201223566 六、發明說明： 【發明所屬之技術領域】 本發明關於一種可用於組織工程之感溫性水膠。 【先前技術】 玻璃體為自然演化的完美結果。玻璃體腔約佔眼球的三分之二， 重量約為4 g’ 容積為 4 mL ( Bishop, Ρ· N· (2000). "Structural macromolecules and supramolecular organisation of the vitreous gel." Prog fezVz Eye及as 19(3): 323-44)，其主要組成為水（約99%)。玻璃體為 透明的膠體，存在於眼球前腔，於水晶體後方且鄰近視網膜。玻璃體 内含有膠原蛋白膠束（collagenmicelle)及玻尿酸，而這些非液狀之物 質可提升玻璃體之機械穩定性。膠原蛋白及玻尿酸為非自源性之物 質。而玻尿酸與膠原蛋白的含量比在眼球前腔水晶體周圍約為2倍，在 眼球中間約為10倍’於視網膜附近約為2〇倍。玻璃體為無血管之組織， 其養份來源來自周遭組織如：脈絡膜、睫狀體及視網膜。且玻璃體是 無法再生的（Soman，N· and R. Baxieijee (2003). "Artificial vitreous replacements." Biomed Mater Eng 13(1): 59-74; Sun, S. and R. Banerjee (2006). In vitro evaluation of in situ gels as short term vitreous substitutes." JBiomed Mater Res A 79(3): 650-64 ) 〇 玻璃體具有以下之物理及光學特性： a.玻璃體之折射率為1.33 ; 201223566 b. 可吸收震盪及維持眼球之形狀； c. 穩固神經及視網膜感光細胞層； d. 可允許代謝產物及養份之流通（Sneu，r. (1995). Head and neck. Clinical Anatomy for Medical Students. New York, Little, Brown and Co: 713-725.)。 人工玻璃體之流變特性 人工玻璃體的材料可分為氣態、液態及膠體三種（Soman,N. andR. Banerjee (2003). "Artificial vitreous replacements." Biomed Mater Eng 13(1): 59-74)。氣體不易儲存於眼腔内而僅能進行短期的填充。故於 現在的玻璃體置換乃藉由液態及膠體物質進行填充。 矽油為最廣泛地應用於治療視網膜剝離之填充物，可用於治療玻 璃體關聯之疾病及視網膜撕裂傷。但是，將矽油植入眼内可能導致術 後的併發症包含了白内障、青光眼及角膜病變。矽油導致的角膜病變 包含了 ：帶狀角膜病變、角膜薄化及角膜内皮喪失(McCuen，B. W.，2nd， S. P. Azen, W. Stem, Μ. Y. Lai, J. S. Lean, K. L. Linton and S. J. Ryan (1993). "Vitrectomy with silicone oil or perfluoropropane gas in eyes with severe proliferative vitreoretinopathy. Silicone Study Report 3." Retina 13(4):279-84)。 矽油為不溶於水且比重較水輕之物質，亦為高黏度之物質，高黏 度之矽油（5000 CS)較低黏度之矽油〇〇〇〇 CS)不易引起乳化作用 201223566 (Lakits, A., T. Nennadal, C. Scholda, S. Knaus and H. Gruber (1999). "Chemical stability of silicone oil in the human eye after prolonged clinical use." 处106(6): 1091-100) 〇矽油脂乳化作用會形成小油滴 並對眼睛組織造成傷害（Κηοιτ，H. L.，A. Seltsam，L. Holbach and G. Ο.201223566 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a temperature sensitive water gel which can be used for tissue engineering. [Prior Art] The vitreous is the perfect result of natural evolution. The vitreous cavity accounts for about two-thirds of the eyeball and weighs about 4 g'. The volume is 4 mL (Bishop, Ρ·N· (2000). "Structural macromolecules and supramolecular organisation of the vitreous gel." Prog fezVz Eye and As 19(3): 323-44), its main component is water (about 99%). The vitreous is a transparent colloid that exists in the anterior chamber of the eyeball behind the crystal and adjacent to the retina. The vitreous body contains collagenic micelles and hyaluronic acid, and these non-liquid substances enhance the mechanical stability of the vitreous. Collagen and hyaluronic acid are non-self-derived substances. The content of hyaluronic acid and collagen is about 2 times around the anterior lens of the eyeball, about 10 times in the middle of the eyeball, and about 2 times in the vicinity of the retina. The vitreous is avascular tissue, and its nutrient source comes from surrounding tissues such as the choroid, ciliary body and retina. And the vitreous is not regenerable (Soman, N. and R. Baxieijee (2003). "Artificial vitreous replacements." Biomed Mater Eng 13(1): 59-74; Sun, S. and R. Banerjee (2006) In vitro evaluation of in situ gels as short term vitreous substitutes." JBiomed Mater Res A 79(3): 650-64 ) The 〇 glass body has the following physical and optical properties: a. The refractive index of the glass body is 1.33; 201223566 b Can absorb shock and maintain the shape of the eye; c. Stabilize the nerve and retinal photoreceptor layer; d. Allow the circulation of metabolites and nutrients (Sneu, r. (1995). Head and neck. Clinical Anatomy for Medical Students. New York, Little, Brown and Co: 713-725.). Rheological properties of artificial vitreous The material of artificial vitreous can be divided into three types: gaseous, liquid and colloidal (Soman, N. and R. Banerjee (2003). "Artificial vitreous replacements." Biomed Mater Eng 13(1): 59-74 ). The gas is not easily stored in the eye cavity and can only be filled for a short period of time. Therefore, the current vitreous replacement is filled by liquid and colloidal substances. Emu oil is the most widely used filler for the treatment of retinal detachment and can be used to treat glass-related diseases and retinal lacerations. However, implantation of eucalyptus oil into the eye may result in postoperative complications including cataracts, glaucoma, and corneal lesions. Corneal lesions caused by eucalyptus oil include: banded keratopathy, corneal thinning, and loss of corneal endothelium (McCuen, BW, 2nd, SP Azen, W. Stem, Μ. Y. Lai, JS Lean, KL Linton and SJ Ryan (1993) "Vitrectomy with silicone oil or perfluoropropane gas in eyes with severe proliferative vitreoretinopathy. Silicone Study Report 3." Retina 13(4): 279-84). Emu oil is a substance that is insoluble in water and light in weight, and is also a high-viscosity substance. High-viscosity eucalyptus oil (5000 CS) has a lower viscosity and is less likely to cause emulsification. 201223566 (Lakits, A., T. Nennadal, C. Scholda, S. Knaus and H. Gruber (1999). "Chemical stability of silicone oil in the human eye after prolonged clinical use." at 106(6): 1091-100) Emulsification can form small oil droplets and cause damage to eye tissue (Κηοιτ, HL, A. Seltsam, L. Holbach and G. Ο.
Naumann (1996). "[Intraocular silicone oil tamponade. A clinico-pathologic study of 36 enucleated eyes]." Ophthalmologe 93(2): 130-8)。而矽油的黏度與生物相容性之相關性是需被研究的。於視網 膜外科手術中，矽油植入體内之長期間之生物相容性是始終伴隨爭 議’許多研究指出’矽油對於角膜内皮細胞具有細胞毒性而導致角膜 病變。MTS生物分析試驗指出矽油會抑制角膜内皮細胞增生(Yang，c s， Κ. Η. Chen, W. Μ. Hsu and Y. S. Li (2008). "Cytotoxicity of silicone oil on cultivated human comeal endothelium.” 办e (Zo㈣ 22(2): 282-8 )。 不論是動物試驗及臨床試驗中’矽油對於眼角膜上皮細胞的影響 都被提出（Sternberg, P.，Jr., D. L. Hatchell，G. N. Foulks and Μ. B. Landers, 3rd (1985). "The effect of silicone oil on the cornea." Arch 103(1): 90-4)。於矽油誘導之角膜病變的動物試驗中，當 於眼睛前房植入70%之矽油’眼角膜内皮細胞密度會下降4〇%，且在接 觸後六天左右會產生氣泡。在動物試驗中，會導致逐漸性的基質變薄。 於臨床實驗中，21位視網膜剝離之病患經歷過玻璃體切除術並填充矽 油，於術後六個月或更長的時間中（Heidenkummer，H p，A Kampik S. Thierfelder (1992). Experimental evaluation of in vitro stability of purified polydimethylsiloxanes (silicone oil) in viscosity ranges from 1000 to 50()() centistokes·" 12(3 Suppl): S28-32)，使用非矽油的填充物 201223566 並無明顯角膜病變的狀況產生。相較於對照組，在填充石夕油後，角膜 細胞的比重迅速降低，且在病理切片中有嚴重的變化（Kard，L，ΜNaumann (1996). "[Intraocular silicone oil tamponade. A clinico-pathologic study of 36 enucleated eyes]." Ophthalmologe 93(2): 130-8). The correlation between the viscosity of eucalyptus oil and biocompatibility is to be studied. In the retinal membrane surgery, the long-term biocompatibility of eucalyptus oil implanted in the body is always accompanied by controversy. Many studies have pointed out that eucalyptus oil is cytotoxic to corneal endothelial cells and causes corneal lesions. MTS bioanalytical experiments indicate that eucalyptus oil inhibits corneal endothelial cell proliferation (Yang, cs, Κ. Η. Chen, W. Μ. Hsu and YS Li (2008). "Cytotoxicity of silicone oil on cultivated human comeal endothelium. (Zo(4) 22(2): 282-8) Both the effects of eucalyptus oil on corneal epithelial cells have been proposed in both animal and clinical trials (Sternberg, P., Jr., DL Hatchell, GN Foulks and Μ. B). Landers, 3rd (1985). "The effect of silicone oil on the cornea." Arch 103(1): 90-4). In animal testing of keratorrhea-induced corneal lesions, when implanted in the anterior chamber of the eye 70% of eucalyptus oil's corneal endothelial cell density will decrease by 4%, and bubbles will be produced about six days after exposure. In animal experiments, it will lead to gradual matrix thinning. In clinical trials, 21 retinal detachment The patient has undergone vitrectomy and is filled with eucalyptus oil for six months or longer after surgery (Heidenkummer, H p, A Kampik S. Thierfelder (1992). Experimental evaluation of in vitro stability of purified polydimethylsiloxan Es (silicone oil) in viscosity ranges from 1000 to 50()() centistokes·" 12(3 Suppl): S28-32), using non-eucalyptus filler 201223566 has no obvious keratopathy. In the control group, after filling with Shixia oil, the specific gravity of corneal cells decreased rapidly and there were serious changes in pathological sections (Kard, L, Μ
Filipec and J. Obenberger (1986). "Specular microscopy 〇f the comeal endothelium after liquid silicone injection into the vitreous in complicated retinal detachments." Graefes Arch Clin Exp Ophthalmol 224(2): 195-200)，這些結果顯示出，當石夕油與角膜内皮細胞接觸後可能導致 細胞異常。然而，矽油導致視網膜内皮細胞的死亡之作用機制尚未被 釐清，推測的原因可能為，眼内的矽油阻擋了水樣液中的養份傳輸而 使細胞死亡（Sternberg，P.，Jr.，D. L. Hatchdl，G. N. Foulks and Μ. B.Filipec and J. Obenberger (1986). "Specular microscopy 〇f the comeal endothelium after liquid silicone injection into the vitreous in complicated retinal detachments." Graefes Arch Clin Exp Ophthalmol 224(2): 195-200), these results show It may cause cell abnormalities when contact with corneal endothelial cells. However, the mechanism by which eucalyptus oil causes death of retinal endothelial cells has not been clarified. The presumed reason may be that oyster oil in the eye blocks the transport of nutrients in the aqueous solution and causes cell death (Sternberg, P., Jr., DL). Hatchdl, GN Foulks and Μ. B.
Landers, 3rd (1985). "The effect of silicone oil 〇n the cornea." Arch 103(1): 90-4)。矽油具有高度安定性、透明度及高界面張 力專特性’因而被應用於玻璃體-視網膜手術過程中之玻璃體填充物， 然而其咼度疏水性特性導致無法適當填補於體液/視網膜之介面，低比 重之特性需於術後六個月内取出避免造成併發症。且石夕油對於角膜内 皮細胞具有細胞毒性。 商品化之人工玻璃體中，除了㈣之外，另有玻尿酸及全敗全氫 化菲（Perfluomperhydrophenanthrene)等商業化產品，而由玻尿酸製備 而成的人JL玻璃體絲大宗。亦有部分人工麵雜轉原蛋白所製 成。這些錢高分子材料具有良好之生物相容性，但長時間存於體内 會降解，而無法長時_置放於玻軸，且目前所制之材料亦 無感溫聚合特性。因此’於臨床上的使用受到限制。 201223566 -般而言，良好的人工玻璃體應具有以下優點： L良好的光學特性：人工_體需要有良好的透光度打會導致視覺 遮蔽之發生’此外,謂要有適當的娜麵免辟織病患之視 力。 ϋ·可注射性及感溫聚合特性：對於外科手術而言，當材料具有可注射 性時’操作者可以容易的將材相主入受試者體内，並藉由其感溫性 使材料於體溫下產生自體聚合，藉此提高材料在體内時之結構強 度。 iii.極佳的生物相容性：若材料無生物相容性，在植入體内後會導致受 試者產生發炎反應及免疫反應，亦可能有嚴重之併發症發生使受試 者具有生命之威脅。 水膠（hydrogel)為一種可於液體及膠體狀態間改變之物質，當經 歷過外部環境的變換如溫度、pH值及光反應，即會從液體轉換成膠體 (Kim, M. R. and T. G. Park (2002). "Temperature-responsive and degradable hyaluronic acid/Pluronic composite hydrogels for controlled release of human growth hormone.”《/Cow/ro/iieZease 80(1-3): 69-77)。水 膠在過去十年來開始被研究及受到重視，目前已廣泛應用於藥物釋 放、組織工程及再生醫學中（Cosgriff-Hemandez，E. and A. G. Mikos (2008). "New biomaterials as scaffolds for tissue engineering." Pharm Res 25(10): 2345-7)。可注射性水膠被研究於細胞傳遞系統（cell delivery system)，其優點為可使細胞及生物分子迅速的結合於膠體基質中 201223566 (Nuttelman, C. R., M. A. Rice, A. E. Rydholm, C. N. Salinas, D. N. Shah and K. S. Anseth (2008). "Macromolecular Monomers for the Synthesis of Hydrogel Niches and Their Application in Cell Encapsulation and TissueLanders, 3rd (1985). "The effect of silicone oil 〇n the cornea." Arch 103(1): 90-4). Emu oil has high stability, transparency and high interfacial tension characteristics. It is therefore applied to vitreous fillers in vitreous-retinal surgery. However, its hydrophobicity makes it impossible to properly fill the body fluid/retina interface, low specific gravity. Characteristics need to be removed within six months after surgery to avoid complications. And Shixia oil is cytotoxic to corneal endothelial cells. Among the artificial vitreous bodies, in addition to (4), there are commercial products such as hyaluronic acid and Perfluomperhydrophenanthrene, and human JL vitreous filaments prepared from hyaluronic acid are bulky. There are also some artificial surface miscellaneous proteins. These money polymer materials have good biocompatibility, but they will degrade in the body for a long time, and cannot be placed on the glass shaft for a long time, and the materials currently prepared have no temperature-sensing properties. Therefore, clinical use is limited. 201223566 In general, a good artificial vitreous body should have the following advantages: L. Good optical properties: artificial _ body needs to have good transparency, which will lead to visual obscuration. In addition, it is necessary to have proper nata noodles. The vision of the patient.可·Injectability and temperature-sensing polymerization characteristics: For surgery, when the material is injectable, the operator can easily introduce the material into the subject and make the material sensitive by temperature. Autogenous polymerization occurs at body temperature, thereby increasing the structural strength of the material as it is in the body. Iii. Excellent biocompatibility: If the material is not biocompatible, it will cause an inflammatory reaction and immune response in the subject after implantation in the body, and there may be serious complications that cause the subject to have life. Threat. Hydrogel is a substance that changes between liquid and colloidal states. It changes from a liquid to a colloid when subjected to changes in the external environment such as temperature, pH, and photoreaction (Kim, MR and TG Park (2002). "Temperature-responsive and degradable hyaluronic acid/Pluronic composite hydrogels for controlled release of human growth hormone." /Cow/ro/iieZease 80(1-3): 69-77. Water gelatin has been in the past decade. It has been studied and valued and is now widely used in drug release, tissue engineering and regenerative medicine (Cosgriff-Hemandez, E. and AG Mikos (2008). "New biomaterials as scaffolds for tissue engineering." Pharm Res 25 ( 10): 2345-7). Injectable water gel is studied in a cell delivery system, which has the advantage of allowing cells and biomolecules to rapidly bind to a colloidal matrix 201223566 (Nuttelman, CR, MA Rice, AE Rydholm, CN Salinas, DN Shah and KS Anseth (2008). "Macromolecular Monomers for the Synthesis of Hydrogel Niches and Their Application In Cell Encapsulation and Tissue
Engineering.” Prag户〇/>7« <SW 3；3(2): 167-179)。天然的可注射水膠在凝膠 (gelation)前具有卓越的能力可輕易地將自體細胞植入於任何大小 及形狀之填充位置。此外，水膠注射後具有良好之生理特性，且對受 術者的侵入性極小（Pratt，A. B.，F. E. Weber, H. G. Schmoekel，R. Muller and I. A. Hubbell (2004). "Synthetic extracellular matrices for in situ tissue engineering." Biotechnol Bioeng 86(1): 27-36 ) 0 ^ (thermosensitive hydrogel)為當水膠經過溫度的改變會隨著其變化使 型態產生改變，此固液態轉移之特性可應用於組織工程中，當水膠受 熱超過臨界液態溫度（lower critical solution temperature，LCST)，會產 生凝膠化而從液態轉換成膠體。 玻尿酸（hyaluronic acid，HA )為由N-乙醯基-D-葡萄胺糖及D-葡 萄糖醛酸所組成之聚合物。玻尿酸為結締組織中最主要的胞外基質， 也豐富的存在於玻璃體及關節液中。玻尿酸在傷口癒合中松演著重要 的角色，可促進細胞分化及維持細胞型態。玻尿酸於眼科及關節外科 的臨床上被視為是一種黏性補充劑（visco_SUpplementati〇nagent)。破 尿酸具有極佳的生物相容性及生物降解性，在組織工程中，經常被用 來製備多孔性的支架或藥物釋放裝置》 玻尿酸是玻璃體中最主要的成份，也是眼科學中最重要的巨分 子。玻尿酸是一種良好的人工玻璃體材料。由於玻尿酸獨特的黏性， 201223566 於進行白内障及水晶體植入之外科手術時可用於保護脆弱的眼組織。Engineering.” Praghu/>7« <SW 3;3(2): 167-179). Natural injectable water gels have excellent ability to easily autologous cells before gelation Implanted in any size and shape filling position. In addition, hydrogel has good physiological properties after injection and is minimally invasive to the subject (Pratt, AB, FE Weber, HG Schmoekel, R. Muller and IA Hubbell ( 2004). "Synthetic extracellular matrices for in situ tissue engineering." Biotechnol Bioeng 86(1): 27-36 ) 0 ^ (thermosensitive hydrogel) is the type of water gel that changes with temperature as it changes. Change, the characteristics of this solid-liquid transfer can be applied to tissue engineering, when the water gel is heated to exceed the critical liquid temperature (LCST), it will gel and convert from liquid to colloid. Hyaluronic acid (HA) It is a polymer composed of N-acetyl-D-glucamine and D-glucuronic acid. Hyaluronic acid is the most important extracellular matrix in connective tissue, and is also abundant in vitreous and joints. Hyaluronic acid plays an important role in wound healing, promoting cell differentiation and maintaining cell type. Hyaluronic acid is clinically regarded as a viscous supplement (visco_SUpplementati〇nagent) in ophthalmology and joint surgery. Excellent biocompatibility and biodegradability, often used in tissue engineering to prepare porous scaffolds or drug delivery devices. Hyaluronic acid is the most important component in the vitreous and the most important macromolecule in ophthalmology. Hyaluronic acid is a good artificial vitreous material. Due to the unique viscosity of hyaluronic acid, 201223566 can be used to protect fragile ocular tissues during cataract and hydrogel implantation.
Pluronic F-127為一種三區域（tri-block )之感溫性 (temperature-sensitive)聚合物，由聚(乙二醇)·聚(丙二醇)聚（乙二醇) 所組成，其結構如下面所示：Pluronic F-127 is a tri-block temperature-sensitive polymer composed of poly(ethylene glycol)·poly(propylene glycol) poly(ethylene glycol) and its structure is as follows Shown as follows:
Pluronic F-127為水溶性及低毒性之物質，並通過美國fda於臨床 使用之認可。Pluronic F-127具有固態-液態轉型之特性。將15%之 Pluronic F-127水溶液置於25°C以下之環境時，此水膠為液態，但將此 水膠置於體内環境中（37。(：，pH=7.2〜7.4)，此水膠會轉型為膠體。 Pluronic F-127具有親水端之聚(乙二醇)及疏水端之聚(丙二醇），可藉由 親水端及疏水端之作用力產生型態的改變。在溫度低於低臨界溶液溫 度（lower critical solution temperature，LCST)時’乙稀及丙浠基團會含 水’且聚氧化乙烯會溶於水中。當溫度提升超過低臨界溶液溫度時， 聚氧化乙浠會逐漸失去水溶性並形成微胞，當膠粒内聚性提高時即會 產生水膠。 近年來，Pluronic F-127已使用於眼科藥物傳遞系統(Lin，H. R.，K. C. Sung and W. J. Vong (2004). "In situ gelling of alginate/pluronic solutions for ophthalmic delivery of pilocarpine.1' Biomacromolecules 5(6): 2358-65)，但於商業上尚無任何玻尿酸/Pluronic f-127之產品應用於人 201223566 工玻璃體或組織工程。 【發明内容】 本發明係使用具有極佳生物相容性之玻尿酸以及FDA所認可之具 有感溫聚合特性之聚合物Pluronic F_127，將兩聚合物以化學接合方法 形成共聚物（c〇-pdymer) ’並藉由其疏水性區域之交互作用產生凝膠 特性’開發出智慧型之感溫性水膠（thermosensitivity hydrogel)，以取 代傳統臨床上廣泛使用之矽油做為人工玻璃體。本發明之共聚物具有 獨特之感溫聚合特性，會隨著溫度之提升而增加其黏彈性，且具有可 注射性，使臨床操作者容易使用。本發明之共聚物並具有良好之光學 特性，與人類視網膜色素細胞具有極佳之生物相容性。因此本發明之 共聚物具有感溫聚合特性、可注射性、良好的光學特性及生物相容性， 與傳統上用來當作人工玻璃體之石夕油相比，本發明之共聚物可作為人 工玻璃體之良好材料，亦有取代石夕油之潛能。 一般而言化學接合之方法是利用環氧化合物（Εχ_81〇)、#_(3_二甲 胺丙基)’6基碳二亞胺鹽酸鹽（EDC)、戊二路及i•經基苯并三唾單 水合物（HQBt · H2Q)等所製備之交麵之—或其組合，其作用機制 係利用Pl_ic F-12?結構上之〇H基與玻尿酸上骨架上之一或多個選 自由CH3、NH3、COOH及OH所組成群組之基團進行接合，而達到形成 共聚物之目的。 本文中所使用的術語意義除非另有特別界定，否則均與熟習此項技 201223566 藝者的普遍認知相同。本㈣案中使用的術語其意義如下所列： 「共聚物」意指-種源自兩個（或更多）單體種類的聚合物，其係 相對於僅有一個單體的均聚物而言。 「交聯劑」意指-種能在線型分子間起架橋作用從而使多個線型分 子相互鍵合交魏網路結_物質，其為—種可促進朗節聚合物分 子鏈間共價鍵或離子鍵形成的物質。本發财所使狀交聯劑包括但 不限於EDC、HOBt、環氧樹脂 '戊二醛及遍8〇中其一或其組合。 因此，本發明提供-種用於組織工程的共聚物，包含玻尿酸與 Pluronic F-127交聯後之聚合^在較佳實施例中，玻尿_ piur_ F-127的重量比範圍為3:丨至丨:6G。在更佳實施例中，玻尿酸與恤他 F-127的重量比為1 : 1、丨：15或丨：2〇。該共聚物會隨溫度改變_ 化型態’低於臨界液態溫麟為雜，高誠界液態溫度制為踢體。 在較佳實施例中，該共聚物具有良好可注射性，可容易地通過打號針 頭，並具有良好之光學特性、生物安定性及生物相容性。在更佳實施 例中’該共聚物係作為人工玻璃體之材料。 本發明亦提供一種製備玻尿酸與Plur〇nic F427共聚物的方法，勹 含使用交聯劑將Pluronic F-127結構上之〇H基與玻尿酸骨架上—曳多 個選自由(¾、NH3、C00H及OH所組成群組之基團進行接合。在較佳 實施例中，該方法包含以下步驟： a.提供一交聯劑、一玻尿酸溶液及一piuronicF-127溶液； 201223566 b. 混合交聯劑、玻尿酸溶液及PluronicF-127溶液，形成一混合溶液； c. 維持該混合溶液之pH值於6到8之間；及 d. 將該混合溶液以透析處理，乾燥後取得乾燥產物。 上述方法在較佳實施例中’步驟a之溶液的溶劑為水；步驟b係先將 交聯劑加入玻尿酸溶液或Pluronic F-127溶液中攪拌均勻後，再加入剩下 之另-溶液進行反應；步驟c之pH值較佳為6.8。在一較佳實施例中，步 驟c之反應時間至少為2小時。在另-較佳實施例中，玻尿酸與piur〇nic F-127的重量比範圍為3 : 1至1 : 6〇。在更佳實施例中，玻尿酸與朽臟化鲁 F-127的重量比為1 :卜1 : 15或1 : 2〇。在另一較佳實施例中，該交聯 劑為EDC及HOBt之組合。在較佳實施例中，該共聚物具有良好可注射 性，可容易地通過27號針頭，並具有良好之光學特性、生物安定性及生 物相容性。在更佳實施例中，該共聚物係作為人工玻璃體之材料。 【實施方式】 本發明可能以不同的形式來實施，並不僅限於下列文中所提及的實 例。下列實施例僅作為本發明不同面向及特點中的代表。 實施例1 感溫性人工玻璃體之製備 本發明之共聚物隨著所接合之Pluronic F_127比例不同可發展出三 種不同的共聚物：共聚物卜共聚物2及共聚物3。 1·秤取玻尿酸及不同比例Pluronic叩7藉由交聯劑合成三種類共 ^ ^ mm (^^/PluronicF-127=0.1 g/〇.i g , w/w). (¾ 12 201223566 尿酸/Pluronic F-127=(U似g，w/w) '共聚物3 (破尿酸跑〇血 F-127=0.1 g/2g，w/w)分別溶於5〇ml去離子水中。 2. m78gEDC、a77gHOBt^^2〇mlDMSO/^*^(m， WV) ’緩慢地加入玻尿酸溶液中，待攪拌均勻再加入水溶 液。 3. 維持pH值於6.8 ’於室溫下反應至少4小時，待其pH穩定後放置整 夜。 4·將玻尿酸/PluromcF-127溶液以透析處理，並以冷;東乾燥之方法 獲得乾燥產物。 本發明之交聯機制如圖1所示。 實施例2流變特性分析 本實驗係以流變儀（Rheometer，AR2000-ex，TA instrument，NewPluronic F-127 is a water-soluble and low-toxic substance approved by the US fda for clinical use. Pluronic F-127 has a solid-liquid transition. When 15% of the aqueous solution of Pluronic F-127 is placed in an environment below 25 ° C, the water gel is in a liquid state, but the water gel is placed in the body environment (37. (:, pH = 7.2 to 7.4), The water gel will be transformed into a colloid. Pluronic F-127 has a hydrophilic end of poly(ethylene glycol) and a hydrophobic end of poly(propylene glycol), which can be changed by the action of the hydrophilic end and the hydrophobic end. At low critical solution temperature (LCST), the 'ethylene and propylene groups will contain water' and the polyethylene oxide will dissolve in water. When the temperature rises above the low critical solution temperature, the polyethylene oxide will gradually Loss of water solubility and formation of micelles, water gel is produced when the cohesiveness of the micelles is increased. In recent years, Pluronic F-127 has been used in ophthalmic drug delivery systems (Lin, HR, KC Sung and WJ Vong (2004). "In situ gelling of alginate/pluronic solutions for ophthalmic delivery of pilocarpine.1' Biomacromolecules 5(6): 2358-65), but there is no commercial hyaluronic acid/Pluronic f-127 product applied to human 201223566 Or tissue engineering SUMMARY OF THE INVENTION The present invention uses a hyaluronic acid having excellent biocompatibility and an FDA-approved polymer Pluronic F_127 having a temperature-sensing property to form a copolymer (c〇-pdymer) by chemical bonding. 'And by the interaction of its hydrophobic regions to produce gel properties' developed a smart thermosensitivity hydrogel to replace the traditional clinically widely used emu oil as an artificial vitreous. The copolymer of the present invention is unique The temperature-sensing polymerization property increases its viscoelasticity with an increase in temperature and is injectable, making it easy for clinical operators to use. The copolymer of the present invention has good optical properties and is extremely polar with human retinal pigment cells. Good biocompatibility. Therefore, the copolymer of the present invention has temperature-sensing polymerization properties, injectability, good optical properties and biocompatibility, compared with the traditionally used as an artificial vitreous stone. The copolymer of the invention can be used as a good material for artificial vitreous body, and also has the potential to replace Shixia oil. Generally speaking, chemistry The method is to use an epoxy compound (Εχ_81〇), #_(3_dimethylaminopropyl) '6-carbodiimide hydrochloride (EDC), pentane and i. The interface prepared by monohydrate (HQBt · H2Q) or the like, or a combination thereof, utilizes one or more of the skeletons on the Pl_ic F-12? structure on the H group and the hyaluronic acid to be selected from CH3, The groups of the group consisting of NH3, COOH and OH are joined to achieve the purpose of forming a copolymer. The meaning of the terms used herein is the same as the general knowledge of those skilled in the art, unless otherwise specifically defined. The terminology used in this (4) case is as follows: "Copolymer" means a polymer derived from two (or more) monomer species, relative to a homopolymer having only one monomer. In terms of. "Crosslinking agent" means a kind of intermolecular intermolecular bridging effect, so that a plurality of linear molecules are bonded to each other to form a network-based substance, which is a kind of covalent bond between the molecular chains of the Langfang polymer. Or a substance formed by an ionic bond. The present invention has a cross-linking agent including, but not limited to, EDC, HOBt, epoxy resin glutaraldehyde, and one or a combination thereof. Accordingly, the present invention provides a copolymer for tissue engineering comprising a polymerization of hyaluronic acid crosslinked with Pluronic F-127. In a preferred embodiment, the weight ratio of hyaluronic_piur_F-127 is 3: To the 丨: 6G. In a more preferred embodiment, the weight ratio of hyaluronic acid to the shirt F-127 is 1:1, 丨:15 or 丨:2〇. The copolymer will change with temperature _ ing form 'below the critical liquid temperature, and the high temperature liquid temperature will be the kick. In a preferred embodiment, the copolymer has good injectability, can be easily passed through a marking needle, and has good optical properties, biostability, and biocompatibility. In a more preferred embodiment, the copolymer is used as a material for artificial glass bodies. The invention also provides a method for preparing a copolymer of hyaluronic acid and Pluur〇nic F427, which comprises using a cross-linking agent to bind a ruthenium H group and a hyaluronic acid skeleton on the Pluronic F-127 structure to a plurality of selected from (3⁄4, NH3, C00H). And the group of the group consisting of OH is joined. In a preferred embodiment, the method comprises the steps of: a. providing a crosslinking agent, a hyaluronic acid solution and a piuronic F-127 solution; 201223566 b. , hyaluronic acid solution and Pluronic F-127 solution, forming a mixed solution; c. maintaining the pH of the mixed solution between 6 and 8; and d. treating the mixed solution by dialysis, drying to obtain a dried product. In the preferred embodiment, the solvent of the solution of step a is water; in step b, the crosslinking agent is added to the hyaluronic acid solution or the Pluronic F-127 solution and stirred uniformly, and then the remaining solution is added to carry out the reaction; The pH is preferably 6.8. In a preferred embodiment, the reaction time of step c is at least 2 hours. In another preferred embodiment, the weight ratio of hyaluronic acid to piur〇nic F-127 is 3: 1 to 1: 6 〇. In a better implementation The weight ratio of hyaluronic acid to turmeric Lu F-127 is 1: 1:15 or 1:2. In another preferred embodiment, the crosslinking agent is a combination of EDC and HOBt. In the examples, the copolymer has good injectability, can easily pass through a 27 gauge needle, and has good optical properties, biostability and biocompatibility. In a more preferred embodiment, the copolymer is artificial. The material of the glass body. [Embodiment] The present invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the different aspects and features of the present invention. Preparation of Artificial Glass Body The copolymer of the present invention can develop three different copolymers according to the ratio of the Pluronic F_127 to be bonded: copolymer copolymer 2 and copolymer 3. 1) Weighing hyaluronic acid and different ratios of Pluronic叩7 Three kinds of compounds were synthesized by cross-linking compound ^^ mm (^^/PluronicF-127=0.1 g/〇.ig, w/w). (3⁄4 12 201223566 uric acid/Pluronic F-127=(U like g, w/w ) 'Copolymer 3 (breaking uric acid running blood F-127 = 0.1 g / 2g, w / w) dissolved separately 5〇ml deionized water 2. m78gEDC, a77gHOBt^^2〇mlDMSO/^*^(m, WV) ' Slowly add hyaluronic acid solution, stir evenly and add water solution. 3. Maintain pH at 6.8 ' The reaction was allowed to proceed for at least 4 hours at room temperature, and allowed to stand overnight after the pH was stabilized. 4. The hyaluronic acid/Pluromc F-127 solution was treated with dialysis, and the dried product was obtained by cold; east drying. The crosslinking mechanism of the present invention is shown in FIG. Example 2 Rheological properties analysis This experiment is a rheometer (Rheometer, AR2000-ex, TA instrument, New
Castle，DE，USA )分析試樣之流變特性，其試驗方法是使用4〇 ^平 盤、溫度坡道步驟（Temperature ramp step)模式、升溫範圍為4_45。〇、 升溫速率為1.5°C/min及壓力百分比（％Strain)為1〇進行觀察。 bit變學分析結果指出共聚物1並無感溫性（圖2)，而共聚物2及共聚 物3則具有極佳之感溫聚合特性（圖3及圖4)。共聚物2之臨界液態溫度 (LCST)約為23°C，且在植入眼内（32°C)後其儲存模數（G，）可達 到804.6 Pa，且損失模數（G”）為448.6。共聚物3共聚物於32t：下儲存 模數（G’）可達到1137 Pa ’且損失模數（G”）為575.7 pa。於臨床應用 上’相較於無感溫聚合特性的石夕油，共聚物2及共聚物3在注入受試者 η 201223566 體内後’會財更佳之結構賊及穩定性。 實施例3可注射性 本試驗係為了驗證本發敗編之可注，其下：製 備重量百姐_〜·之錄財純，熟技雜，將試樣移置 針筒中，並裝置27號針頭進行試驗。 實驗結果指出，共聚物2及共聚物3可在室溫下料騎過27號針 頭（圖5)’此結果碰了共聚物之可注祕。諸料具有可注射性於 臨床使用上’操作者更容易將材料注人受試者體内，且可以使用較小 的針頭。較小的針頭可減少植入時之傷口面積。 實施例4透光度分析 以紫外 *T 見光分光光度計（UY/Yisibie spectrophotometer ) (Ultrospec 2100 pro, Amersham Bioscience，Sweden )债測試樣在可見光 (400-700 nm)之穿透率（Surf，s. and R，Baneijee (2006)· "In vitro evaluation of in situ gels as short term vitreous substitutes." J Biomed J 79(3): 650-64)。折射率分析則是以阿貝折射計（ATAGO, NAR-1T，Kirkland，WA，USA)於室溫下進行測定（Swindle-Reilly，Κ. Ε·, M. Shah, P. D. Hamilton, T. A. Eskin, S. Kaushal and N. Ravi (2009). "Rabbit study of an in situ forming hydrogel vitreous substitute." Invest 1¾ 5b/ 5〇(l〇): 484〇_6)。 玻璃體之折射率約為 1.33 ( Snell, R. (1995). Head and neck. Clinical Anatomy for Medical Students. New York, Little, Brown and Co: 201223566 713-725)，而本發明所合成之共聚物的折射率之平均值為I %”（表 1) ’相較於矽油之折射率L405 (McCartney，D L，κ M m版，w」Castle, DE, USA) Analyze the rheological properties of the sample by using a 4 〇 ^ plate, Temperature ramp step mode, and a temperature rise range of 4_45. 〇, the heating rate was 1.5 ° C / min and the pressure percentage (% Strain) was 1 〇 for observation. The results of the bit variation analysis indicated that the copolymer 1 was not temperature sensitive (Fig. 2), while the copolymer 2 and the copolymer 3 had excellent temperature-sensing polymerization characteristics (Fig. 3 and Fig. 4). The critical liquid temperature (LCST) of Copolymer 2 is about 23 ° C, and its storage modulus (G,) can reach 804.6 Pa and the loss modulus (G") after implantation in the eye (32 ° C). 448.6. Copolymer 3 copolymer at 32t: storage modulus (G') can reach 1137 Pa 'and loss modulus (G") is 575.7 pa. In the clinical application, compared with the non-temperature-sensing polymerization characteristics of the copolymer, the copolymer 2 and the copolymer 3 are better in structural thieves and stability after being injected into the subject η 201223566. Example 3 Injectability This test is to verify the success of this failure, under the following: preparation of the weight of the hundred sisters _ ~ · of the pure, skilled, the sample is placed in the syringe, and the device 27 needle experimenting. The experimental results indicate that Copolymer 2 and Copolymer 3 can be passed over a 27 gauge needle at room temperature (Fig. 5). The ingredients are injectable for clinical use. The operator is more likely to inject material into the subject and a smaller needle can be used. Smaller needles reduce the wound area at the time of implantation. Example 4 Transmittance Analysis The transmittance of visible light (400-700 nm) in a UV*T spectroscopy (UY/Yisibie spectrophotometer) (Ultrospec 2100 pro, Amersham Bioscience, Sweden) bond test (Surf, s. and R, Baneijee (2006) · "In vitro evaluation of in situ gels as short term vitreous substitutes." J Biomed J 79(3): 650-64). The refractive index analysis was carried out at room temperature using an Abbe refractometer (ATAGO, NAR-1T, Kirkland, WA, USA) (Swindle-Reilly, Κ. Ε·, M. Shah, PD Hamilton, TA Eskin, S Kaushal and N. Ravi (2009). "Rabbit study of an in situ forming hydrogel vitreous substitute." Invest 13⁄4 5b/ 5〇(l〇): 484〇_6). The refractive index of the vitreous is about 1.33 (Snell, R. (1995). Head and neck. Clinical Anatomy for Medical Students. New York, Little, Brown and Co: 201223566 713-725), and the copolymer synthesized by the present invention The average value of the refractive index is I %" (Table 1) 'Compared to the refractive index L405 of eucalyptus oil (McCartney, DL, κ M m version, w"
Stark, D. L. Guyton and R. G Michels (1987). -Intraocular lens style and refraction in eyes treated with silicone oil." Arch Ophthalmol 105(10): 1385-7)更接近天然玻璃體。 表1共聚物之折射率 折射率（nD) "" - 樣本 1.3573 1.3536 1.3537 1.3543 # ^玻尿酸 共聚物1 共聚物2 共聚物3_ 透光度分析指出（圖6)，共聚物1之透光度極低，在可見光範圍 中，波長皆小於1%。另一方面，共聚物2及共聚物3此兩共聚物之透 光度皆在70%以上’而在波長別肺，其透光度高達·以上。於折 參 射率及透光度兩方面來評估，共聚物2及共聚物3此兩共聚物為一良 好之人工玻璃體材料。 實施例5聚合程度分析 以三硝基苯續酸（TNBS)與試樣結構中的自由胺基結合，若結構 中自由胺基之存在越少則代表此結構之聚合程度較佳。其分析程序係 修飾Nam等人之方法（Nam，Κ·, T. Kimura，S. Funamoto and A. Kishida "Preparation of a collagen/polymer hybrid gel designed f〇r tissue membranes. Part I： controlling the polymer-collagen cross-linking process 15 201223566 using an ethanol/water co-solvent." Acta Biomater 6(2): 403-8 )： 1. 將lOmg試樣浸置於4%碳酸氫鈉（s〇diumbicarb〇nate)及TNBS 中。 2. 以40°C水浴反應2小時，並以5號濾紙過濾反應液。並將濾紙部 份及濾液進行冷凍乾燥。 3·以6MHC1將濾紙部份上之以反應之TNBS回溶，並以〇D 345邮 (Epoch, Bio-tek instruments，USA )測定其吸光值。 4.以下列公式計算共聚物之聚合程度： 聚合程度（％)=Stark, D. L. Guyton and R. G Michels (1987). -Intraocular lens style and refraction in pixels treated with silicone oil.&Arch; Arch Ophthalmol 105(10): 1385-7) is closer to the natural glass body. Table 1 refractive index refractive index (nD) of the copolymer "" - sample 1.3573 1.3536 1.3537 1.3543 # ^ hyaluronic acid copolymer 1 copolymer 2 copolymer 3_ transmittance analysis pointed out (Figure 6), the transmission of copolymer 1 Very low, in the visible range, the wavelength is less than 1%. On the other hand, the copolymers 2 and 3 have a transmittance of 70% or more in both of the copolymers, and the transmittance in the lungs is as high as or higher than the above. The two copolymers of copolymer 2 and copolymer 3 were evaluated as a good artificial vitreous material in terms of refractive index and transmittance. Example 5 Analysis of Degree of Polymerization Trinitrobenzene acid (TNBS) was combined with a free amine group in the structure of the sample. If the presence of free amine groups in the structure is less, the degree of polymerization of the structure is preferred. The analysis program is a modification of the method of Nam et al. (Nam, T. Kimura, S. Funamoto and A. Kishida " Preparation of a collagen/polymer hybrid gel designed f〇r tissue membranes. Part I: controlling the polymer -collagen cross-linking process 15 201223566 using an ethanol/water co-solvent." Acta Biomater 6(2): 403-8 ): 1. Immerse lOmg sample in 4% sodium bicarbonate (s〇diumbicarb〇) Nate) and TNBS. 2. The reaction was carried out in a 40 ° C water bath for 2 hours, and the reaction solution was filtered through a No. 5 filter paper. The filter paper portion and the filtrate were freeze-dried. 3. Re-dissolve the TNBS on the filter paper portion with 6 MHC1 and measure the absorbance at 〇D 345 (Epoch, Bio-tek instruments, USA). 4. Calculate the degree of polymerization of the copolymer by the following formula: Degree of polymerization (%) =
V聚合樣本的吸光度^ ,未聚合樣本的吸光度J 理論上，玻尿酸接合PluronicF-127之比例越高，聚合程度亦越高。 TNBS分析指出，隨著玻尿酸所接合之比例越高，試樣所 測得之自由胺基越低’亦表示其聚合程度較高（圖7)。於本實驗中驗 證共聚物2及共聚物3具有極佳的聚合程度，此結果揭示出此兩共聚物 具有良好的結構強度及穩定性。當共聚物具有良好之結構強度及穩定 性，當共聚物應用於體内試驗時，可避免自由基釋放而對生物造成影 響。故本發明所揭示之共聚物應具有良好之生物安定性，應可適用於 眼科臨床。 實施例6生物相容性分析 16 201223566 玻璃體為透明的膠體存在於眼球前腔，位於水晶體後方且大部 份之面積皆與鄰近之視網膜接觸。有鑑於此，此生物相容性之體外試 驗係以人類視網膜色素上皮細胞株（ARPE-19，BCRC編號為60383) 進行共聚物之細胞毒性分析。由於需模擬體内試驗之情形，故先將 ARPE-19細胞培養至嵌入式培養皿（Insert well) (Millicell，Millipore corporation，Billerica，MA，USA)上，再加入共聚物與細胞接觸，而培 養孤中之培養基可由培養孤底部之半通透膜經擴散作用傳遞營養物 質，猶如模擬玻璃體為無血管之組織，其養份來源來自周遭組織如： 脈絡膜、睫狀體及視網膜（Suri，S. and R. Banerjee (2006). "In vitro evaluation of in situ gels as short term vitreous substitutes." J Biomed Maier細j 79(3): 650-64) ’若具細胞毒性之物質將可透過半通透膜並 對ARPE-19細胞造成毒殺作用。其操作程序如下（圖8): 1.將ARPE-19細胞置入插入式培養皿中（每格2xl〇4個細胞），於 37 C、5% C〇2之環境下培養24小時。 2·將試樣加入插入式培養皿中，並於c〇2培養箱作用3曰。 3·進行MTT分析：加入10%體積之NOT溶液（5mg/ml於PBS中） 並於37它、5%C〇2之環境下反應4小時。 夺液體去除’並以一曱亞礙（DMSO)將MTT甲腹（formazan) 回浴，待晶體完全溶解，以OD.570nm測定其吸光值。 本式驗係利用插入式培養皿以模擬共聚物填充至玻璃體腔與視網 膜接觸之If形’生物相容性絲指出，轉明所製㈣三種共聚物對 17 201223566 於類視網膜色素上皮細胞具有極佳之生物相容性（圖9)。本發明之共 聚物相對於控制組、單獨玻尿酸、單獨Pluronic F-127及石夕油，具有更 高的存活率，且具有顯著差異（户^.05)。此結果揭示，爾後將共聚物 進行體内移植，並避免生物相容性不佳所造成之細胞毒性，可大幅降 低造成發炎反應及併發症之產生。 一個熟知此領域技藝者能很快體會到本發明可很容易達成目標，並 獲得所提到之結果及優點，以及那些存在於其中的東西。本發明中之 共聚物、水膠、人工玻璃體及其製造程序與方法乃較佳實施例的代表， 其為示範性且；Ϊ；僅侷限於本個領域。熟知此鋪者將會想到其中可 修改之處及其個途。這些修_齡在本發_精神巾，並在申請 專利範圍中界定。 本發明的魄錢與實施綱揭科細，得使倾熟習此技藝者能 夠製造及使用本發明，即使其中有各種不同的改變、_、及進步之 處，仍應視為不脫離本發明之精神及範圍。 說月田中提及之所有專利及出版品，都肺發财關領域之一般技 藝為準。所有專利和出版品都在此被納人相_參考程度，就如同每 -個侧都被具體且侧麟㈣入參考。 在此所適當地舉例說明之發明，可能得以在缺乏任何要件，或許多 要件、限制條件或並非狀為本文中所揭示的限制情況下實施。所使 用的名詞及表達是作為朗書之描述㈣_，_並無賴使用這 類排除任何等同於所示及說明之特點或其部份之名詞及表達，但需認 18 201223566 '月的是’在本發_專利巾請範圍内有可能出現各種不同的改變。因 此應了解到雖然已根據較佳實施例及任意的特點來具體揭示本發 明，但是熟知此技藝者仍會修改和改變其中所揭示的内容，諸如此類 的修改和變化仍在本發明之申請專利範圍内。 【圖式簡單說明】 圖1、玻尿酸與Pluronic F_127之共聚物合成機制示意圖。（A):玻尿 酸、（B) : Pluronic F-127、（C):共聚物。『{丨』符號中間的部 份表示其為疏水性區域。 圖2、共聚物1之流變圖。 圖3、共聚物2之流變圖。 圖4、共聚物3之流變圖。 圖5、共聚物可注射性示意圖。 圖6、透光度分析結果。 圖7、聚合程度分析結果》 圖8、生物相容性分析示意圖。 圖9、生物相容性分析結果。Absorbance of V-polymerized sample^, absorbance of unpolymerized sample J Theoretically, the higher the proportion of hyaluronic acid-bonded Pluronic F-127, the higher the degree of polymerization. TNBS analysis indicated that the higher the proportion of hyaluronic acid bound, the lower the free amine group measured by the sample, which also indicates a higher degree of polymerization (Fig. 7). It was confirmed in the experiment that the copolymer 2 and the copolymer 3 had an excellent degree of polymerization, and the results revealed that the two copolymers had good structural strength and stability. When the copolymer has good structural strength and stability, when the copolymer is applied to an in vivo test, it can avoid the release of free radicals and affect the organism. Therefore, the copolymer disclosed in the present invention should have good bio-safeness and should be applicable to ophthalmology. Example 6 Biocompatibility Analysis 16 201223566 The vitreous is a transparent colloid present in the anterior chamber of the eye, behind the crystal and most of the area is in contact with the adjacent retina. In view of this, the in vitro test for biocompatibility was performed on a human retinoic pigment epithelial cell line (ARPE-19, BCRC No. 60383) for cytotoxicity analysis of the copolymer. Since it is necessary to simulate the in vivo test, the ARPE-19 cells are first cultured on an Insert Well (Millicell, Millipore corporation, Billerica, MA, USA), and then the copolymer is added to the cells for culture. The medium in the solitary medium can be used to transfer nutrients through the diffusion of the semi-permeable membrane at the bottom of the cell, as if the simulated vitreous is avascular tissue, and the nutrient source is from surrounding tissues such as: choroid, ciliary body and retina (Suri, S. And R. Banerjee (2006). "In vitro evaluation of in situ gels as short term vitreous substitutes." J Biomed Maier 细j 79(3): 650-64) 'If cytotoxic substances will pass through half The membrane is permeable and causes a toxic effect on ARPE-19 cells. The procedure was as follows (Fig. 8): 1. Place ARPE-19 cells in an insert culture dish (2xl〇4 cells per cell) and incubate for 24 hours in a 37 C, 5% C〇2 environment. 2. Add the sample to the Petri dish and apply 3 于 in the c〇2 incubator. 3. Perform MTT analysis: 10% by volume of NOT solution (5 mg/ml in PBS) was added and reacted for 4 hours in an environment of 37 5% C 2 . The liquid was removed and the MTT formazan was returned to the bath with a glimpse of DMSO. The crystals were completely dissolved and the absorbance was measured at OD.570 nm. This type of laboratory uses a plug-in culture dish to simulate the copolymer-filled If-shaped biocompatible silk that is in contact with the retina in the vitreous cavity. It is indicated that the three copolymers produced by the transgenic method (4) have a pole on the retinal pigment epithelial cells of 17 201223566. Good biocompatibility (Figure 9). The copolymer of the present invention has a higher survival rate than the control group, hyaluronic acid alone, Pluronic F-127 alone, and Shixia oil, and has a significant difference (Hua.05). This result reveals that the copolymer is then transplanted in vivo and avoids cytotoxicity caused by poor biocompatibility, which can greatly reduce the incidence of inflammatory reactions and complications. A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those that are present. The copolymers, water gels, artificial glass bodies, and processes and methods for their manufacture in the present invention are representative of the preferred embodiments, which are exemplary and; Ϊ; are limited to the field. Those who are familiar with this shop will think of the modifications and their ways. These revisions are in the hair of the present invention and are defined in the scope of the patent application. The present invention can be made and used by those skilled in the art, even if there are various changes, _, and improvements therein, it should be considered as not departing from the invention. Spirit and scope. All patents and publications mentioned in Yuetianzhong are subject to the general skills in the field of finance. All patents and publications are hereby referenced, as if each side was specifically and side-by-side (four) reference. The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description of the book (4) _, _ does not rely on the use of such terms and expressions that exclude any features or parts thereof as shown and described, but need to recognize 18 201223566 'Month' is in There may be various changes in the scope of this patent _ patent towel. Therefore, it is to be understood that the present invention has been described in detail and in accordance with the preferred embodiments of the present invention. Inside. [Simple description of the diagram] Figure 1. Schematic diagram of the synthesis mechanism of the copolymer of hyaluronic acid and Pluronic F_127. (A): hyaluronic acid, (B): Pluronic F-127, (C): copolymer. The part in the middle of the 『{丨』 symbol indicates that it is a hydrophobic area. Figure 2. Rheological diagram of Copolymer 1. Figure 3. Rheological diagram of Copolymer 2. Figure 4. Rheological diagram of copolymer 3. Figure 5. Schematic diagram of the injectability of the copolymer. Figure 6. Transmittance analysis results. Figure 7. Results of polymerization degree analysis. Figure 8. Schematic diagram of biocompatibility analysis. Figure 9. Results of biocompatibility analysis.