臺灣博碩士論文加值系統

下背痛是一種普遍的慢性疾病，造成的主因是椎間盤退化與突出，進而壓迫神經產生疼痛。一般通常以低風險的椎間盤手術進行治療，但椎間盤手術後卻有降低椎間盤高度與症狀復發等缺點。本篇研究中，我們使用化學修飾改質幾丁聚醣和透明質酸，利用改質後的水溶性幾丁聚醣和氧化透明質酸可自我交聯的特性，希望藉此注入於椎間盤術後的缺口中，原位形成水膠填補空洞，減少椎間盤高度的降低，也避免椎間盤突出術後復發的機率。
研究中以不同分子量(39k、220k、900k、1390k)之透明質酸做為調整水膠性質的參數，使其交聯時間介於13~15分鐘；力學強度介於19.59 ± 2.7~11 ± 2.9 kpa之間；組織黏著性介於39.75 ± 3.476 ~19.675± 4.989 kpa之間；膨潤率介於1.3~1.9倍之間。與人體髓核細胞外基質的材料力學強度相近，且符合手術使用上的需求並具有初期固定的能力。
研究結果顯示，Aldehyde hyaluronic acid與N-Succinyl-chitosan所製備的水膠具有高含水性與人體髓核細胞外基質類似的材料力學性質，且有固化時間短與初期固定的黏著性，具有發展成經由微創傷口運送原位形成膠體至創傷部位的潛力，值得繼續深入研究與發展。

Low back pain is a general chronic disease, one of the leading causes is nerve compression resulting from the degeneration and hernition of intervertebral disc. A microdiscectomy is typically performed for a herniated lumbar disc, but the surgery have problems like disc height loss and herniation recurrence. In this study, we use chitosan and hyaluronic acid to form in-situ injectable hydrogels by chemical modification. Hoping that we can fill up the surgical space, reduce disc height loss and decrease the risk of herniation recurrence by injecting in-situ forming hydrogels. By using different molecular weight of hyaluroic acid(39k、220k、900k、1390k) to adjusting the characteristic of hydrogel. It's gelation time is 13~15min, compressive modulus is between 19.59 ± 2.7~11 ± 2.9 kpa, tissue adhesive is between 39.75 ± 3.476 ~19.675± 4.989 kpa, swelling ratio is between 1.3~1.9. The michinal property of this hydrogel is similier to nucleus pulposus. Besides, it has short gelation time and initial fixation strength.
From this research, the michinal property of high water contain AHA-SCS hydrogel is similiar to human nucleus pulposus. Base on the characteristic of short gelation time and ability of initial fixation, there is a potential to be a injectable in situ forming hydogel delivered by minimally invasive surgery. It is worth to development and continuous research.

目錄
致謝 ................................................................................................ i
摘要 ............................................................................................... ii
ABSTRACT .............................................................................................. iii
目錄 .............................................................................................. iv
圖目錄 ............................................................................................ viii
表目錄 .............................................................................................. xi
第一章 研究動機與目標 .................................................................... 1
第二章 文獻回顧 ................................................................................ 3
2-1 椎間盤之結構位置與功能 .................................................... 3
2-2 椎間退化成因 ....................................................................... 5
2-3 椎間盤退化突出之治療方法 ................................................ 6
2-3-1 手術去除疼痛來源 ...................................................... 6
2-3-2 透過組織工程修復椎間盤 .......................................... 9
2-4 水膠 ..................................................................................... 11
2-4-1 共聚合交聯 ............................................................... 12
2-4-2 網狀互穿水膠 ............................................................ 13
2-4-3 雙網路水膠 ............................................................... 14
2-4-4 奈米複合型水膠 ........................................................ 15
2-5 注射型水膠 ......................................................................... 17
2-5-1 注射型物理交聯水膠 ................................................ 18
2-5-1-1 溫感型水膠 ...................................................... 19
2-5-1-2 酸鹼誘發水膠 .................................................. 20
2-5-1-3 離子介質誘發水膠 .......................................... 21
2-5-1-4 自組裝型水膠 .................................................. 22
2-5-2 注射型化學交聯水膠 ................................................ 24
2-5-2-1 自由基聚合反應 .............................................. 25
2-5-2-2 Schiff-base 交聯 .............................................. 26
2-5-2-3 酵素觸發交聯 .................................................. 27
2-6 水膠的天然高分子材料 ...................................................... 28
2-6-1 纖維蛋白(Fibrin) ....................................................... 28
2-6-2 膠原蛋白(Collagen) ................................................... 29
2-6-3 透明質酸(Hyaluronic acid) ........................................ 30
2-6-4 幾丁聚醣(Chitosan) ................................................... 31
第三章 實驗材料與方法 .................................................................. 33
3-1 實驗藥品 ............................................................................. 33
3-2 實驗儀器與裝置 .................................................................. 34
3-3 Succinyl-chitosan(SCS)之合成與鑑定 ................................ 35
3-3-1 Succinyl-chitosan(SCS)之合成步驟 .......................... 35
3-3-2 檢測Succinyl-chitosan(SCS)之Carboxyl group含量 ................................................................................................. 35
3-3-3 Succinyl-chitosan(SCS)之1H-NMR結構鑑定 .......... 36
3-3-4 Succinyl-chitosan(SCS)之FTIR ................................ 37
3-4 Aldehyde hyaluronic acid(AHA)之合成 .............................. 37
3-3-1 Aldehyde hyaluronic acid(AHA)之合成步驟 ............ 37
3-3-2 檢測Aldehyde hyaluronic acid(AHA)之aldehyde group含量 ............................................................................... 38
3-3-3 Aldehyde hyaluronic acid(AHA)之紫外光-可見光光譜鑑定 ......................................................................................... 39
3-5 SCS-AHA水膠製備 ............................................................ 39
3-6 SCS-AHA水膠力學性質之鑑定 ........................................ 39
3-7 SCS-AHA水膠膨潤性質之鑑定 ........................................ 40
3-8 SCS-AHA水膠成膠時間之鑑定 ........................................ 40
3-9 SCS-AHA水膠溶解時間之鑑定 ........................................ 41
3-10 椎間盤髓核細胞、環狀纖維與纖維母細胞細胞培養 ....... 41
3-11 SCS-AHA水膠細胞毒性之鑑定 ........................................ 43
3-12 SCS-AHA水膠細胞貼附性之鑑定 .................................... 45
3-13 SCS-AHA水膠組織黏著性之鑑定 .................................... 46

第四章 結果與討論 .............................................................................. 47
4-1 Succinyl-chitosan合成之鑑定與性質分析 ........................... 47
4-1-1 Succinyl-chitosan之1H-NMR (D2O)........................... 47
4-1-2 Succinyl-chitosan之FTIR ........................................... 48
4-1-3 Succinyl-chitosan之TNBS assay ................................ 49
4-2 Aldehyde hyaluronic acid之合成鑑定與性質分析 ............... 51
4-2-1 Aldehyde hyaluronic acid之aldehyde group含量鑑定 ................................................................................................. 51
4-3 SCS-AHA水膠之製作與成膠型態 ...................................... 54
4-4 SCS-AHA水膠成膠時間 ...................................................... 57
4-5 SCS-AHA水膠膨潤性質 ...................................................... 58
4-6 SCS-AHA水膠之力學性質 .................................................. 60
4-7 SCS-AHA水膠溶解時間 ...................................................... 62
4-8 SCS-AHA水膠細胞毒性 ...................................................... 63
4-9 SCS-AHA水膠組織黏著性 .................................................. 66
第五章 結論................................................................................. 69
第六章 參考文獻 ......................................................................... 70

圖目錄
第一章
圖 1- 1 以水膠填塞移除的髓核缺口 ................................... 2
第二章
圖 2- 1 脊椎側面圖 .............................................................. 3
圖 2- 2 椎間盤組成 .............................................................. 4
圖 2- 3 椎間盤突出壓迫神經 .............................................. 5
圖 2- 4 各種人工椎間盤 ...................................................... 7
圖 2- 5 椎間盤顯微切除手術(Microdiscectomy) ................. 8
圖 2- 6 半網狀互穿水膠網路模型(左) 網狀互穿水膠網路模型(右) ............................................................... 13
圖 2- 7 奈米複合型水膠網路模型 ..................................... 16
圖 2- 8 PEG-nHAp奈米複合型水膠 ................................. 17
圖 2- 9 褐藻酸G單元與鈣離子鍵結 ................................ 22
圖 2- 10 Coiled-coil motif示意圖 ........................................ 23
圖 2- 11 S-CS與A-HA反應示意圖 ................................... 27
圖 2- 12 透明質酸結構式 .................................................... 30

圖 2- 13 幾丁聚醣結構式 .................................................... 32
第三章
圖 3- 1 Succinyl- Chitosan 之改質示意圖 ........................ 35
圖 3- 2 TNBS與一級胺之反應 ......................................... 36
圖 3- 3 Aldehyde hyaluronic acid之改質示意圖 ............... 37
圖 3- 4 tert-Butyl carbazate與醛基之反應 ........................ 38
圖 3- 5 鐵氟龍模具 ............................................................ 40
圖 3- 6 SD大鼠椎間盤細胞原代培養(primary culture) .... 43
圖 3- 7 MTT開環反應 ....................................................... 44
第四章
圖 4- 1 Succinyl-chitosan之1H-NMR (D2O) ..................... 48
圖 4- 2 Succinyl-chitosan之FTIR ..................................... 49
圖 4- 3 Chitosan之amino group標準曲線 ....................... 50
圖 4- 4 Aldehyde hyaluronic acid之UV/Vis吸收光譜 ..... 52
圖 4- 5 tert-Butyl carbazate標準曲線 ................................ 53
圖 4- 6 水膠注射混合器 .................................................... 55
圖 4- 7 SCS-AHA水膠外觀 .............................................. 57
圖 4- 8 SCS-AHA水膠成膠時間 ...................................... 58
圖 4- 9 SCS-AHA水膠膨潤性質 ...................................... 59

圖 4- 10 SCS-AHA水膠之力學性質................................... 61
圖 4- 11 SCS-AHA水膠溶解時間 ....................................... 63
圖 4- 12 爬出組織塊的髓核細胞與環狀纖維細胞 ........... 64
圖 4- 13 椎間盤髓核與環狀纖維細胞(左)、L929纖維母細胞(右) ................................................................... 64
圖 4- 14 髓核細胞與環狀纖維細胞與水膠浸泡液共培養之細胞活性 .............................................................. 65
圖 4- 15 纖維母細胞與水膠浸泡液共培養之細胞活性 .... 65
圖 4- 16 水膠組織黏著性測試 .......................................... 67
圖 4- 17 SCS-AHA水膠組織黏著性強度 ......................... 68

表目錄
表 1 髓核組織工程三要素 ................................................... 9
表 2 注射型水膠的類型 .................................................... 18
表 3 不同條件下Chitosan的改質比率 ............................. 50
表 4 不同分子量透明質酸開環比例 ................................. 54
表 5 不同SCS濃度成膠狀況的結果 ................................ 56
表 6 不同SCS濃度成膠狀況的結果 ................................ 56
表 7 SCS-AHA的 交聯時間與機械性質整理 ................. 61
表 8 SCS-AHA水膠組織黏著性強度 ............................... 67
表 9 不同成分SCS-AHA水膠性質比較 .......................... 68
表 10 SCS-AHA與目標條件的比較 ................................... 69

1. Urban, J.P.G. and S. Roberts, Arthritis Research &; Therapy, 2003. 5(3): p. 120.
2. O'Halloran, D.M. and A.S. Pandit, Tissue-engineering approach to regenerating the intervertebral disc. Tissue Eng, 2007. 13(8): p. 1927-54.
3. Umehara, S., et al., Effects of degeneration on the elastic modulus distribution in the lumbar intervertebral disc. Spine (Phila Pa 1976), 1996. 21(7): p. 811-9; discussion 820.
4. Cloyd, J.M., et al., Material properties in unconfined compression of human nucleus pulposus, injectable hyaluronic acid-based hydrogels and tissue engineering scaffolds. Eur Spine J, 2007. 16(11): p. 1892-8.
5. Mercuri, J.J. and D.T. Simionescu, Advances in Tissue Engineering Approaches to Treatment of Intervertebral Disc Degeneration: Cells and Polymeric Scaffolds for Nucleus Pulposus Regeneration. 2011. 247: p. 201-231.
6. Zhang, Y., et al., Biological treatment for degenerative disc disease: implications for the field of physical medicine and rehabilitation. Am J Phys Med Rehabil, 2008. 87(9): p. 694-702.
7. P. Prithvi Raj, M., FIPP, ABIPP, Intervertebral disc anatomy physiology pathophysiology treatment. World Institute of Pain, 2008. Pain Practice, Volume 8( Issue 1): p. 18–44.
8. Diwan, A.D.P., H K; Khan, S N; Sandhu, H S; Girardi, F P; Cammisa, F P, Current concepts in intervertebral disc restoration. Orthopedic clinics of North America, 2000. 31(3): p. 453-464.
9. Bao, Q.B., et al., The artificial disc: theory, design and materials. Biomaterials, 1996. 17(12): p. 1157-67.
10. Burkus, J.K., et al., Anterior lumbar interbody fusion for the management of chronic lower back pain: current strategies and concepts. Orthop Clin North Am, 2004. 35(1): p. 25-32.
11. Sagi, H.C., Q.B. Bao, and H.A. Yuan, Nuclear replacement strategies. Orthop Clin North Am, 2003. 34(2): p. 263-7.
12. Robert G. Watkins, I., MD, Lytton A. Williams, MD, Robert G. Watkins, III, MD, Microscopic lumbar discectomy results for 60 cases in professional and Olympic athletes. The Spine Journal, 2003: p. 100–105.
13. Jensdottir, M., et al., 20 years follow-up after the first microsurgical lumbar discectomies in Iceland. Acta Neurochir (Wien), 2007. 149(1): p. 51-8; discussion 57-8.
14. Yang, X. and X. Li, Nucleus pulposus tissue engineering: a brief review. Eur Spine J, 2009. 18(11): p. 1564-72.
15. Paesold, G., A.G. Nerlich, and N. Boos, Biological treatment strategies for disc degeneration: potentials and shortcomings. Eur Spine J, 2007. 16(4): p. 447-68.
16. Matsunaga, S., et al., Age-related changes in expression of transforming growth factor-beta and receptors in cells of intervertebral discs. J Neurosurg, 2003. 98(1 Suppl): p. 63-7.
17. Gruber, H.E., H.J. Norton, and E.N. Hanley, Jr., Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro. Spine (Phila Pa 1976), 2000. 25(17): p. 2153-7.
18. Wei, A., et al., Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J, 2008. 8(3): p. 466-74.
19. Masuda, K., et al., Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model. Spine (Phila Pa 1976), 2006. 31(7): p. 742-54.
20. Gilbertson, L., et al., The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells. Spine J, 2008. 8(3): p. 449-56.
21. Tsai, T.T., et al., Fibroblast growth factor-2 maintains the differentiation potential of nucleus pulposus cells in vitro: implications for cell-based transplantation therapy. Spine (Phila Pa 1976), 2007. 32(5): p. 495-502.
22. Nomura, T., et al., Nucleus pulposus allograft retards intervertebral disc degeneration. Clin Orthop Relat Res, 2001(389): p. 94-101.
23. Takada, T., et al., Fas ligand exists on intervertebral disc cells: a potential molecular mechanism for immune privilege of the disc. Spine (Phila Pa 1976), 2002. 27(14): p. 1526-30.
24. Gaetani, P., et al., Adipose-derived stem cell therapy for intervertebral disc regeneration: an in vitro reconstructed tissue in alginate capsules. Tissue Eng Part A, 2008. 14(8): p. 1415-23.
25. Lu, Z.F., et al., Influence of collagen type II and nucleus pulposus cells on aggregation and differentiation of adipose tissue-derived stem cells. J Cell Mol Med, 2008. 12(6B): p. 2812-22.
26. Tapp, H., et al., Adipose-derived mesenchymal stem cells from the sand rat: transforming growth factor beta and 3D co-culture with human disc cells stimulate proteoglycan and collagen type I rich extracellular matrix. Arthritis Res Ther, 2008. 10(4): p. R89.
27. Xie, L.W., et al., Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro. Chin J Traumatol, 2009. 12(2): p. 98-103.
28. Lu, Z.F., et al., Differentiation of adipose stem cells by nucleus pulposus cells: configuration effect. Biochem Biophys Res Commun, 2007. 359(4): p. 991-6.
29. Masuda K, O.T.J., An HS:, Growth factors and treatment of intervertebral disc degeneration. Spine J, 2004. 29: p. 2757–69.
30. Jeong, B., S.W. Kim, and Y.H. Bae, Thermosensitive sol-gel reversible hydrogels. Adv Drug Deliv Rev, 2002. 54(1): p. 37-51.
31. Kopeček, J. and J. Yang, Hydrogels as smart biomaterials. Polymer International, 2007. 56(9): p. 1078-1098.
32. Yang-Ho Na, T.K., Yoshinori Katsuyama,Hiroyuki Tsukeshiba,, Y.O. Jian Ping Gong, Satoshi Okabe,Takeshi Karino,and, and M. Shibayama, Structural Characteristics of Double Network Gels with Extremely High Mechanical Strength. Macromolecules, 2004. 37: p. 5370-5374.
33. Kazutoshi Haraguchi, T.T., Nanocomposite Hydrogels A unique organic-inorganic network structure with extraordinary mechanical optical and swelling De-swelling properities. Advanced Materials, 2002. 14(16): p. 1120-1124.
34. Kris Kostanski, L., et al., Interpenetrating polymer networks as a route to tunable multi-responsive biomaterials: development of novel concepts. J Biomater Sci Polym Ed, 2009. 20(3): p. 271-97.
35. A.D. Jenkins, P.K., R.F.T. Stepto, U. W. Suter, GLOSSARY OF BASIC TERMS IN POLYMER SCIENCE. INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY, 1996. 68: p. 2287-2311.
36. B. Suthar, H.X.X., D. Klempner and K. C. Frisch, A Review of Kinetic Studies on the Formation of Interpenetrating Polymer Networks. Polym. Adv. Technol, 1996. 7: p. 211-213.
37. Gong, J.P., Why are double network hydrogels so tough? Soft
Matter, 2010. 6(12): p. 2583.
38. Daisaku Kaneko, T.T., Takayuki Kurokawa, Jian P. Gong, and Yosjofito Osada, Mechanically strong hydrogels with ultra-low frictional coefficients. Advanced Materials, 2005. 17(5).
39. Tsukeshiba, H., et al., Effect of polymer entanglement on the toughening of double network hydrogels. J Phys Chem B, 2005. 109(34): p. 16304-9.
40. Zhang, H., A. Qadeer, and W. Chen, In situ gelable interpenetrating double network hydrogel formulated from binary components: thiolated chitosan and oxidized dextran. Biomacromolecules, 2011. 12(5): p. 1428-37.
41. Haraguchi, K. and H.J. Li, Control of the coil-to-globule transition and ultrahigh mechanical properties of PNIPA in nanocomposite hydrogels. Angew Chem Int Ed Engl, 2005. 44(40): p. 6500-4.
42. Gaharwar, A.K., et al., Highly extensible, tough, and elastomeric nanocomposite hydrogels from poly(ethylene glycol) and hydroxyapatite nanoparticles. Biomacromolecules, 2011. 12(5): p. 1641-50.
43. Overstreet, D.J., et al., Injectable hydrogels. Journal of Polymer Science Part B: Polymer Physics, 2012. 50(13): p. 881-903.
44. McLemore, R., M.C. Preul, and B.L. Vernon, Controlling delivery properties of a waterborne, in-situ-forming biomaterial. J Biomed Mater Res B Appl Biomater, 2006. 79(2): p. 398-410.
45. Pollock, J.F. and K.E. Healy, Mechanical and swelling characterization of poly(N-isopropyl acrylamide -co- methoxy poly(ethylene glycol) methacrylate) sol-gels. Acta Biomater, 2010. 6(4): p. 1307-18.
46. Baroli, B., Hydrogels for tissue engineering and delivery of tissue-inducing substances. J Pharm Sci, 2007. 96(9): p. 2197-223.
47. Packhaeuser, C.B., et al., In situ forming parenteral drug delivery systems: an overview. Eur J Pharm Biopharm, 2004. 58(2): p. 445-55.
48. Berger, J., et al., Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur J Pharm Biopharm, 2004. 57(1): p. 19-34.
49. Cho, J., et al., Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature. Biomacromolecules, 2005. 6(6): p. 3267-75.
50. Rinaudo, M., Periodate Oxidation of Methylcellulose: Characterization and Properties of Oxidized Derivatives. Polymers, 2010. 2(4): p. 505-521.
51. Jain, A., et al., In situ gelling hydrogels for conformal repair of spinal cord defects, and local delivery of BDNF after spinal cord injury. Biomaterials, 2006. 27(3): p. 497-504.
52. Gan, T., Y. Zhang, and Y. Guan, In situ gelation of P(NIPAM-HEMA) microgel dispersion and its applications as injectable 3D cell scaffold. Biomacromolecules, 2009. 10(6): p. 1410-5.
53. Bao, H., et al., Thermo-responsive association of chitosan-graft-poly(N-isopropylacrylamide) in aqueous solutions. J Phys Chem B, 2010. 114(32): p. 10666-73.
54. Chen, J.P. and T.H. Cheng, Thermo-responsive chitosan-graft-poly(N-isopropylacrylamide) injectable hydrogel for cultivation of chondrocytes and meniscus cells. Macromol Biosci, 2006. 6(12): p. 1026-39.
55. Shim, W.S., et al., Biodegradability and biocompatibility of a pH- and thermo-sensitive hydrogel formed from a sulfonamide-modified poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol)-poly(epsilon-caprolactone-co-lactide) block copolymer. Biomaterials, 2006. 27(30): p. 5178-85.
56. Jeong, B., Y.H. Bae, and S.W. Kim, In situ gelation of PEG-PLGA-PEG triblock copolymer aqueous solutions and degradation thereof. J Biomed Mater Res, 2000. 50(2): p. 171-7.
57. Kumar, M.N., et al., Chitosan chemistry and pharmaceutical perspectives. Chem Rev, 2004. 104(12): p. 6017-84.
58. Joshi, R., D.H. Robinson, and K.J. Himmelstein, In vitro properties of an in situ forming gel for the parenteral delivery of macromolecular drugs. Pharm Dev Technol, 1999. 4(4): p. 515-22.
59. Drury, J.L., R.G. Dennis, and D.J. Mooney, The tensile properties of alginate hydrogels. Biomaterials, 2004. 25(16): p. 3187-99.
60. Shapiro, L. and S. Cohen, Novel alginate sponges for cell culture and transplantation. Biomaterials, 1997. 18(8): p. 583-90.
61. Kim, W.S., et al., Adipose tissue engineering using injectable, oxidized alginate hydrogels. Tissue Eng Part A, 2012. 18(7-8): p. 737-43.
62. Zhao, L., M.D. Weir, and H.H. Xu, An injectable calcium
phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering. Biomaterials, 2010. 31(25): p. 6502-10.
63. Lima, E.G., et al., The effect of applied compressive loading on tissue-engineered cartilage constructs cultured with TGF-beta3. Conf Proc IEEE Eng Med Biol Soc, 2006. 1: p. 779-82.
64. Vandermeulen, G.W. and H.A. Klok, Peptide/protein hybrid materials: enhanced control of structure and improved performance through conjugation of biological and synthetic polymers. Macromol Biosci, 2004. 4(4): p. 383-98.
65. Wang, C., J. Kopecek, and R.J. Stewart, Hybrid hydrogels cross-linked by genetically engineered coiled-coil block proteins. Biomacromolecules, 2001. 2(3): p. 912-20.
66. Chunyu Xu, L.J., Chun Wang, Michal Pechar, Jindřich Kopeček, The Influence of Fusion Sequences on the Thermal Stabilities of Coiled-Coil Proteins. 2002. 2(8): p. 395-401.
67. Kopecek, J. and J. Yang, Peptide-directed self-assembly of hydrogels. Acta Biomater, 2009. 5(3): p. 805-16.
68. Li, B., D.O. Alonso, and V. Daggett, The molecular basis for the inverse temperature transition of elastin. J Mol Biol, 2001. 305(3): p. 581-92.
69. Aggeli, A., et al., Responsive gels formed by the spontaneous self-assembly of peptides into polymeric beta-sheet tapes. Nature, 1997. 386(6622): p. 259-62.
70. Nowak, A.P., et al., Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature, 2002. 417(6887): p. 424-8.
71. Hatefi, A. and B. Amsden, Biodegradable injectable in situ forming drug delivery systems. J Control Release, 2002. 80(1-3): p. 9-28.
72. Nickerson, M.T., et al., Kinetic and mechanistic considerations in the gelation of genipin-crosslinked gelatin. Int J Biol Macromol, 2006. 39(4-5): p. 298-302.
73. Elisseeff, J., et al., Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semi-interpenetrating networks. J Biomed Mater Res, 2000. 51(2): p. 164-71.
74. Varghese, S., et al., Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells. Matrix Biol, 2008. 27(1): p. 12-21.
75. Leach, J.B., et al., Development of photocrosslinkable hyaluronic acid-polyethylene glycol-peptide composite hydrogels for soft tissue engineering. J Biomed Mater Res A, 2004. 70(1): p. 74-82.
76. DeLong, S.A., A.S. Gobin, and J.L. West, Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration. J Control Release, 2005. 109(1-3): p. 139-48.
77. Rice, M.A. and K.S. Anseth, Encapsulating chondrocytes in copolymer gels: bimodal degradation kinetics influence cell phenotype and extracellular matrix development. J Biomed Mater Res A, 2004. 70(4): p. 560-8.
78. Shin, H., et al., In vivo bone and soft tissue response to injectable, biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels. Biomaterials, 2003. 24(19): p. 3201-11.
79. Temenoff, J.S., et al., In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels. J Biomed Mater Res A, 2004. 70(2): p. 235-44.
80. Lee, W.K., et al., Novel poly(ethylene glycol) scaffolds crosslinked by hydrolyzable polyrotaxane for cartilage tissue engineering. J Biomed Mater Res A, 2003. 67(4): p. 1087-92.
81. Tan, H., et al., Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering. Biomaterials, 2009. 30(13): p. 2499-506.
82. Huaping Tan, J.P.R.a.K.G.M., Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for adipose tissue regeneration. Organogenesis, 2010. 6(3).
83. Maia, J., et al., Synthesis and characterization of new injectable and degradable dextran-based hydrogels. Polymer, 2005. 46(23): p. 9604-9614.
84. Wang, D.A., et al., Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration. Nat Mater, 2007. 6(5): p. 385-92.
85. Stabenfeldt, S.E., et al., Engineering fibrin polymers through engagement of alternative polymerization mechanisms. Biomaterials, 2012. 33(2): p. 535-44.
86. Johnson, P.J., et al., Controlled release of neurotrophin-3 and platelet-derived growth factor from fibrin scaffolds containing neural progenitor cells enhances survival and differentiation into neurons in a subacute model of SCI. Cell Transplant, 2010. 19(1): p.
89-101.
87. Litvinov, R.I., et al., Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level. Blood, 2005. 106(9): p. 2944-51.
88. Yang, S.H., et al., Three-dimensional culture of human nucleus pulposus cells in fibrin clot: comparisons on cellular proliferation and matrix synthesis with cells in alginate. Artif Organs, 2008. 32(1): p. 70-3.
89. Wei, Y., et al., A novel injectable scaffold for cartilage tissue engineering using adipose-derived adult stem cells. J Orthop Res, 2008. 26(1): p. 27-33.
90. Tan, H., et al., Microscale control over collagen gradient on poly(L-lactide) membrane surface for manipulating chondrocyte distribution. Colloids Surf B Biointerfaces, 2008. 67(2): p. 210-5.
91. Lee, C.R., A.J. Grodzinsky, and M. Spector, The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. Biomaterials, 2001. 22(23): p. 3145-54.
92. Lu, Z., et al., Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape. Tissue Eng Part A, 2010. 16(1): p. 81-90.
93. Halloran, D.O., et al., An injectable cross-linked scaffold for nucleus pulposus regeneration. Biomaterials, 2008. 29(4): p. 438-47.
94. Dechert, T.A., et al., Hyaluronan in human acute and chronic dermal wounds. Wound Repair Regen, 2006. 14(3): p. 252-8.
95. Shen, B., et al., Hyaluronan : its potential application in intervertebral disc regeneration. Orthopedic Research and Reviews, 2010. 2: p. 17-26.
96. Christopher A. Maxwell, J.J.K., Andrew R. Belch, Linda M. Pilarski, and Tony Reiman, Receptor for Hyaluronan-Mediated Motility Correlates with Centrosome Abnormalities in Multiple Myeloma and Maintains Mitotic Integrity. Cancer Res, 2005. 65(3): p. 850-860.
97. Burdick, J.A. and G.D. Prestwich, Hyaluronic acid hydrogels for biomedical applications. Adv Mater, 2011. 23(12): p. H41-56.
98. Vanderhooft, J.L., B.K. Mann, and G.D. Prestwich, Synthesis and characterization of novel thiol-reactive poly(ethylene glycol)
cross-linkers for extracellular-matrix-mimetic biomaterials. Biomacromolecules, 2007. 8(9): p. 2883-9.
99. Pouyani, T. and G.D. Prestwich, Functionalized derivatives of hyaluronic acid oligosaccharides: drug carriers and novel biomaterials. Bioconjug Chem, 1994. 5(4): p. 339-47.
100. Darr, A. and A. Calabro, Synthesis and characterization of tyramine-based hyaluronan hydrogels. J Mater Sci Mater Med, 2009. 20(1): p. 33-44.
101. Gangurde, H., et al., Biodegradable chitosan-based ambroxol hydrochloride microspheres: effect of cross-linking agents. J Young Pharm, 2011. 3(1): p. 9-14.
102. de Alvarenga, E.S., C. Pereira de Oliveira, and C. Roberto Bellato, An approach to understanding the deacetylation degree of chitosan. Carbohydrate Polymers, 2010. 80(4): p. 1155-1160.
103. Fujimoto, T., et al., Antibacterial effects of chitosan solution against Legionella pneumophila, Escherichia coli, and Staphylococcus aureus. Int J Food Microbiol, 2006. 112(2): p. 96-101.
104. Chenite, A., et al., Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials, 2000. 21(21): p. 2155-61.
105. Emilia Curotto, F.A., Quantitative Determination of Chitosan and the Percentage of Free Amino Groups. ANALYTICAL BIOCHEMISTRY, 1993. 211: p. 240-241.
106. Kamal H. Bouhadira, D.S.H., David J. Mooneya, Synthesis of cross-linked poly(aldehyde guluronate) hydrogels. Polymer, 1999. 40: p. 3575-3584.
107. Bilgic, S., et al., A new approach for the estimation of intervertebral disc volume using the Cavalieri principle and computed tomography images. Clin Neurol Neurosurg, 2005. 107(4): p. 282-8.
108. Sun, S. and A. Wang, Adsorption properties of N-succinyl-chitosan and cross-linked N-succinyl-chitosan resin with Pb(II) as template ions. Separation and Purification Technology, 2006. 51(3): p. 409-415.
109. Asako Hirai, H.O., and Akio Nakajima, Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy. Polymer Bulletin, 1991. 26: p. 87-94.
110. O'Brien, P.J., A.G. Siraki, and N. Shangari, Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit Rev Toxicol, 2005. 35(7): p. 609-62.
111. Ryou, M. and C.C. Thompson, Tissue Adhesives: A Review. Techniques in Gastrointestinal Endoscopy, 2006. 8(1): p. 33-37.
112. Poitout, D.G., Biomechanics and Biomaterials in Orthopedics, ed. 12004.
113. Ehud Raanani, M., David A. Latter, MD, Lee E. Errett, MD, Daniel B. Bonneau, MD,Yves Leclerc, MD, and Gary C. Salasidis, MD, Use of BioGlue in Aortic Surgical Repair. The Annals of Thoracic Surgery, 2001. 72: p. 638-640.