TWI413535B - Thremosensitivity hydrogel for tissue engineering and use thereof - Google Patents

Abstract

The present invention relates to a co-polymer for tissue engineering, which comprises a polymer made by cross-linking hyaluronic acid and Pluronic F-127. The present invention also relates to a method of making a co-polymer of hyaluronic acid and Pluronic F-127, comprising cross-linking OH group on Pluronic F-127 structure with CH3, NH3, COOH or OH group on hyaluronic acid backbone by cross-linking agent.

Description

用於組織工程之感溫性水膠及其應用Temperature sensitive water glue for tissue engineering and its application

本發明關於一種可用於組織工程之感溫性水膠。The invention relates to a temperature sensitive water gel which can be used for tissue engineering.

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,P. N. (2000). "Structural macromolecules and supramolecular organisation of the vitreous gel."Prog Retin Eye Res 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。玻璃體內含有膠原蛋白膠束(collagen micelle)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20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. Banerjee(2003). "Artificial vitreous replacements."Biomed Mater Eng 13 (1): 59-74;Suri,S. and R. Banerjee (2006). "In vitro evaluation of in situ gels as short term vitreous substitutes."J Biomed Mater Res A 79 (3): 650-64)。The vitreous is the perfect result of natural evolution. The vitreous cavity accounts for about two-thirds of the eyeball, weighs about 4 g, and has a volume of 4 mL (Bishop, PN (2000). "Structural macromolecules and supramolecular organisation of the vitreous gel." Prog Retin Eye Res 19 (3): 323-44), whose 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 contains collagen 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 20 times in the vicinity of the retina. The vitreous is avascular tissue with nutrients derived from surrounding tissues such as the choroid, ciliary body and retina. And the vitreous is not regenerable (Soman, N. and R. Banerjee (2003). "Artificial vitreous replacements." Biomed Mater Eng 13 (1): 59-74; Suri, S. and R. Banerjee (2006). In vitro evaluation of in situ gels as short term vitreous substitutes." J Biomed Mater Res A 79 (3): 650-64).

玻璃體具有以下之物理及光學特性：The vitreous has the following physical and optical properties:

a.　玻璃體之折射率為1.33；a. The refractive index of the vitreous is 1.33;

b.　可吸收震盪及維持眼球之形狀；b. absorb shock and maintain the shape of the eye;

c.　穩固神經及視網膜感光細胞層；c. Stabilizing the nerve and retinal photoreceptor layer;

d.　可允許代謝產物及養份之流通(Snell,R.(1995). Head and neck.Clinical Anatomy for Medical Students. New York,Little,Brown and Co: 713-725.)。d. Allow the circulation of metabolites and nutrients (Snell, 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Rheological properties of artificial vitreous

人工玻璃體的材料可分為氣態、液態及膠體三種(Soman,N. and R. Banerjee(2003). "Artificial vitreous replacements."Biomed Mater Eng 13 (1): 59-74)。氣體不易儲存於眼腔內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。Artificial vitreous materials can be classified 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.

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，將矽油植入眼內可能導致術後的併發症包含了白內障、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、角膜薄化及角膜內皮喪失(McCuen,B. W.,2nd,S. P. Azen,W. Stern,M. 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)。Emu oil is the most widely used filler for the treatment of retinal detachment and can be used to treat vitreous-associated 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. Stern, MY 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).

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(1000 CS)不易引起乳化作用(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."Ophthalmology 106 (6): 1091-100)。矽油脂乳化作用會形成小油滴並對眼睛組織造成傷害(Knorr,H. L.,A. Seltsam,L. Holbach and G. O. 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，矽油植入體內之長期間之生物相容性是始終伴隨爭議，許多研究指出，矽油對於角膜內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內皮細胞增生(Yang,C. S.,K. H. Chen,W. M. Hsu and Y. S. Li(2008). "Cytotoxicity of silicone oil on cultivated human corneal endothelium."Eye(Lond) 22 (2): 282-8)。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) viscous oil of lower viscosity (1000 CS) is less likely to cause emulsification (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." Ophthalmology 106 (6): 1091-100). Epoxy oil emulsification can form small oil droplets and cause damage to eye tissue (Knorr, HL, A. Seltsam, L. Holbach and GO Naumann (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 retinal surgery, the long-term biocompatibility of eucalyptus oil implanted in the body is always controversial, and many studies indicate that eucalyptus oil is cytotoxic to corneal endothelial cells and causes corneal lesions. MTS bioassay experiments indicate that eucalyptus oil inhibits corneal endothelial cell proliferation (Yang, CS, KH Chen, WM Hsu and YS Li (2008). "Cytotoxicity of silicone oil on cultivated human corneal endothelium." Eye (Lond) 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 M. B. Landers,3rd(1985). "The effect of silicone oil on the cornea."Arch Ophthalmol 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，眼角膜內皮細胞密度會下降40%，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 and S. Thierfelder(1992). "Experimental evaluation of in vitro stability of purified polydimethylsiloxanes(silicone oil) in viscosity ranges from 1000 to 5000 centistokes."Retina 12 (3 Suppl): S28-32)，使用非矽油的填充物並無明顯角膜病變的狀況產生。相較於對照組，在填充矽油後，角膜細胞的比重迅速降低，且在病理切片中有嚴重的變化(Karel,I.,M. Filipec and J. Obenberger(1986). "Specular microscopy of the corneal 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，當矽油與角膜內皮細胞接觸後可能導致細胞異常。然而，矽油導致視網膜內皮細胞的死亡之作用機制尚未被釐清，推測的原因可能為，眼內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. Hatchell,G. N. Foulks and M. B. Landers,3rd(1985). "The effect of silicone oil on the cornea."Arch Ophthalmol 103 (1): 90-4)。矽油具有高度安定性、透明度及高界面張力等特性,因而被應用於玻璃體-視網膜手術過程中之玻璃體填充物，然而其高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內取出避免造成併發症。且矽油對於角膜內皮細胞具有細胞毒性。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 MB Landers, 3rd (1985). "The effect of silicone oil on The cornea." Arch Ophthalmol 103 (1): 90-4). In an animal test of keratoderma-induced corneal lesions, when 70% of eucalyptus oil is implanted in the anterior chamber of the eye, the corneal endothelial cell density decreases by 40%, and bubbles are generated about six days after the contact. In animal testing, it leads to a gradual thinning of the matrix. In clinical trials, 21 patients with retinal detachment underwent vitrectomy and were filled with sputum oil for six months or longer after surgery (Heidenkummer, HP, A. Kampik and S. Thierfelder (1992). Experimental evaluation of in vitro stability of purified polydimethylsiloxanes (silicone oil) in viscosity ranges from 1000 to 5000 centistokes. " Retina 12 (3 Suppl): S28-32), using a non-anchovy filler without significant keratopathy. Compared with the control group, the specific gravity of corneal cells decreased rapidly after filling with eucalyptus oil, and there were serious changes in pathological sections (Karel, I., M. Filipec and J. Obenberger (1986). "Specular microscopy of the corneal Endothelium after liquid silicone injection into the vitreous in complicated retinal detachments. " Graefes Arch Clin Exp Ophthalmol 224 (2): 195-200), these results show that when eucalyptus oil is contacted with corneal endothelial cells, it may cause cell abnormalities. 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). Hatchell, GN Foulks and MB Landers, 3rd (1985). "The effect of silicone oil on the cornea." Arch Ophthalmol 103 (1): 90-4). Emu oil has high stability, transparency and high interfacial tension, so it is applied to vitreous filler during vitreore-retina surgery. However, its highly hydrophobic nature makes it impossible to properly fill the body fluid/retina interface, low specific gravity. Need to be removed within six months after surgery to avoid complications. And eucalyptus oil is cytotoxic to corneal endothelial cells.

商品化之人工玻璃體中，除了矽油之外，另有玻尿酸及全氟全氫化菲(Perfluoroperhydrophenanthrene)等商業化產品，而由玻尿酸製備而成的人工玻璃體為最大宗。亦有部分人工玻璃體係由膠原蛋白所製成。這些天然高分子材料具有良好之生物相容性，但長時間存於體內會降解，而無法長時間的置放於玻璃體腔內，且目前所使用之材料亦無感溫聚合特性。因此，於臨床上的使用受到限制。Commercially available artificial glass bodies, in addition to eucalyptus oil, are also commercially available products such as hyaluronic acid and perfluoroperhydrophenanthrene, and artificial vitreouss prepared from hyaluronic acid are the largest. There are also some artificial glass systems made of collagen. These natural polymer materials have good biocompatibility, but they will degrade in the body for a long time, and cannot be placed in the vitreous cavity for a long time, and the materials used at present have no temperature-sensing properties. Therefore, clinical use is limited.

一般而言，良好的人工玻璃體應具有以下優點：In general, a good artificial vitreous should have the following advantages:

i.　良好的光學特性：人工玻璃體需要有良好的透光度才不會導致視覺遮蔽之發生，此外亦需要有適當的折射率避免影響術後病患之視力。i. Good optical properties: artificial vitreous requires good transparency so as not to cause visual obscuration. In addition, proper refractive index is needed to avoid affecting the vision of postoperative patients.

ii.　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料注入受試者體內，並藉由其感溫性使材料於體溫下產生自體聚合，藉此提高材料在體內時之結構強度。Ii. Injectability and temperature-sensing polymerization characteristics: For surgery, when the material is injectable, the operator can easily inject the material into the subject, and the temperature is used to make the material at body temperature. Autogenous polymerization is produced thereby increasing the structural strength of the material as it is in the body.

iii.　極佳的生物相容性：若材料無生物相容性，在植入體內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。Iii. Excellent biocompatibility: If the material is not biocompatible, it will cause an inflammatory reaction and immune response in the body after implantation, and there may be serious complications that cause the subject to have a life threat. .

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."J Control Release 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-Hernandez,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(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 Tissue Engineering."Prog Polym Sci 33 (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 J. A. Hubbell(2004). "Synthetic extracellular matrices for in situ tissue engineering."Biotechnol Bioeng 86 (1): 27-36)。感溫性水膠(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。Hydrogel is a substance that changes between liquid and colloidal states. When subjected to changes in the external environment such as temperature, pH and photoreaction, it is converted from liquid to colloid (Kim, MR and TG Park (2002). "Temperature-responsive and degradable hyaluronic acid/Pluronic composite hydrogels for controlled release of human growth hormone." J Control Release 80 (1-3): 69-77). Water gel has been researched and valued in the past decade and is now widely used in drug release, tissue engineering and regenerative medicine (Cosgriff-Hernandez, E. and AG Mikos (2008). "New biomaterials as scaffolds for tissue engineering. " Pharm Res 25 (10): 2345-7). Injectable water gels have been studied in cell delivery systems, which have the advantage of allowing cells and biomolecules to rapidly bind to colloidal matrices (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." Prog Polym Sci 33 (2): 167-179). Natural injectable water gels have excellent capabilities before gelation to easily implant autologous cells into filling positions of any size and shape. 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 JA Hubbell (2004). "Synthetic extracellular matrices for in situ tissue Engineering." Biotechnol Bioeng 86 (1): 27-36). Thermosensitive hydrogel is a change in the shape of a water gel as it changes with temperature. The properties of this solid-liquid transfer can be applied to tissue engineering. When the water gel is heated, it exceeds the critical liquid temperature. Solution temperature, LCST), which gels and converts from liquid to colloid.

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扮演著重要的角色，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-supplementation agent)。玻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。Hyaluronic acid (HA) 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 synovial fluid. Hyaluronic acid plays an important role in wound healing, promoting cell differentiation and maintaining cell type. Hyaluronic acid is clinically regarded as a visco-supplementation agent in ophthalmology and joint surgery. Hyaluronic acid has excellent biocompatibility and biodegradability and is often used in tissue engineering to prepare porous scaffolds or drug delivery devices.

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，於進行白內障及水晶體植入之外科手術時可用於保護脆弱的眼組織。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, it 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). Its structure is as follows Shown as:

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℃以下之環境時，此水膠為液態，但將此水膠置於體內環境中(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，乙烯及丙烯基團會含水，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，聚氧化乙烯會逐漸失去水溶性並形成微胞，當膠粒內聚性提高時即會產生水膠。Pluronic 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 Pluronic F-127 aqueous solution is placed in an environment below 25 ° C, the water gel is liquid, but the water gel is placed in the body environment (37 ° C, pH = 7.2 ~ 7.4), the water gel will transform For colloids. Pluronic F-127 has a hydrophilic end of poly(ethylene glycol) and a hydrophobic end of poly(propylene glycol), which can be altered by the force of the hydrophilic end and the hydrophobic end. When the temperature is lower than the lower critical solution temperature (LCST), the ethylene and propylene groups will contain water, and the polyethylene oxide will dissolve in the water. When the temperature rises above the temperature of the low critical solution, the polyethylene oxide will gradually lose water solubility and form micelles, and when the colloidal cohesiveness is increased, water gel will be produced.

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."Biomacromolecules 5 (6): 2358-65)，但於商業上尚無任何玻尿酸/Pluronic F-127之產品應用於人工玻璃體或組織工程。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." Biomacromolecules 5 (6): 2358-65), but there is no commercial hyaluronic acid/Pluronic F-127 product for artificial vitreous or tissue engineering.

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(co-polymer)，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矽油相比，本發明之共聚物可作為人工玻璃體之良好材料，亦有取代矽油之潛能。The present invention uses a hyaluronic acid having excellent biocompatibility and a Pluronic F-127 which is an FDA-approved polymer having temperature-sensing properties, and forms a copolymer (co-polymer) by chemical bonding. The gel properties were generated by the interaction of its hydrophobic regions, and a smart thermosic hydrogel was developed to replace the traditionally widely used emu oil as an artificial vitreous. The copolymer of the present invention has unique temperature-sensing polymerization characteristics, 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 has excellent biocompatibility with human retinal pigment cells. Therefore, the copolymer of the present invention has temperature-sensing polymerization characteristics, injectability, good optical properties and biocompatibility, and the copolymer of the present invention can be used as an artificial vitreous body compared with emu oil which is conventionally used as an artificial vitreous body. Good materials also have the potential to replace oyster sauce.

一般而言化學接合之方法是利用環氧化合物(EX-810)、N -(3-二甲胺丙基)-N' -乙基碳二亞胺鹽酸鹽(EDC)、戊二醛及1-羥基苯并***單水合物(HOBt‧H₂ O)等所製備之交聯劑之一或其組合，其作用機制係利用Pluronic F-127結構上之OH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。Generally the chemical is bonded with an epoxy compound (EX-810), N - (3- dimethylaminopropyl) - N '- ethylcarbodiimide hydrochloride (EDC), and glutaraldehyde One of the cross-linking agents prepared by 1-hydroxybenzotriazole monohydrate (HOBt‧H ₂ O) or the like, the mechanism of action of which utilizes the OH group on the Pluronic F-127 structure and the hyaluronic acid on the skeleton One or more groups selected from the group consisting of CH ₃ , NH ₃ , COOH, and OH are joined to achieve the purpose of forming a copolymer.

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藝者的普遍認知相同。本申請案中使用的術語其意義如下所列：The meaning of the terms used herein is the same as commonly recognized by those skilled in the art, unless specifically defined otherwise. The meanings of the terms used in this application are as follows:

「共聚物」意指一種源自兩個(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。"Copolymer" means a polymer derived from two (or more) monomer species relative to a homopolymer having only one monomer.

「交聯劑」意指一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聯成網路結構的物質，其為一種可促進或調節聚合物分子鏈間共價鍵或離子鍵形成的物質。本發明中所使用之交聯劑包括但不限於EDC、HOBt、環氧樹脂、戊二醛及DMSO中其一或其組合。"Crosslinking agent" means a substance capable of intermolecular intermolecular bridging so that a plurality of linear molecules are bonded to each other to form a network structure, which is a substance which promotes or regulates covalent bonds between polymer molecular chains or A substance formed by ionic bonds. The crosslinking agent used in the present invention includes, but is not limited to, one of EDC, HOBt, epoxy resin, glutaraldehyde, and DMSO, or a combination thereof.

因此，本發明提供一種用於組織工程的共聚物，包含玻尿酸與Pluronic F-127交聯後之聚合物。在較佳實施例中，玻尿酸與Pluronic F-127的重量比範圍為3：1至1：60。在更佳實施例中，玻尿酸與Pluronic F-127的重量比為1：1、1：15或1：20。該共聚物會隨溫度改變而變化型態，低於臨界液態溫度時為液體，高於臨界液態溫度時則為膠體。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。Accordingly, the present invention provides a copolymer for tissue engineering comprising a polymer crosslinked with hyaluronic acid and Pluronic F-127. In a preferred embodiment, the weight ratio of hyaluronic acid to Pluronic F-127 ranges from 3:1 to 1:60. In a more preferred embodiment, the weight ratio of hyaluronic acid to Pluronic F-127 is 1:1, 1:15 or 1:20. The copolymer will change shape with changes in temperature, liquid below the critical liquid temperature, and colloid above the critical liquid temperature. In a preferred embodiment, 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 used as a material for an artificial vitreous.

本發明亦提供一種製備玻尿酸與Pluronic F-127共聚物的方法，包含使用交聯劑將Pluronic F-127結構上之OH基與玻尿酸骨架上一或多個選自由CH₃ 、NH₃ 、COOH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：The invention also provides a method for preparing a hyaluronic acid and a Pluronic F-127 copolymer, comprising using a crosslinking agent to bind one or more of the OH group and the hyaluronic acid skeleton of the Pluronic F-127 structure from CH ₃ , NH ₃ , COOH and The groups of the group consisting of OH are joined. In a preferred embodiment, the method comprises the steps of:

a.　提供一交聯劑、一玻尿酸溶液及一Pluronic F-127溶液；a. providing a crosslinking agent, a hyaluronic acid solution and a Pluronic F-127 solution;

b.　混合交聯劑、玻尿酸溶液及Pluronic F-127溶液，形成一混合溶液；b. mixing a crosslinking agent, a hyaluronic acid solution and a Pluronic F-127 solution to form a mixed solution;

c. 　維持該混合溶液之pH值於6到8之間；及 c. maintaining the pH of the mixed solution between 6 and 8;

d. 　將該混合溶液以透析處理，乾燥後取得乾燥產物。 d. The mixed solution was subjected to dialysis treatment, and dried to obtain a dried product.

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，再加入剩下之另一溶液進行反應；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。在另一較佳實施例中，玻尿酸與Pluronic F-127的重量比範圍為3：1至1：60。在更佳實施例中，玻尿酸與Pluronic F-127的重量比為1：1、1：15或1：20。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。In the preferred embodiment, the solvent of the solution of step a is water; and step b is first added to the hyaluronic acid solution or the Pluronic F-127 solution and stirred uniformly, and then the other solution is added to carry out the reaction. The pH of step c 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 Pluronic F-127 ranges from 3:1 to 1:60. In a more preferred embodiment, the weight ratio of hyaluronic acid to Pluronic F-127 is 1:1, 1:15 or 1:20. In another preferred embodiment, the crosslinking agent is a combination of EDC and HOBt. In a preferred embodiment, 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 used as a material for an artificial vitreous.

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。The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the various aspects and features of the present invention.

實施例1　感溫性人工玻璃體之製備Example 1 Preparation of Temperature Sensitive Artificial Glass Body

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1、共聚物2及共聚物3。The copolymer of the present invention can develop three different copolymers depending on the ratio of the Pluronic F-127 to be bonded: copolymer 1, copolymer 2 and copolymer 3.

1.　秤取玻尿酸及不同比例Pluronic F-127藉由交聯劑合成三種類共聚物；共聚物1(玻尿酸/Pluronic F-127=0.1 g/0.1 g，w/w)、共聚物2(玻尿酸/Pluronic F-127=0.1 g/1.5 g，w/w)、共聚物3(玻尿酸/Pluronic F-127=0.1 g/2 g，w/w)分別溶於50 ml去離子水中。1. Weighing hyaluronic acid and different ratios of Pluronic F-127 to synthesize three kinds of copolymers by cross-linking agent; copolymer 1 (hyaluronic acid/Pluronic F-127=0.1 g/0.1 g, w/w), copolymer 2 (hyaluronic acid) /Pluronic F-127 = 0.1 g / 1.5 g, w / w), copolymer 3 (hyaluronic acid / Pluronic F-127 = 0.1 g / 2 g, w / w) were dissolved in 50 ml of deionized water.

2.　將0.78 g EDC、0.77 g HOBt溶解於20ml DMSO/去離子水(1/1，v/v)，緩慢地加入玻尿酸溶液中，待攪拌均勻再加入Pluronic F-127水溶液。2. Dissolve 0.78 g EDC, 0.77 g HOBt in 20 ml DMSO/deionized water (1/1, v/v), slowly add to the hyaluronic acid solution, and add Pluronic F-127 aqueous solution while stirring.

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。3. Maintain pH at 6.8, react at room temperature for at least 4 hours, and allow to stand overnight after the pH has stabilized.

4.　將玻尿酸/Pluronic F-127溶液以透析處理，並以冷凍乾燥之方法獲得乾燥產物。4. The hyaluronic acid/Pluronic F-127 solution was dialyzed and lyophilized to obtain a dried product.

本發明之交聯機制如圖1所示。The crosslinking mechanism of the present invention is shown in FIG.

實施例2　流變特性分析Example 2 Rheological property analysis

本實驗係以流變儀(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40 mm平盤、溫度坡道步驟(Temperature ramp step)模式、升溫範圍為4-45℃、升溫速率為1.5℃/min及壓力百分比(% Strain)為10進行觀察。In this experiment, the rheological properties of the samples were analyzed by a rheometer (Rheometer, AR2000-ex, TA instrument, New Castle, DE, USA) using a 40 mm flat disk, temperature ramp step (Temperature ramp step). The mode, the temperature rise range was 4-45 ° C, the heating rate was 1.5 ° C / min, and the pressure percentage (% Strain) was 10 for observation.

流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℃，且在植入眼內(32℃)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32℃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矽油，共聚物2及共聚物3在注入受試者體內後，會具有更佳之結構強度及穩定性。The rheological analysis results indicate that the copolymer 1 has no temperature sensitivity (Fig. 2), while the copolymer 2 and the copolymer 3 have excellent temperature-sensing polymerization characteristics (Fig. 3 and Fig. 4). The critical liquid temperature (LCST) of Copolymer 2 was about 23 ° C, and its storage modulus (G') reached 804.6 Pa and the loss modulus (G") was 448.6 after implantation in the eye (32 ° C). The copolymer 3 copolymer had a storage modulus (G') of 1137 Pa at 32 ° C and a loss modulus (G") of 575.7 Pa. In clinical applications, copolymer 2 and copolymer 3 have better structural strength and stability after being injected into a subject than eucalyptus oil having no temperature-sensitive polymerization characteristics.

實施例3　可注射性Example 3 Injectability

本試驗係為了驗證本發明之共聚物之可注射性，其方法如下：製備重量百分比為10~20%之共聚物水溶液，待其完全溶解，將試樣移置針筒中，並裝置27號針頭進行試驗。This test is to verify the injectability of the copolymer of the present invention by the following method: preparing an aqueous solution of a copolymer having a weight percentage of 10 to 20%, and when it is completely dissolved, the sample is placed in a syringe, and a 27-gauge needle is placed. test.

實驗結果指出，共聚物2及共聚物3可在室溫下輕易的通過27號針頭(圖5)，此結果驗證了共聚物之可注射性。當材料具有可注射性於臨床使用上，操作者更容易將材料注入受試者體內，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。The experimental results indicate that Copolymer 2 and Copolymer 3 can easily pass through a 27 gauge needle at room temperature (Fig. 5), and the results verify the injectability of the copolymer. When the material is injectable for clinical use, it is easier for the operator to inject the material into the subject and a smaller needle can be used. Smaller needles reduce the wound area at the time of implantation.

實施例4　透光度分析Example 4 Transmittance Analysis

以紫外-可見光分光光度計(UV/Visible spectrophotometer)(Ultrospec 2100 pro,Amersham Bioscience,Sweden)偵測試樣在可見光(400-700 nm)之穿透率(Suri,S. and R. Banerjee(2006). "In vitro evaluation of in situ gels as short term vitreous substitutes."J Biomed Mater Res A 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,K. E.,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 Ophthalmol Vis Sci 50 (10): 4840-6)。The transmittance of the sample in visible light (400-700 nm) was measured by a UV/Visible spectrophotometer (Ultrospec 2100 pro, Amersham Bioscience, Sweden) (Suri, S. and R. Banerjee (2006) "In vitro evaluation of in situ gels as short term vitreous substitutes." J Biomed Mater Res A 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, KE, M. Shah, PD Hamilton, TA Eskin, S. Kaushal and N. Ravi (2009). "Rabbit study of an in situ forming hydrogel vitreous substitute." Invest Ophthalmol Vis Sci 50 (10): 4840-6).

玻璃體之折射率約為1.33(Snell,R.(1995). Head and neck.C1inical Anatomy for Medical Students . New York,Little,Brown and Co: 713-725)，而本發明所合成之共聚物的折射率之平均值為1.3547(表1)，相較於矽油之折射率1.405(McCartney,D. L.,K. M. Miller,W. J. 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。The refractive index of the vitreous is about 1.33 (Snell, R. (1995). Head and neck. C1inical Anatomy for Medical Students . New York, Little, Brown and Co : 713-725), and the refraction of the copolymer synthesized by the present invention. The average of the rates is 1.3547 (Table 1) compared to the refractive index of enamel 1.405 (McCartney, DL, KM Miller, WJ Stark, DL Guyton and RG Michels (1987). "Intraocular lens style and refraction in pixels treated with silicone Oil." Arch Ophthalmol 105 (10): 1385-7) is closer to the natural glass body.

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570 nm，其透光度高達90%以上。於折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。The transmittance analysis indicated (Fig. 6) that the transmittance of the copolymer 1 was extremely low, and the wavelength was less than 1% in the visible light range. On the other hand, the copolymers 2 and 3 have a transmittance of more than 70%, and at a wavelength of 570 nm, the transmittance is as high as 90% or more. The two copolymers of copolymer 2 and copolymer 3 were evaluated as a good artificial glass material in terms of both refractive index and transmittance.

實施例5　聚合程度分析Example 5 Analysis of polymerization degree

以三硝基苯磺酸(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,K.,T. Kimura,S. Funamoto and A. Kishida "Preparation of a collagen/polymer hybrid gel designed for tissue membranes. Part I: controlling the polymer-collagen cross-linking process using an ethanol/water co-solvent."Acta Biomater 6 (2): 403-8)：The combination of trinitrobenzenesulfonic acid (TNBS) with a free amine group in the sample structure, if the presence of free amine groups in the structure is less, represents a better degree of polymerization of the structure. The analysis procedure is a modification of the method of Nam et al. (Nam, K., T. Kimura, S. Funamoto and A. Kishida "Preparation of a collagen/polymer hybrid gel designed for tissue membranes. Part I: controlling the polymer-collagen cross -linking process using an ethanol/water co-solvent." Acta Biomater 6 (2): 403-8):

1.　將10 mg試樣浸置於4%碳酸氫鈉(Sodium bicarbonate)及TNBS中。1. Dip 10 mg sample in 4% sodium bicarbonate and TNBS.

2.　以40℃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。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.　以6M HCl將濾紙部份上之以反應之TNBS回溶，並以OD.345 nm(Epoch,Bio-tek instruments,USA)測定其吸光值。3. The filter paper portion was back-dissolved with the reacted TNBS with 6 M HCl, and its absorbance was measured at OD.345 nm (Epoch, Bio-tek instruments, USA).

4.　以下列公式計算共聚物之聚合程度：4. Calculate the degree of polymerization of the copolymer by the following formula:

理論上，玻尿酸接合Pluronic F-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Pluronic F-127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，當共聚物應用於體內試驗時，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。In theory, the higher the proportion of hyaluronic acid-bonded Pluronic F-127, the higher the degree of polymerization. TNBS analysis indicated that the higher the ratio of Pluronic F-127 bound by hyaluronic acid, the lower the free amine group measured by the sample, indicating 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 in vivo tests, it can avoid the release of free radicals and affect the organism. Therefore, the copolymer disclosed in the present invention should have good bio-safety and should be applicable to ophthalmology.

實施例6　生物相容性分析Example 6 Biocompatibility Analysis

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。由於需模擬體內試驗之情形，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，而培養皿中之培養基可由培養皿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 Mater Res A 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)：The vitreous is a transparent colloid present in the anterior chamber of the eyeball, behind the lens and most of the area is in contact with the adjacent retina. In view of this, this biocompatibility in vitro test was performed on a human cytotoxicity analysis of a human retinal pigment epithelial cell line (ARPE-19, BCRC No. 60383). Since it is necessary to simulate the in vivo test, the ARPE-19 cells were first cultured on an insert well (Millicell, Millipore corporation, Billerica, MA, USA), and then the copolymer was contacted with the cells, and the culture dish was cultured. The medium can be transferred by diffusion through the semi-permeable membrane at the bottom of the culture dish, as if the simulated vitreous is avascular tissue, and its 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 Mater Res A 79 (3): 650-64), if cytotoxic substances will pass through the semi-permeable membrane It also caused a poisoning effect on ARPE-19 cells. The operating procedure is as follows (Figure 8):

1.　將ARPE-19細胞置入***式培養皿中(每格2×10⁴ 個細胞)，於37℃、5% CO₂ 之環境下培養24小時。1. Place ARPE-19 cells in an insert culture dish (2 × 10 ⁴ cells per division) and incubate for 24 hours at 37 ° C in a 5% CO ₂ atmosphere.

2.　將試樣加入***式培養皿中，並於CO₂ 培養箱作用3日。2. The sample was placed in a Petri dish and allowed to act in a CO ₂ incubator for 3 days.

3.　進行MTT分析：加入10%體積之MTT溶液(5 mg/ml於PBS中)並於37℃、5% CO₂ 之環境下反應4小時。3. Perform MTT assay: Add 10% by volume of MTT solution (5 mg/ml in PBS) and react for 4 hours at 37 ° C, 5% CO ₂ .

4.　將液體去除，並以二甲亞碸(DMSO)將MTT甲臢(formazan)回溶，待晶體完全溶解，以OD.570 nm測定其吸光值。4. The liquid was removed and the MTT formazan was dissolved back in dimethyl hydrazine (DMSO) until the crystals were completely dissolved, and the absorbance was measured at OD.570 nm.

本試驗係利用***式培養皿以模擬共聚物填充至玻璃體腔與視網膜接觸之情形，生物相容性結果指出，本發明所製備出三種共聚物對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及矽油，具有更高的存活率，且具有顯著差異(p≦0.05 )。此結果揭示，爾後將共聚物進行體內移植，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。This test uses a plug-in culture dish to simulate the filling of the copolymer into the vitreous cavity and the retina. The biocompatibility results indicate that the three copolymers prepared by the present invention have excellent biocompatibility for retinal pigment epithelial cells. Sex (Figure 9). The copolymer of the present invention has a higher survival rate and a significant difference ( p ≦ 0.05 ) relative to the control group, hyaluronic acid alone, Pluronic F-127 alone, and eucalyptus oil. 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.

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，其為示範性且不僅侷限於本發明領域。熟知此技藝者將會想到其中可修改之處及其他用途。這些修改都蘊含在本發明的精神中，並在申請專利範圍中界定。A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. 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 not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

本發明的內容敘述與實施例均揭示詳細，得使任何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、修飾、及進步之處，仍應視為不脫離本發明之精神及範圍。The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope.

說明書中提及之所有專利及出版品，都以和發明有關領域之一般技藝為準。所有專利和出版品都在此被納入相同的參考程度，就如同每一個個別出版品都被具體且個別地指出納入參考。All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

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、限制條件或並非特定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說明書之描述而非限制，同時並無意圖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，但需認清的是，在本發明的專利申請範圍內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內容，諸如此類的修改和變化仍在本發明之申請專利範圍內。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 and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described, but Various changes are possible within the scope of the patent application of the invention. Therefore, it is to be understood that the present invention has been disclosed and described herein in accordance with the preferred embodiments and the features of the present invention. Within the scope.

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。『{}』符號中間的部份表示其為疏水性區域。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 middle part of the 『{}』 symbol indicates that it is a hydrophobic area.

圖2、共聚物1之流變圖。Figure 2. Rheological diagram of Copolymer 1.

圖3、共聚物2之流變圖。Figure 3. Rheological diagram of Copolymer 2.

圖4、共聚物3之流變圖。Figure 4. Rheological diagram of copolymer 3.

圖5、共聚物可注射性示意圖。Figure 5. Schematic diagram of the injectability of the copolymer.

圖6、透光度分析結果。Figure 6. Transmittance analysis results.

圖7、聚合程度分析結果。Figure 7. Results of polymerization degree analysis.

圖8、生物相容性分析示意圖。Figure 8. Schematic diagram of biocompatibility analysis.

圖9、生物相容性分析結果。Figure 9. Results of biocompatibility analysis.

Claims (9)

一種用於組織工程的共聚物，包含玻尿酸與Pluronic F-127交聯後之聚合物，其中玻尿酸與Pluronic F-127的重量比範圍為1：15至1：20。 A copolymer for tissue engineering comprising a polymer crosslinked with hyaluronic acid and Pluronic F-127, wherein the weight ratio of hyaluronic acid to Pluronic F-127 ranges from 1:15 to 1:20. 如申請專利範圍第1項之共聚物，其中玻尿酸與Pluronic F-127的重量比為1：15或1：20。 The copolymer of claim 1 wherein the weight ratio of hyaluronic acid to Pluronic F-127 is 1:15 or 1:20. 如申請專利範圍第1項之共聚物，其會隨溫度改變而變化型態，低於臨界液態溫度時為液體，高於臨界液態溫度時則為膠體。 For example, the copolymer of the first application of the patent range, which changes with the temperature, is a liquid below the critical liquid temperature, and is a colloid above the critical liquid temperature. 如申請專利範圍第1項之共聚物，其係作為人工玻璃體之材料。 The copolymer of claim 1 is used as a material for artificial vitreous. 一種製備玻尿酸與Pluronic F-127共聚物的方法，包含以下步驟：a. 提供一交聯劑、玻尿酸溶液及一Pluronic F-127；b. 混合交聯劑、玻尿酸溶液及Pluronic F-127溶液，使交聯劑將Pluronic F-127結構上之OH基與玻尿酸骨架上一或多個選自由CH₃ 、NH₃ 、COOH及OH所組成群組之基團進行接合，形成一混合溶液；c.維持該混合溶液之pH值與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。A method for preparing a hyaluronic acid and a Pluronic F-127 copolymer, comprising the steps of: a. providing a crosslinking agent, a hyaluronic acid solution, and a Pluronic F-127; b. a mixed crosslinking agent, a hyaluronic acid solution, and a Pluronic F-127 solution, The crosslinking agent is bonded to the OH group on the Pluronic F-127 structure and one or more groups selected from the group consisting of CH ₃ , NH ₃ , COOH and OH to form a mixed solution; c. Maintaining the pH of the mixed solution between 6 and 8; and d. treating the mixed solution with dialysis and drying to obtain a dried product. 如申請專利範圍第5項之方法，其中步驟c之pH值為6.8。 The method of claim 5, wherein the pH of step c is 6.8. 如申請專利範圍第5項之方法，其中玻尿酸與Pluronic F-127的重量比範圍為3：1至1：60。 The method of claim 5, wherein the weight ratio of hyaluronic acid to Pluronic F-127 ranges from 3:1 to 1:60. 如申請專利範圍第7項之方法，其中玻尿酸與Pluronic F-127的重量比為1：1、1：15或1：20。 The method of claim 7, wherein the weight ratio of hyaluronic acid to Pluronic F-127 is 1:1, 1:15 or 1:20. 如申請專利範圍第5項之方法，其中交聯劑為EDC及HOBt之組合。 The method of claim 5, wherein the crosslinking agent is a combination of EDC and HOBt.