本研究將聚乙二醇甲醚末端改質，並環化左旋丙氨酸後，開環聚合形成雙嵌段共聚物mPEG-polyalanine，此溫感型水膠雖具有良好的生物相容性，但其機械性質較弱，而作為硬骨組織工程的支架需具有一定的耐壓程度，因此實驗中將溫感型水膠與陶瓷材料―三鈣磷酸鹽結合為複合材料，其具有良好的生物相容性、骨傳導性，並結合PRP於體外活化成膠，活化的血小板可釋放生長因子，亦能增加其材料之彈性，探討其應用在骨組織之再生。
藉由核磁共振光譜儀、傅立葉反射式紅外線、凝膠滲透層析儀及掃描式電子顯微鏡分析mPEG-polyalanine之結構與性質，並透過相轉變及流變性質求得高分子成膠時之最低溫度與濃度。機械強度測試可得溫感型水膠及PRP之抗壓性隨著濃度增加而增加，而添加β-TCP則隨著比例增加使得材料較為硬脆。後續探討材料對於細胞毒性、與細胞共培養之生物相容性，由MTT assay及Live&Dead結果可知細胞存活率皆在90%以上，證明此兩種材料具有良好的生物相容性。
透過添加低分子量肝素探討是否能延緩PRP釋放，於PRP活化成膠後加入藥物可達到緩釋效果但藥效僅能維持四小時，仍須探討其他可能性。後續以酵素免疫分析法及定量聚合酶反應確認MC3T3與水膠、β-TCP共培養後，仍具有成骨細胞分化的功能，且β-TCP亦具有骨誘導性促進成骨化作用，並可推測已形成成熟的造骨細胞。而MC3T3與水膠、PRP的實驗組僅有第一天的較高表現量，但根據生長因子釋放曲線可得知第一天釋放已達80%，導致表現量隨著時間並無明顯增加，需延長PRP釋放週期才可證實是否能促進成骨細胞分化，進而達到硬骨修復的效果。
本研究最終期望此溫度敏感型水膠，具有低黏度可注射性、原位成膠及良好之生物相容性等特性，且能透過高分子修飾或結合其他生醫材料提高機械強度，並延長PRP釋放時間，使其成為具有優勢的骨組織工程之細胞支架。

In this study, the end of methoxy-polyethylene glycol was modified, and L-alanine was cyclize, then ring-opening polymerization was carried out to form the two-block copolymer mPEG-polyalanine. The thermosensitive hydrogel has good biocompatibility, but its mechanical properties are weak, and as a scaffold for bony tissue engineering needs to have a certain extent of pressure resistance. Therefore, in the experiment, the thermosensitive hydrogel and the ceramic material-tricalcium phosphate are combined into a composite material, which has good biocompatibility and osteoconductivity. Combined with PRP activated in vitro to form a gel, the activated platelets can release growth factors and increase the elasticity of the material. It explore the application in the regeneration of bone tissue.
Analyze the structure and properties of mPEG-polyalanine by nuclear magnetic resonance spectrometer, Fourier reflection infrared, gel permeation chromatography and scanning electron microscope. Through the phase transition and rheological properties obtain the lowest temperature and concentration of polymer gelation. The mechanical strength test shows that the pressure resistance of the thermosensitive hydrogel and PRP increases with the increase of the concentration, and the addition of β-TCP makes the material harder and more brittle with the increase of the proportion. We will discuss the cytotoxicity and the biocompatibility of material co-cultured with cells. The results of MTT assay and Live&Dead show that the cell viability is above 90%, which proves that the two materials have good biocompatibility.
By adding low-molecular-weight heparin to explore whether it can defer the release of PRP. Adding drugs after PRP is activated to form a gel can achieve a sustained release effect, but the efficacy can only be maintained for four hours. Other possibilities must be explored. Subsequent use of enzyme-linked immunosorbent assay and quantitative polymerase chain reaction to confirm that MC3T3 still has the function of osteoblast differentiation after co-cultured with hydrogel and β-TCP. Moreover, β-TCP also has osteoinductive and ossification-promoting effects, and it can be inferred that mature osteoblasts have formed. However, the experimental group of MC3T3, hydrogel and PRP only had a high level of expression on the first day. According to the growth factor release curve, it can be known that the release has reached 80% on the first day, resulting in no significant increase in performance over time. It is necessary to extend the release cycle of PRP to confirm whether it can promote osteoblast differentiation and achieve the effect of bone repair and regeneration.
This research finally hopes that this thermosensitive hydrogel has the characteristics of low viscosity, injectability, in-situ gel formation and good biocompatibility. And it can improve the mechanical strength through polymer modification or combining with other biomedical materials. By prolonging the release time of PRP, it becomes an advantageous cell scaffold for bone tissue engineering.

摘要 I
Abstract III
致謝 V
目錄 VI
圖目錄 X
表目錄 XII
第一章 文獻回顧 1
1.1 高分子水膠簡介 1
1.1.1 水膠的類型與相關性質 2
1.1.2 水膠之應用 4
1.2 三鈣磷酸鹽（β-tricalcium phosphate, β-TCP） 7
1.3 高濃度血小板血漿（Platelet-Rich Plasma, PRP） 9
1.4 低分子量肝素（Low-molecular weight heparin, LMWH） 11
1.5 骨組織 13
1.5.1 骨再生 13
1.5.2 骨組織工程 15
第二章 研究動機與目的 16
第三章 實驗藥品與設備儀器 17
3.1 實驗藥品 17
3.2 儀器設備 19
第四章 研究方法與步驟 20
4.1 mPEG-poly（L-alanine）（mPA）嵌段共聚物製備 20
4.1.1 聚乙二醇甲醚末端改質（mPEG-OH基→mPEG-NH2基） 20
4.1.2 丙氨酸環化反應（N-carboxyl anhydride of L-alanine, NCA-Ala） 21
4.1.3 mPEG-poly（L-alanine）開環聚合反應 21
4.2 mPEG-p（L-alanine）嵌段共聚物之結構鑑定與物性分析 22
4.2.1 核磁共振光譜儀（1H-NMR） 22
4.2.2 傅立葉反射式紅外線光譜儀（ATR-FTIR） 22
4.2.3 凝膠滲透層析儀（Gel Permeation Chromatography, GPC） 23
4.2.4 溶膠‒凝膠相轉變測試（Sol-gel transition） 23
4.2.5 流變儀（Rheology） 23
4.2.6 場發射掃描式電子顯微鏡（FESEM） 24
4.2.7 機械性質測試 24
4.3 Platelet-Rich Plasma（PRP）製備與應用 24
4.3.1 PRP製備與凝膠 24
4.3.2 PRP與mPEG-p（Ala）、β-TCP混合製備 25
4.3.3 PRP與mPEG-p（Ala）、LMWH混合製備 25
4.4 材料之生物相容性（Biocompatibility of the hydrogel） 25
4.4.1 細胞培養（Cell culture） 25
4.4.2 材料與細胞共培養（Cell cultured with hydrogel） 26
4.5 細胞毒性測試 26
4.5.1 MTT assay 26
4.5.2 Live/Dead螢光染色 27
4.6 基因表現定量分析 27
4.6.1 引子基因序列[36,37] 27
4.6.2 RNA萃取與定量 28
4.6.3 RNA反轉錄cDNA 28
4.6.4 定量聚合酶連鎖反應(Quantitative Polymerase Chain Reaction) 29
4.7 酵素免疫分析法（Enzyme-linked immunosorbent assay） 29
4.7.1 生長因子之釋放 29
4.7.2 生長因子之定量 30
第五章 實驗結果與討論 31
5.1 mPEG-poly（L-alanine）嵌段共聚物之鑑定分析 31
5.1.1 共聚物氫原子光譜之結構分析 31
5.1.2 共聚物之FT-IR結構分析 32
5.1.3 共聚物分子量分析 32
5.1.4 溶膠‒凝膠相轉變測試 33
5.1.5 流變性質測試 34
5.1.6 共聚物水膠三維結構分析 35
5.1.7 機械性質測試 36
5.2 生物相容性測試 37
5.3 生長因子之釋放與定量 39
5.4 基因表現 41
第六章 結論與未來展望 45
第七章 參考文獻 47

1. Yu, K.-M., Ping, Q.-N., Sun, M.-J., Application and development trend of implantable drug delivery system. Progress in Pharmaceutical Sciences. 2020, 5, 361-370.
2. Parhi, R., Cross-linked hydrogel for pharmaceutical applications: a review. Adv Pharm Bull. 2017, 7(4), 515–530.
3. Yahia, L.-H., Chirani, N., Gritsch, L., History and applications of hydrogels. Journal Biomedical Science. 2015, 4(2).
4. Amin, G.-N., Nureddin, A., Wu, X.-Y., Ali K.-H., Crosslinking strategies for 3D bioprinting of polymeric hydrogels. Nano Micro small. 2020, 16(35).
5. George, J.-L., Hsu, C.-C., Nguyen, L.T.B., Ye, H., Cui, Z.-F., Neural tissue engineering with structured hydrogels in CNS models and therapies. Biotechnology Advances. 2020, 42, 107370.
6. Wei, W.-P., Li, H.-F., Yin, C.-C., Tang, F.-S., Research progress in the application of in situ hydrogel system in tumor treatment. Drug Delivery. 2020, 27(1), 460-468.
7. Cui, S.-Q., Yu, L., Ding, J.-D., Injectable thermally induced hydrogel based on moderately amphiphilic block copolymer. Acta Polym Sin. 2018, 8, 997-1015.
8. Caló, E., Khutoryanskiy, V.-V., Biomedical applications of hydrogels: A review of patents and commercial products. European Polymer Journal. 2015, 65, 252-267.
9. Hollister, S.-J., Porous scaffold design for tissue engineering. Nature Materials. 2005, 4, 518–524.
10. Blanchard, C.-R., Timmons, S.-F., Smith, R.-A., Keratin-based hydrogel for biomedical applications and method of production. United States Patent. 2002.
11. Martino, M.-M., Tortelli, F., Mochizuki, M., Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing. Science Translational Medicine. 2011, 3(100), 100ra89.
12. Qasim, M., Chae, D.-S., Lee, N.-Y., Advancements and frontiers in nano-based 3D and 4D scaffolds for bone and cartilage tissue engineering. Int J Nanomedicine. 2019, 14, 4333-4351.
13. Jeong, J., Kim, J,-H., Shim, J.-H., Bioactive calcium phosphate materials and applications in bone regeneration. Biomaterials Research. 2019, 23(4).
14. Liu, B., Lun, D.-X., Current application of β‐tricalcium phosphate composites in orthopaedics. Orthop Surg. 2012, 4(3), 139-144.
15. Marx, R.-E., Platelet-Rich Plasma (PRP): What is PRP and What is not PRP? Implant Dentistry. 2001, 10(4), 225-228.
16. Ehrenfest, D.-M.-D., Andia, I., Zumstein, M.-A., Zhang, C.-Q., Pinto, N.-R., Bielecki, T., Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives. Muscles Ligaments Tendons J. 2014, 4(1), 3-9.
17. LaBaze, D., Li, H.-S., Platelet rich plasma: biology and clinical usage in orthopedics. Orthopedic Biomaterials. 2018, 243-286.
18. Mohan, S.-P., Jaishangar, N., Devy, S., Narayanan, A., Cherian, D., Madhavan, S.-S., Platelet-rich plasma and platelet-rich fibrin in periodontal regeneration: A review. J Pharm Bioallied Sci. 2019, 11(2), 126-130
19. Yin, W.-J., Qi, X., Zhang, Y.-L., Xu, Z.-L., Tao, S.-C., Xie, X.-T., Li, X.-L., Zhang, C.-Q., Advantages of pure platelet-rich plasma compared with leukocyte- and platelet-rich plasma in promoting repair of bone defects. J Transl Med. 2016, 14.
20. Relievus company, Platelet rich plasma injection treatment. https://stemcellprp.com/prp.html
21. Garcia, D., Libby, E., Crowther, M.-A., The new oral anticoagulants. Blood. 2010, 115(1), 15-20.
22. Brey, R.-L., Coull, B.-M., Chapter 34 Coagulation abnormalities in stroke. Stroke (Fourth Edition) Pathophysiology, Diagnosis, and Management. 2004, 707-724.
23. Fareed, J., Hoppensteadt, D., Walenga, J., Iqbal, O., Ma, Q., Jeske, W., Sheikh, T., Pharmacodynamic and pharmacokinetic properties of enoxaparin. Clinical Pharmacokinetics. 2003, 42, 1043-1057.
24. Muiz-Lozano, A., Rollini, F., Franchi, F., Angiolillo, D.-J., Anticoagulation therapy. Heparins, Factor II and Factor Xa inhibitors. Pharmacological Treatment of Acute Coronary Syndromes. 2013, 59-122.
25. Lai, S., Coppola, B., Use of enoxaparin in end-stage renal disease. Kidney International. 2013, 84(3), 433-436.
26. Tripathy, N., Perumal, E., Ahmad, R., Song, J.-E., Khang, G., Chapter 40 Hybrid composite biomaterials. Principles of Regenerative Medicine (Third Edition). Academic Press. 2019, 695-714.
27. Wubneh, A., Tsekoura, E.-K., Ayranci, C., Uludağ, H., Current state of fabrication technologies and materials for bone tissue engineering. Acta Biomater. 2018, 80, 1-30.
28. Nieden, N.-I.-Z., Kempka G, Ahr H.-J., In vitro differentiation of embryonic stem cells into mineralized osteoblasts. Differentiation. 2003, 71(1), 18-27.
29. Bose, S., Roy, M., Bandyopadhyay, A., Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology. 2012, 30(10), 546-554.
30. Tortora, G.-J., Dickinson, B.-H., Principles of anatomy and physiology(6th edition). HarperCollins publishers. 2010, 217(5), 97-161.
31. Stevens, M.-M., Biomaterials for bone tissue engineering. Materials Today. 2008, 11(5), 18-25.
32. Burg, K.-J.-L., Potter, S., Kellam, J.-F., Biomaterial developments for bone tissue engineering. Biomaterials. 2000, 21(23), 2347-2359.
33. Rezwan, K., Chen, Q.-Z., Blaker, J.-J., Boccaccini, A.-R., Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials. 2006, 27(18), 3413-3431.
34. Salgado, A.-J., Coutinho, O.-P., Reis, R.-L., Bone tissue engineering: state of the art and future trends. Macromolecular Bioscience. 2004, 4(8), 743-765.
35. Black, C.-R.-M., Goriainov, V., Gibbs, D., Kanczler, J., Tare, R.-S., Oreffo, R.-O.-C., Bone tissue engineering. Curr Mol Bio Rep. 2015, 1(3), 132-140.
36. Chou, Y.-F., Huang, W.-B., Dunn, J.-C.-Y., Miller, T.-A., Wu, B.-M., The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression. Biomaterials. 2005, 26(3), 285-295.
37. Saito, T., Hayashi, H., Kameyama, T., Hishida, M., Nagai, K., Teraoka, K., Kato, K., Suppressed proliferation of mouse osteoblast-like cells by a rough-surfaced substrate leads to low differentiation and mineralization. Materials Science and Engineering: C. 2010, 30(1), 1-7.
38. Wang, X.-Y., Wang, G., Liu, L., Zhang, D.-Y., The mechanism of a chitosan-collagen composite film used as biomaterial support for MC3T3-E1 cell differentiation. Sci Rep. 2016, 6.
39. Kim, I.-R., Kim, S.-E., Baek, H.-S., et al., The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells. BMC Complement Altern Med. 2016, 16(1).
40. Rousseau, M., Boulzaguet, H., Biagianti, J., Duplat, D., Milet, C., Lopez, E., Bédouet, L., Low molecular weight molecules of oyster nacre induce mineralization of the MC3T3‐E1 cells. J Biomed Mater Res A. 2007, 85(2), 487-497.