多孔狀幾丁聚醣-動物明膠/聚乳酸複合支架之製備及其特性探討

Abstract

由冷凍凝膠法或快速原型技術製備的多孔狀基材，可以建構出適合細胞生長的環境，故在發展組織工程支架的領域上具有潛力。冷凍凝膠法是結合非溶劑相分離法和熱誘導相分離法，而快速原型技術中的熔融沉積法是將設計的三維圖樣透過機器將材料以層層堆疊的方式建構出實體，並且經由調控列印的參數，可方便且有效地控制實體的結構和孔隙度。本研究擬選用具有優異機械強度的聚乳酸(P)以快速原型技術中的熔融沉積法製備組織工程的支架，再利用冷凍凝膠法將生物相容性良好的天然高分子幾丁聚醣(C)和明膠(G)在聚乳酸支架大孔洞中形成孔洞較小由C或CG所組成的多孔狀結構(稱為C/P複合支架或CG/P複合支架)，開發適用於硬組織的組織工程支架，此外含有明膠的支架會以EDC/NHS(EN)交聯以提升其穩定性。 本研究以掃瞄式電子顯微鏡(SEM)觀察支架的結構，除了研究熔融沉積法的製程參數外，也在冷凍凝膠法中使用快速冷卻模式(FC)或慢速冷卻模式(SC)造成不同的多孔狀結構，進而影響複合支架的水通透率，例如支架填充密度在25% 且測量時水位高差在100 cm時，CG/P-FC支架和C/P-FC支架的水通透率約在20 x 10-6 (mm2)，而CG/P-SC支架和C/P-SC支架的水通透率約在13 x 10-6 (mm2)，這是因為快速冷卻模式會造成層狀的方向性孔洞結構，水分子較容易通過支架，而慢速冷卻會造成等向性的孔洞結構，水分子通過支架時較為不易，此外，透過機械性質的測定可以得到支架的最大抗壓強度和楊氏係數，其數值分別大約落在6~13(MPa) 和20~70(MPa)，綜合各項分析得知在填充密度為25%情況下製備的多孔狀複合支架為最佳製備結果。由氣泡接觸角測定可以驗證P支架、C/P-FC複合支架和C/P-SC複合支架為疏水性材料，而CG/P-FC複合支架和CG/P-SC複合支架較為親水，因為添加了親水性的明膠。由傅立葉紅外線光譜儀(FT-IR)可以驗證製備出的支架的成分以及有無交聯，在熱性質的測定中，由熱重示差同步掃描分析儀(TGA)和差式掃描熱量分析(DSC)可以得知支架的熱性質。最後使用間葉幹細胞(KP-hMSC)進行細胞相容性的測定，由實驗結果可知無論冷卻模式和支架的組成成分，多孔狀複合支架皆具有良好的細胞相容性且無毒性，其中又以培養在由快速冷卻模式下製備且含有明膠的CG/P-FC-EN複合支架上的細胞具有最快的增殖速率，在細胞培養7天後，若與細胞數量及活性最低的P支架組別相比較，可以看到其細胞數量提升了5倍，細胞活性提升了4.5倍，具有顯著差異。由支架崩解性測試的結果驗證有被EDC/NHS交聯的CG/P-FC-EN 複合支架在浸泡於PBS溶液7天後仍然可以維持孔洞的型態，且浸泡於PBS溶液21天後重量剩餘達97.5 (%)以上，具良好的穩定性，有助於維持多孔狀的結構，故綜合以上實驗結果，本研究製備出的CG/P-FC-EN複合支架具有優異的機械強度、穩定性和良好的多孔狀結構，利於細胞移入及養分提供，所以其細胞相容性優於其他支架，故可嘗試將其應用作為硬組織的多孔狀組織工程支架。

Porous matrices fabricated by freeze-gelation method or rapid prototyping technology can provide environments for cell growth. Thus matrices have great potential in the tissue engineering-related scaffold applications. The freeze-gelation method combines non-solvent induced phase separation method and thermally-induced phase separation method. The fused deposition modeling (FDM) is a type of rapid prototyping technology. It’s a manufacturing process that can produce complex structures in a layer-by-layer manner via computer-aided design 3D models. Besides, the porosity and structure of the object can be easily controlled by adjusting printing parameters. In this study, the FDM technology was applied to build polylactic acid (P) scaffolds which have great mechanical strength. Then the porous structures inside the large pores of the polylactic acid scaffolds were fabricated with chitosan (C) and gelatin (G) which have excellent biocompatibility via the freeze-gelation method. Thus, the hard tissue engineering scaffolds with high porosity, namely C/P composite scaffold or CG/P composite scaffold, were developed by combining FDM and freeze-gelation methods. During the freeze-gelation process, either fast cooling (FC) or slow cooling (SC) mode was used to create different pore structures. Besides, CG/P-FC scaffold and CG/P-SC scaffold were crosslinked by EDC/NHS (EN) in order to enhance their stability. In this study, SEM was used to observe the morphology of the scaffolds and the process parameters for FDM wree also optimized. In the freeze-gelation method, FC or SC caused different pore structures in the scaffolds and thus altered the permeability of the scaffolds. The water permeabilities of CG/P-FC, C/P-FC, CG/P-SC and C/P-SC composite scaffolds were 19.93 ± 0.46, 19.74 ± 0.82, 13.31 ± 0.48 and 12.69 ± 0.38 (mm2) respectively when infill density was 25% and the water head was 100 cm. The difference in permeabilities was caused by different cooling modes. FC mode caused layered pore structures that could make water molecules pass through scaffold easily and SC mode caused isotropic pore structures to make water molecules pass through scaffold more difficultly. The mechanical testing of scaffolds showed that maximum compressive strengths were between 6~13 MPa and Young’s moduli were between 20~70 MPa. The contact angle measurements showed that P scaffolds were hydrophobic and CG/P-FC and CG/P-SC composite scaffolds were more hydrophilic than C/P-FC and C/P-SC composite scaffolds because of the presence of hydrophilic gelatin. The compositions of the scaffolds and the effectiveness of crosslinking were verified by FT-IR analysis. The thermal properties of scaffolds were investigated by TGA and DSC. In cytocompatibility tests, mesenchymal stem cells (KP-hMSC) were cultured on different scaffolds. The results showed that all the porous composite scaffolds fabricated by FDM and freeze-gelation method had excellent cytocompatibility and were non-toxic to cells. Among the porous composite scaffolds, KP-hMSC in the CG/P-FC scaffolds exhibited the highest proliferation rate. Compared to KP-hMSC in P scaffolds which exhibited the lowest proliferation rate, the amounts of cells were 5 times and the activities of cells in CG/P-FC scaffolds were 4.5 times more than those in P scaffolds after 7 days in culture. The testing of scaffolds stability in PBS solution showed that the morphology of the pore structures of CG/P-FC-EN composite scaffolds could be maintained after being immersed in PBS solution for 7 days and the remaining weight of CG/P-FC-EN composite scaffolds could be maintained at 97.5 (%) after immersion in PBS solution for 21 days. These results implied that EDC/NHS-mediated cross linking could increase the stability of scaffolds and could maintain pore structures of scaffolds. To sum up, the CG/P-FC-EN composite scaffolds fabricated via FDM and freeze-gelation method in this study not only had excellent mechanical properties, stability and pore structures but also had the most excellent cytocompatibility among all scaffolds. Therefore, the CG/P-FC-EN composite scaffolds could be utilized in hard tissue engineering-related applications in the future.