幾丁聚醣-明膠-羥基磷灰石/聚己內酯之多孔複合支架製備及其特性探討

Preparation and Characterization of Chitosan-Gelatin-Hydroxyapatite / Polycaprolactone Porous Composite Scaffolds

許宸誌(CHEN-CHIH HSU)

指導教授 ： 謝學真

國立臺灣大學/工學院/化學工程學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902434

摘要

本研究利用研究室先前製備幾丁聚醣多孔基質的冷凍凝膠技術結合熔融沉積法所製備具有生物可降解性的聚己內酯(polycaprolactone, P)三維支架進行研究討論。透過將幾丁聚醣複合溶液灌於聚己內酯支架孔洞中進行冷凍凝膠的製程步驟，使聚己內酯三維支架的孔洞中形成以幾丁聚醣複合材料所組成的微小多孔基質，其中幾丁聚醣(chitosan, C)形成多孔基質的主要結構，添加明膠(gelatin, G)可提升中細胞在支架上細胞相容性，而添加羥基磷灰石(hydroxyapatite, H)使多孔複合支架更適合細胞貼附，本研究製備的CGH/P多孔複合支架具有較佳的機械強度且形狀可自由調整，因此可做為開發未來骨組織修復的組織工程支架。
本研究透過掃瞄式電子顯微鏡(SEM)觀察支架的結構，分別對不同比例之幾丁聚醣(C)、明膠(G)溶液與幾丁聚醣、膠原蛋白肽溶液在冷凍凝膠法製備支架，觀察各組支架孔洞形態差異並篩選出適合的材料及比例，並微調三維列印時的參數，接續觀察幾丁聚醣、明膠以不同比例之溶液灌注在不同填充密度的聚己內酯三維支架，進一步篩選出孔洞形態合適之組別並加入羥基磷灰石(H) 研究其對形態上造成的影響，最後找到多孔複合支架的最佳參數，其中C:G:H的最後濃度比例為2wt% : 0.7wt% : 0.3wt%。同時，冷凍凝膠法將以兩種不同降溫程序進行，分別為快速冷卻(fast-cooling, fc)以及慢速冷卻(slow-cooling, sc)兩種模式，接續利用掃描式電子顯微鏡觀察兩種冷卻模式中冷凍凝膠法製備多孔基質與三維列印支架的結合形態並分析其孔洞大小及方向性，選定以填充密度為30%的支架組別進行後續測定。
最終完成測試的多孔複合支架分別有P, C/P-sc, C/P-fc, CG/P-sc, CG/P-fc,CGH/P-sc, CGH/P-fc支架，在支架透水率測定中，當壓力為100cm水柱高且聚幾內酯填充密度為30%時，CGH/P-sc的透水率為23×10-6 cm2而CGH/P-fc的透水率為26×10-6 cm2，其原因為兩種不同降溫程序形成的孔洞形態差異，快速冷卻模式所製備出的截面結構為垂直於支架表面的層狀的孔洞，而慢速冷卻模式所製備的截面結構是等向性圓孔狀的孔洞，故相同條件下，快速冷卻模式組別CGH/P-fc的透水率在透水率測定中皆高於慢速冷卻模式組別CGH/P-sc。再以利用吸水率測定了解材料對液體之吸收能力，因多孔基質的比例隨聚幾內酯支架填充密度上升而減少，導致多孔複合支架的吸水率會隨著支架填充密度的上升而下降。透過壓縮強度試驗機測試支架的機械強度，可測得支架的最大抗壓強度與楊氏模數，分別落於2-7 MPa和15-35 MPa，並綜合先前分析選出填充密度於30%時為多孔複合支架的最佳製備情況並以此組別作為後續之測定。接著進行支架的礦化改質，將支架浸入模擬體液(simulated body fluid, SBF)，比較各組別支架礦化程度，添加羥基磷灰石的CGH/P支架礦化後增加約12%的重量，具有最好的礦化改質效果。接續利用接觸角測定(捕泡法測定)多孔複合支架的表面親水性質，發現將多孔基質填充於P支架中能明顯提升其親水性，於多孔基質成分中如含有親水性成分，例如明膠，更可進一步的提升多孔複合支架的親水性。多孔複合支架的的細胞相容性則使用間葉幹細胞進行利用細胞蛋白質總量測定(細胞增殖測定)和MTT測定(細胞活性測定)顯示CGH/P-fc支架的細胞相容性最佳，於第七天與P支架相比，其細胞總量提升2.2倍，細胞活性提升5.2倍。最終利用穩定性測定探討添多孔複合支架的穩定性，浸於PBS溶液21天後，所有支架組別的殘餘重量百分比皆在99%以上，表示所有組別皆具備良好的重量穩定性。
綜合以上測定結果，本研究結合冷凍凝膠法與熔融沉積法所製備的CGH/P-fc支架具有良好的孔洞結構、透水率、吸水率、機械性質、礦化程度、親水性以及穩定性，除了利於細胞遷移及養分傳遞，同時也具有良好的細胞相容性，故可嘗試將CGH/P-fc支架開發成為未來硬組織修復的組織工程支架。

關鍵字

冷凍凝膠法 ； 三維列印 ； 聚己內酯 ； 幾丁聚醣 ； 明膠 ； 羥基磷灰石

並列摘要

This research combined the freeze-gelation method which was used to fabricate the porous chitosan-based matrix and fused deposition modeling (FDM) method which was used to produce the biodegradable 3D polycaprolactone (P) scaffold. The microporous chitosan-based matrix was formed in the pores of the polycaprolactone scaffold by pouring the chitosan composite solution into the pores of the polycaprolactone scaffold to perform the freeze-gelation. Chitosan (C) formed the main structure of the porous matrix while gelatin (G) enhanced the cell compatibility of the scaffold, and hydroxyapatite (H) improved cell attachment to the scaffold. The CGH/P porous composite scaffold possessed enhenced mechanical strength and can be freely adjusted in shape. Thus CGH/P porous composite scaffold can be developed into a tissue engineering scaffold for hard tissue repairing-related applications in the future.
In this study, SEM was used to observe the morphology of the scaffolds which were fabricated with different proportions of chitosan, gelatin or collegan peptide for optimized materials selection. The process parameters for FDM were also improved. Beside, H was added to chitosan composite solution. The optimized ratio of C:G:H was 2 wt% : 0.7 wt% : 0.3 wt%. During the freeze-gelation process, different pore structures were created by slow cooling (sc) mode or fast cooling (fc) mode and the pore sizes were measured by ImageJ software.
The P, C/P-sc, C/P-fc, CG/P-sc, CG/P-fc, CGH/P-sc and CGH/P-fc scaffolds were prepared and characterized. When the pressure different was 100 cm water head and the infill density was 30%, the water permeability of CGH/P-sc scaffold was 23×10-6 cm2 and CGH/P-fc scaffold was 26×10-6 cm2. The isotropic pores in the cross-sectional structure of scaffold were created by sc mode and the directional pores in the cross-sectional structure of scaffold were created by fc mode, thus increasing the water permeability of CGH/P-fc scaffold. The water absorption of scaffolds decreased while the infill density increased. The mechanical testing of scaffolds indicated that the maximan compressive strength and Young’s modulus were in the range of 2-7 MPa and 15-35 MPa, respectively. Finally, the scaffolds with infill density of 30% were selected to perform the following charaterization.
To modifiy the scaffold surfaces by mineralization, the scaffolds were immersed in simulated body fluid (SBF). The degree of mineralization of the CGH/P scaffolds was the highest with the weight increase of 12% after mineralization. The hydrophilicity of CGH/P scaffolds was enhanced. The cytocompatibility tests were performed using mesenchymal stem cells cultured on different scaffolds. The results showed that the CGH/P-fc scaffolds exhibited the best cytocompatibility. On the seventh day, compared with the P scaffold, the total cell number increased by 2.2 times and the cell activity increased by 5.2 times. Finally, the stability testing of scaffolds showed that all of the scaffolds maintained over 99 % of their weights after immersion in PBS solution for 21 days.
In summary, the CGH/P-fc scaffolds fabricated via freeze-gelation method and FDM method not only have good porous structure, permeability, water absorption, mechanical properties, mineralization, hydrophilicity and stability but also possess excellent cytocompatibility. Therfore, the CGH/P-fc scaffolds can serve as tissue engineering scaffolds for hard tissue repairing-related applications in the future.

並列關鍵字

freeze-gelation ； 3D printing ； polycaprolactone ； chitosan ； gelatin ； hydroxyapatite

參考文獻

1. Langer, R. and J.P. Vacanti, Tissue engineering. Science, 1993. 260(5110): p. 920-927.

2. Kim, N.J., S.J. Lee, and A. Atala, 1 - Biomedical nanomaterials in tissue engineering, in Nanomaterials in Tissue Engineering, A.K. Gaharwar, et al., Editors. 2013, Woodhead Publishing: Sawston. p. 1-25.

3. Kramschuster, A. and L.-S. Turng, 17 - Fabrication of Tissue Engineering Scaffolds, in Handbook of Biopolymers and Biodegradable Plastics, S. Ebnesajjad, Editor. 2013, William Andrew Publishing: Boston. p. 427-446.

4. 鄭農哲, 多孔狀幾丁聚醣-動物明膠/聚乳酸複合支架之製備及其性質探討. 國立台灣大學工學院化學工程學研究所碩士論文, 2018.

5. Ho, M.-H., P.-Y. Kuo, H.-J. Hsieh, T.-Y. Hsien, L.-T. Hou, J.-Y. Lai, and D.-M. Wang, Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterials, 2004. 25(1): p. 129-138.