| 摘要: | 本研究目的是利用幾丁聚醣與海藻膠、硫酸軟骨素及肝素等聚醣體來形成人工細胞外基質之支架，以利於組織培養與傷口修復。硫酸軟骨素與肝素於生物體內具有調控生長因子活性的能力；海藻膠則是海洋中源源不絕的產物。兩類型態的陰電性多醣體的差異性在於：硫酸軟骨素與肝素擁有調控生長因子活性的磺酸官能基，而海藻膠則是便宜量多等優點。但由於聚陰電性多醣體因電荷性質排斥效應，無法自身形成穩定架構體。所以研究希望利用帶正電荷幾丁聚醣與海藻膠、硫酸軟骨素及肝素陰電性聚醣體產生聚電荷反應，並探討此電荷複合反應機制之架構體是否可以藉由三種不同型態的交聯機制之交聯劑（戊二醛、EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide）、鈣離子）利用共價鍵與離子鍵來產生更穩定的架構體。 過程中此複合聚醣體之水膠體利用相分離技術製造出立體孔洞狀之聚醣體複合膜。進一步，此孔洞膜材利用電荷反應與可誘導血管增生及促進細胞分化生長因子（上皮生長因子，IP=4.6、鹼性纖維母細胞生長因子，IP=9.6）結合，來瞭解聚醣體複合膜對於不同等電點生長因子之吸附與釋放之控制能力。此結合生長因子聚醣體複合膜經由人類纖維母細胞增生程度測試來確定其生長因子之活性，最後將結合生長因子聚醣體複合膜移植至老鼠體內，經由60天修復過程，來觀察複合膜孔洞內部之誘導組織再生的能力。 相關的物性結果呈現，藉由冷凍乾燥所得到的聚醣體複合膜型態為孔洞交錯之結構體，且孔洞大小分佈介於100?200μm。由穩定性結果分析，發現未經交聯處理的聚醣體複合膜，其重量損失隨著官能基莫爾數比例的趨近1，而損失率降低。經一週的穩定度測試結果分析：幾丁聚醣/肝素(重量比2：1)與幾丁聚醣/海藻膠複合膜之重量損失可維持40％以下，而幾丁聚醣/肝素(重量比1：1)與幾丁聚醣/ 硫酸軟骨素呈現不穩定型態（重量損失高達80％）。隨著胺基質子化程度的增加雖然可與陰電性聚醣體增加電荷反應性質，但是由於複合鹽類的殘留，使的損失加速提升，而無法達到原有調控穩定的效果。另一方面，藉由酸鹼調控質子化的胺基或是離子化的羧基與磺酸基還原，來增加材料穩定，重量損失結果發現藉由還原質子化胺基的重量損失可降至30％以下，而還原離子化羧基穩定性質的重量損失依舊維持60％以上。 聚醣體複合膜經戊二醛交聯後，隨著交聯時間的增加而陰電性多醣體流失明顯增加，且材料性質隨交聯程度的提升而材質變的硬脆，這是因為戊二醛醛基與幾丁聚醣胺基反應形成半穿網結構（semi-IPN），而釋出陰電性多醣體。所以隨交聯時間的增加而有較高的重量損失率。EDC交聯的結果雖然為互穿網模式，但交聯的過程中易會造成材料的不穩定性，而於初期會造成材料的流失。且隨時間增加而重量損失增加，顯示EDC交聯劑於聚醣體複合膜系統中，反應速率過慢以致反應不完全而造成流失。但此系列條件的優點為可保有較高比例的負電荷聚醣體，優於調控帶正電荷的生長因子。鈣離子交聯可有效控制負電荷聚醣體的穩定，相對於幾丁聚醣多醣體，可能會因鈣離子競爭反應的條件下而造成流失。交聯與未交聯系列的穩定多醣體複合膜藉由溶菌酵素的加入而增加其降解效率，顯示複合膜生物可降解性之特色。 未經交聯處理的多醣體複合膜，整體上吸附生長因子含量(EGF、Basic-FGF)相較於經交聯處理實驗組有較高的吸收。原因是交聯會消耗電荷官能基，間接導致吸附率下降。經由戊二醛交聯系列之聚醣體複合膜隨交聯改質時間增加而生長因子吸附降低。藉由EDC交聯系列多醣體複合膜，隨著交聯時間的增加，Basic-FGF吸附增加；而EGF卻是隨交聯提升而降低吸附量。鈣離子交聯系列多醣體複合膜，如幾丁聚醣/海藻膠與幾丁聚醣/硫酸軟骨素就有大致上相同的模式，隨鈣離子交聯時間的增加，而降低Basic-FGF與EGF吸附。而幾丁聚醣/肝素卻是隨交聯時間增加而Basic-FGF與EGF吸附增加，這是因為幾丁聚醣/肝素可形成較穩定材料，而保有兩者多醣體官能基性質所致。 生長因子控制釋放方面，EGF(1~12%)的整體釋放量都低於Basic-FGF(5~20%)釋放，顯示多醣體複合膜會因生長因子電荷性質而吸附有所不同。生長因子釋放量依據吸附量與材料穩定性調控，且於體外細胞實驗得知：生長因子(EGF、Basic-FGF)與多醣體複合膜經由電荷吸附結合確實能保有其活性。 由動物實驗30天結果呈現，包覆mEGF與Basic-FGF生長因子之多聚醣架構體可以促進微血管的增生，與加速膠原蛋白束與細胞外基質的形成。相較於控制組60天發炎反應的結果，包覆生長因子的實驗組可以加速細胞的遷入以增進多聚醣架構體的分解而形成新生的組織。 In the present study, a novel method was designed to prepare chitosan-alginate, chondroitin sulfate and heparin complex based artificial extracellular matrix (scaffolds) for wound repairing. Tissue engineering is a newly developed specialty involves the construction of temporary scaffolds to serve as a three-dimensional (3-D) template for initial cell attachment and subsequent tissue formation. Ideally, a scaffold should be fabricated from biocompatible and bioresorbable materials conducive to cell attachment, proliferation, and differentiation. It should also be high porosity with an interconnected pore network for cell growth and transport of nutrients/metabolic waste. However, even if cells are distributed throughout a scaffold, there is a need for a vascular supply to nourish the cells in the interior of the scaffold. Thus, stimulation of blood vessel ingrowth into the scaffold would assure tissue survival and function. It was reported that basic fibroblast growth factors (bFGF) played an important role for angiogenesis. The vascularization could be promoted by bFGF to provide sufficient nutrient transport for the transplanted cells. One promising way to enhance in vivo efficacy of growth factors is the controlled release at the site of action over an extended time period by incorporating the growth factor into an appropriate bFGF-binding material. Heparin and chondroitin sulfate, the sulfated glycosaminoglycans, can stabilize an active conformation of bFGF to protect them from proteolysis and enhance their interaction with specific cellular receptors. Alginate is a negatively charged polysaccharider. The carboxyl groups on alginate appear to bind with basic amino acid residues in the FGFs. Chitosan is a copolymer of glucosamine and N-acetylglucosamine obtained by N-deacetylation of chitin, which has structural characteristics similar to extracellular glucosaminoglycan. Chitosan-based biomaterials have been noted for its wound-healing acceleration, cartilage repairing and bone-forming ability in several studies. It is believed that the combination of chitosan-alginate, chondroitin sulfate and heparin is of benefit to binding bFGF for tissue repairing. Since alginate, chondroitin sulfate and heparin were very soluble in water, we want to couple the polysaccharides to chitosan respectively using glutaraldehyde, EDC and calcium ion for the preparation of stable polysaccharides complex scaffolds. The process involves the construction of three-dimensional (3-D) porous films based on polysaccharides complex. To evaluate the interaction of growth factors (EGF, IP=4.6; bFGF, IP=9.6) with the polysaccharides complex scaffolds, the adsorption and release properties of EGF and bFGF-conjugated scaffolds are examined by ELISA studies. The bFGF or EGF releasing from the polysaccharides complex scaffolds retain its biological activity as examined by the in vitro proliferation of human fibroblast and in vitro histological examination of regenerative tissue. The lyophilized product of the polysaccharides complex scaffolds show interconnected porous structures with pore size of 100-200μm. After one weeks of soaking in PBS solution, the weight loss of non-crosslinked polysaccharides complex scaffolds approach to 100%. Less than 40% of weight loss is observed from the chitosan-heparin (chitosan/heparin=2:1) and chitosan-alginate scaffold; however, almost 80% of weight loss can be found from the chitosan-heparin (chitosan/heparin=1:1) and chitosan-chondroitin sulfate scaffold. The protonation of amino groups and ionization of the carboxylic acid and sulfonate groups are responsible for the stability of prepared polysaccharides complex scaffolds. Weight loss of the polysaccharides complex scaffolds were reduced to less than 30% after deprotonation of amino groups. On the contrary, the polysaccharides complex scaffolds still retain more than 60% of weight loss after reduction of carboxylic ions. After crosslinked by glutaraldehyde, all serious of chitosan/anion polysaccharide scaffolds were appeared anion polysaccharide losses seriously and brittle properties obvious with crosslinking time increased. The phenomenon due to amine group reaction with aldehyde to form semi-IPN and then anion polysaccharide were liberated. From literature report the crosslinked type of EDC was showed into full interpenetrating, but using in chitosan/ anion polysaccharide scaffolds will induce unstable during the initial stage due to its polyion chains destroyed by carboxyl group reaction to imime group of EDC. This phenomenon indicated both chitosan and anion polysaccharide with losses through EDC reaction, and weight loss increased obvious in three type chitosan/anion polysaccharide scaffolds by EDC reaction time increased through one week. But in this case, we can find that sulfate element content were raised after EDC crosslinked 24hours in chitosan/ chondroitin sulfate and chitosan/heparin scaffolds, the higher sulfate element content will benefit to absorption basic fibroblast growth factor. The third type crosslinked agent is calcium ion. Even though divalent calcium ion will stable aion polysaccharides (carboxyl and sulfuric group), but its competition reactions with amine on chitosan would resulting into chitosan and anion polysaccharide weight losses during the primary stage. Due to the decrease of electronic groups, the efficiencies for the adsorption of growth factors (EGF and bFGF) to the cross-linked polysaccharides complex scaffolds were less than that of the non-crosslinked polysaccharides complex scaffolds. By cross-linked with glutaraldehyde, the bFGF- and EGF-adsorption efficiencies of the polysaccharides complex scaffolds decrease with the increase of reaction time for crosslinking. The bFGF-adsorption efficiencies of the polysaccharides complex scaffolds increase with the increase of reaction time for crosslinking; however, the EGF-adsorption efficiencies decrease with the increase of reaction time for crosslinking. In case of EDC crosslinked, the Basic-FGF absorption efficiencies increase through EDC reaction time increase; on the contrary, the EGF absorption amounts decrease with EDC reaction time increase. In other hand, divalent calcium ion crosslinked type polysaccharide scaffolds, such as chitosan/alginate and chitosan/chondroitin sulfate had same absorption model in Basic-FGF and EGF which efficiencies increase with the increase of reaction time for crosslinking. But both growth factors absorption efficiencies increase with the increase of reaction time for calcium ion crosslinking under chitosan/heparin scaffolds. The release of growth factors (EGF and bFGF) determined by ELISA assay indicates that the release profiles are dependent on the electrostatic interaction between the polysaccharides complex scaffolds and growth factors. There are 1~12% of EGF release and 5~20% of bFGF release from the polysaccharides complex scaffolds. The biological activity of released EGF and bFGF are examined by the continued proliferation of human fibroblasts. The result indicates that the released bFGF retained its biological activity to enhance the proliferation of human fibroblasts, within one week of incubation. By the animal experiment results show that the polysaccharide scaffold combination with mEGF and Basic-FGF will promote capillaries newborn and than modulate the fibroblast to accelerate the collagen hyperplasia in period of 30days. Compare with the control(inflammation reaction) in 60 days, the scaffold combine with growth factor can also accelerate the cell move into scaffold and then decompose the polysaccharide by enzyme and replace with newborn one, this phenomenon can be utilized to tissue repair or induce new-born organ regenerates of damaging. Final effects will benefit in the development of the tissue engineering research. |
