，在植入體內後，會產生組織纖維化、硬化及鈣化的現象。 為了克服化學合成交聯劑所產生的問題，我們實驗室著手使用了一種從中藥梔子果實裡萃取出來的天然交聯劑genipin，來交聯處理生物組織材料。因此本論文主要是評估，以天然交聯劑genipin交聯處理生物組織，以做為製造人工器官生醫材料的可行性。主要的目的是期望能以傳統中藥的特色，找尋新一代製造人工器官的生物組織材料。 在第一部份的實驗裡我們以從中藥店裡購回的梔子果實，萃取純化出geniposide，接著應用酵素固定化的技術來將geniposide轉換成genipin。在本實驗裡，我們成功的從中藥梔子果實中萃取純化出純度達98%以上的genipin。 在第二部份的實驗裡，我們評估了以天然交聯劑genipin交聯處理的生物組織的交聯性質，在本部份的實驗裡，我們證實了genipin能有效的交聯生物組織材料，並且我們也進一步證實了genipin主要是與生物組織中的lysine、hydroxylysine與arginine上的自由胺基產生交聯反應。另外，我們也發現以genipin交聯處理的生物組織，其機械性質與抗酵素分解能力，皆與glutaraldehyde交聯的生物組織相當。 在第三部份的實驗裡，我們以體外細胞培養的方式，評估了天然交聯劑genipin本身與其交聯處理的生物組織的細胞毒性，而glutaraldehyde與其交聯處理的生物組織為我們的對照組。本部份的實驗結果顯示，genipin本身的細胞毒性比glutaraldehyde低約5000~10000倍。並且以glutaraldehyde交聯處理的生物組組織釋放出來的物質有明顯的細胞毒性，而genipin交聯處理的生物組織釋放出來的物質則沒有明顯的細胞毒性。另外，在glutaraldehyde交聯處理的生物組織本身的細胞相容性的測試結果，genipin交聯處理的生物組織的細胞相容性明顯的比glutaraldehyde交聯處理的生物組織要來得好。 而在第四部份的實驗裡，我們以體外細胞培養的方式，來進一步評估天然交聯劑genipin的遺傳毒性，而glutaraldehyde為我們的實驗對照組。根據細胞微小核及姊妹染色體互換的實驗結果，我們發現glutaraldehyde (0.001 ppm、0.005 ppm、0.01 ppm)在未經S-9酵素活化的系統以及經S-9酵素活化的系統中，都呈現了弱遺傳毒性的反應。而genipin在未經S-9酵素活化的系統以及經S-9酵素活化的系統中，除了在高濃度(50 ppm)下有弱遺傳毒性的反應外，在其他的測試濃度(1 ppm、10 ppm)下，genipin都沒有呈現遺傳毒性的反應。 在第五部份的實驗裡，我們評估了以天然交聯劑genipin交聯處理的生物組織，在生物體內的生物相容性質。實驗裡，我們分別以glutaraldehyde、epoxy compound以及未經交聯處理的生物組織當做對照組，植入老鼠背部的皮層底下。然後分別於1週、4週與12週後，將這些植入的測試組織取出來，分別比較它們的生物相容性質，包括了：測試組織的免疫反應、機械強度的變化、變性溫度的變化、鈣化程度、組織形態的變化情形等。實驗結果顯示，genipin交聯處理的組織的生物相容性，很明顯的比glutaraldehyde或epoxy compound交聯處理的組織要來得好。另外，genipin交聯處理的生物組織在生物體內的抗酵素分解能力，與glutaraldehyde相當，而比epoxy compound要來得好。 在第六部份的實驗裡，我們利用去細胞後的牛心包膜再經genipin交聯處理，來發展新型的人工補綴片。實驗裡，我們分別以genipin及glutaraldehyde交聯處理未去細胞的牛心包膜，以及經glutaraldehyde交聯處理的去細胞的牛心包膜當做對照組，植入老鼠背部的皮層底下。然後分別於3天、1週、4週、12週、24週以及52週後，將這些植入的測試組織取出來，分別比較了它們的生物相容性質，包括了：測試組織的免疫反應、機械強度的變化、變性溫度的變化、鈣化程度等。實驗結果顯示， genipin交聯處理的組織的生物相容性，很明顯的比glutaraldehyde交聯處理的組織要來得好。而且經去細胞後的生物組織能提供細胞遷入的自然的組織結構，並以genipin交聯處理的去細胞組織, 其組織的再生率比glutaraldehyde交聯處理的去細胞組織要來得快。 在第七部份的實驗裡，我們進一步的評估以genipin來交聯處理牛頸靜脈做為人工肺動脈，來重建狗的肺動脈的可行性，而我們的對照組是以glutaraldehyde交聯處理的牛頸靜脈。在植入狗體內4週及24週之後，我們分別取出植入的人工肺動脈（每組4隻動物），並分別評估及比較它們的免疫反應、變性溫度、機械強度、鈣化程度、及血管裡的血栓程度與血管外的沾黏程度等性質。實驗的結果顯示， genipin交聯處理的valved conduit的免疫反應程度比glutaraldehyde交聯處理的valved conduit來得輕微。並且以genipin交聯處理的valved conduit在植入24星期後，在血管內壁及瓣膜表面都有內皮細胞遷入，而在血管內壁及瓣膜表面形成一相當完整的新生內皮細胞層。相對的，glutaraldehyde交聯處理的valved conduit在植入24星期後，不但沒有發現到新生的內皮細胞層，且在瓣膜表面還有一些血栓存在。 綜合以上體外及體內的實驗結果，genipin比glutaraldehyde較適合用來當做生物組織交聯劑。 Bioprostheses derived from biological tissues can be used to repair or replace defect tissues or organs in the clinical applications. These biological tissues, however, must be chemically modified by a crosslinking agent before they can be implanted in humans. Various synthetic crosslinking agents have been used in modifying biological tissues. Nevertheless, these synthetic crosslinking agents are all highly cytotoxic. It is therefore desirable to provide a crosslinking agent for use in biomedical applications that is of low cytotoxicity and that forms stable and biocompatible crosslinked products. In an attempt to achieve this goal, a naturally occurring crosslinking agent (genipin) was used by our group to fix biological tissues. Genipin and its related iridoid glucosides extracted from the fruits of Gardenia jasminoides ELLIS have been widely used as an antiphlogistic and cholagogue in herbal medicine. In this dissertation, we present the results obtained in our feasibility, cytotoxicity, genotoxicity, and biocompatibility (cellular tissue, acellular tissue, and valved conduit) studies of this naturally occurring crosslinking agent for biological tissue fixation. In the study, we successfully purified a naturally occurring crosslinking reagent (genipin) from gardenia fruits, a traditional Chinese medicine. The purity of genipin obtained was approximately 98%. In the feasibility study, the crosslinking characteristics of the genipin-fixed biological tissues was investigated. It was found that the amino acid residues in the porcine pericardium that may react with genipin were lysine, hydroxylysine, and arginine. Additionally, the genipin-fixed tissue had comparable mechanical strength and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. This suggested that genipin can form stable crosslinked products. The results of this in vitro study demonstrated that genipin is an effective crosslinking agent for biological tissue fixation. In the cytotoxicity study, the cytotoxicity of genipin was assessed in vitro using 3T3 fibroblasts (BALB/3T3 C1A31-1-1). Glutaraldehyde, the most commonly used crosslinking agent for tissue fixation, was used as a control. The cytotoxicity of the glutaraldehyde- and genipin-fixed tissues and their residues was also evaluated and compared. The results of the study demonstrated that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde. Also, the cellular compatibility of the genipin-fixed tissue was superior to its glutaraldehyde-fixed counterpart. Finally, the residues from the glutaraldehyde-fixed tissue markedly reduced the population of the cultured cells, while those released from the genipin-fixed tissue had no toxic effect on the seeded cells. The genotoxicity study was to evaluate the genotoxicity of genipin in vitro using Chinese hamster ovary (CHO-K1) cells. Glutaraldehyde was employed as a reference chemical. The selected procedures for this evaluation were the micronucleus (MN) and sister chromatid exchange (SCE) assays with or without the addition of metabolic activation system (S9 mix). Before starting the genotoxicity assays, the maximum noncytotoxic amounts of glutaraldehyde and genipin were determined using the MTT assay. The results obtained in the study revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde with or without the addition of metabolic activation system (S9 mix). Additionally, a weakly clastogenic response of glutaraldehyde in CHO-K1 cells was found in the genotoxic assays. In contrast, no clastogenic effect from genipin in CHO-K1 cells was observed, except that drugged with the highest dose (50 ppm). In the biocompatibility study, a genipin-fixed porcine pericardium was implanted subcutaneously in a growing rat model. Fresh, and the glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was noted that the inflammatory reaction of the genipin-fixed tissue was significantly less than its glutaraldehyde- and epoxy-fixed counterparts. Also, the genipin-fixed tissue had comparable tensile strength and resistance against in vivo degradation as the glutaraldehyde-fixed tissue. In the In the acellular tissue study, the biocompatibility of cellular and acellular bovine pericardia fixed with genipin were evaluated in implanted subcutaneously in a growing rat model and to investigate the cell migration rate into cellular and acellular bovine pericardia fixed with genipin. The glutaraldehyde-fixed counterparts were used as controls. The results indicated that the inflammatory reactions of the cellular and acelluar genipin-fixed tissues were significantly less than their glutaraldehyde-fixed counterparts. The acellular tissues provided a scaffold that be used for cell migration for tissue regeneration. The tissue regenerating rate for the acellular genipin-fixed tissue was faster than its glutaraldehyde-fixed counterpart. In the valved conduit study, the biocompatibility of bovine jugular vein fixed with genipin was to reconstruct the right ventricular outflow tract implanted in a canine model. The glutaraldehyde-fixed counterparts were used as controls. It was noted that the inflammatory reaction of the genipin-fixed valved conduit was significantly less than its glutaraldehyde-fixed counterpart. Also, in the blood flow surface of the genipin-fixed valved conduit was covered by flattened endothelial cells. In contrast, in flow surface of glutaraldehyde-fixed valved conduits, no endothelial cell were observed. The results obtained in the aforementioned studies indicated that genipin is an effective crosslinking agent for biological tissue fixation. Additionally, as far as cytotoxicity, biocompatibility, and genotoxicity are concerned, genipin is a promising crosslinking agent for biological tissue fixation. |
