CN103333349A - Hyaluronic acid-collagen composite hydrogel for injection and preparation method thereof - Google Patents

Hyaluronic acid-collagen composite hydrogel for injection and preparation method thereof Download PDF

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CN103333349A
CN103333349A CN 201310264062 CN201310264062A CN103333349A CN 103333349 A CN103333349 A CN 103333349A CN 201310264062 CN201310264062 CN 201310264062 CN 201310264062 A CN201310264062 A CN 201310264062A CN 103333349 A CN103333349 A CN 103333349A
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collagen
hyaluronic acid
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hydrogel
preparation
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CN 201310264062
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范代娣
马晓轩
张婧婧
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陕西巨子生物技术有限公司
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Abstract

The invention relates to hyaluronic acid-collagen composite hydrogel for injection and a preparation method thereof. Current hyaluronic acid cross-linked hydrogel cannot remove toxicity residue through cleaning in a preparation process, thus having a potential safety hazard. The preparation method comprises the steps of mixing and dissolving sodium hyaluronate and collagen in an alkaline solution, uniformly stirring and adding a cross-linking agent to a reaction system, uniformly mixing and reacting to obtain gel in a block form, processing the gel to remove residual cross-linking agent, and grinding to obtain granular hydrogel. Due to introduction of the collagen, the prepared hydrogel is more ideal, and low in residue of the cross-linking agent and relatively low in damage to a living organism; the collagen can improve the mechanical strength of the hydrogel, and bring about positive influence to cell adhesion and cell growth promotion property of the product, so that the hydrogel, as a soft tissue filling material, shows a greater advantage.

Description

一种注射用透明质酸-胶原蛋白复合水凝胶及其制备方法 An injection of hyaluronic acid - collagen composite hydrogel and preparation method

技术领域 FIELD

[0001] 本发明涉及一种水凝胶,具体涉及一种注射用透明质酸-胶原蛋白复合水凝胶及其制备方法。 [0001] The present invention relates to a hydrogel, particularly relates to an injection of hyaluronic acid - hydrogel and method for preparing collagen composite.

背景技术 Background technique

[0002] 透明质酸是一种酸性粘多糖,又称糖醛酸、玻尿酸,是由两个双糖单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的天然高分子聚合物。 [0002] Hyaluronic acid is a mucopolysaccharide acid, also known as uronic acid, hyaluronic acid, a natural polymer consisting of two disaccharide units of D- glucuronic acid and N- acetylglucosamine thereof. 它的分子可携带500倍以上的水分,是一种效果优良的保湿成分,被称为理想的天然保湿因子。 It may carry more than 500 times the molecular moisture, is excellent in an effect moisturizing ingredients, natural moisturizing factor is referred desirable. 透明质酸、其盐类以及衍生物具有良好的生物可吸收性、生物相容性、粘弹性和保水性,因此被广泛应用于美容、医药等领域。 Hyaluronic acid, its salts and derivatives have good bioabsorbable, biocompatible, viscoelasticity and water retentivity, and therefore are widely used in cosmetic, pharmaceutical and the like. 临床上,透明质酸可用于眼科手术、外科手术中预防术后粘连、促进伤口的愈合和美容整形等方面,把一些药物结合到透明质酸上形成的化合物还可以发挥药物控制缓释的作用,从而达到药物定时和定点释放的目的,但由于透明质酸存在降解速度快、机械强度较低等缺陷,而限制了其应用范围,因此在实际应用中,经常通过交联改性等方法使透明质酸分子内或分子间发生交联制备成为透明质酸水凝胶,从而延长其在生物体内的作用时间。 Clinically, hyaluronic acid can be used in ophthalmic surgery, in the prevention of postoperative surgical adhesions, to promote wound healing and cosmetic plastic surgery, etc., the number of drugs bound to hyaluronic acid compound formed may also play a role in controlling release of the drug to achieve the purpose and timing of the drug release point, but because of rapid degradation rate, mechanical strength, low defect hyaluronic acid, its scope of application is limited, so in practice, it is often that the modification by cross-linking or the like preparation of crosslinked hyaluronic acid molecules or between the molecules become hyaluronic acid hydrogel, thereby extending its time action in vivo.

[0003] 关于透明质酸交联水凝胶的制备已有专利报道。 [0003] Preparation of hyaluronic acid cross-linked hydrogel on have been reported in the patent. 专利CN 101538377 A公开了一种交联透明质酸凝胶的制备方法。 Patent CN 101538377 A discloses a method for preparing a crosslinked hyaluronic acid gel. 该发明中,采用1,4_ 丁二醇二缩水甘油醚作为交联剂在碱性条件下与透明质酸中的羟基发生反应,再用生理平衡液清洗除去未反应的交联剂从而制备得到产品。 The invention adopts 1,4_-butanediol diglycidyl ether as a crosslinking agent react with the hydroxyl groups of hyaluronic acid under basic conditions, and then was washed physiological balance thereby removing unreacted crosslinker prepared product. 但在反应过程中,有的丁二醇二缩水甘油醚只有一个环氧基团参与了反应,这些交联剂无法通过清洗除去,因此还会存在残留毒性的问题。 However, during the reaction, there is only a butanediol diglycidyl ether epoxy group involved in the reaction, these crosslinking agents can not be removed by washing, and therefore also the presence of residual toxicity problems.

[0004] 专利CN 101724164 B公开了一种制造交联透明质酸的方法。 [0004] Patent CN 101724164 B discloses a method of producing crosslinked hyaluronic acid. 该方法中,使透明质酸溶液在10-30°C下进行交联反应超过48h。 In this method, hyaluronic acid solution was subjected to a crosslinking reaction than 48h at 10-30 ° C. 此低反应温度下进行的交联反应,所形成的交联透明质酸不会因碱的水解作用而快速劣化,在使交联剂消耗至合理的含量后即终止反应。 The crosslinking reaction was carried out at this low reaction temperature, the formed crosslinked hyaluronic acid hydrolysis will not deteriorate rapidly and a base, the reaction was terminated crosslinking agent after consumption to a reasonable amount. 方法中未除去残留的BDDE,会存·在安全隐患。 The method not remove the remaining BDDE, will be kept in a safe-hidden.

[0005] 胶原蛋白是一种高分子蛋白,一般呈白色、不透明的纤维状。 [0005] Collagen is a protein polymer, generally white, opaque fiber. 它是人体组成结构的主要成分之一,也是延缓人体衰老所必须的物质,广泛地分布于人体的皮肤、骨骼、软骨、肌肉、关节和头发组织内,能够起到支撑器官、保护肌体和修复的功能。 It is one of the main components of the structure of the human body, but also delay aging substances essential, widely distributed in the body's skin, bone, cartilage, muscle, joint tissue and hair, can play a supporting organ, protection and restoration of the body function. 类人胶原蛋白是将人体胶原蛋白的mRNA逆转录成cDNA,经酶切后的一段基因重组于E.coli(大肠杆菌)内,经过高密度发酵、分离、复性、纯化工艺生产的一种高分子生物蛋白。 Like collagen is human collagen mRNA was reverse transcribed into the cDNA, after a period of digestion recombinant gene in E. coli (E. coli), through high-density fermentation, separation, renaturation and purification process for producing a biological macromolecular protein. 它具有良好的生物吸收性、细胞粘附性和生物相容性,并且可明显促进细胞生长,因此非常适合应用于医学、整形等方面。 It has excellent biological absorptivity, biocompatibility and cell adhesion, and can significantly promote cell growth, making it ideal for medical applications, plastic and so on.

[0006] 环氧化合物类作为交联剂被普遍应用于透明质酸凝胶的制备。 [0006] The epoxy compounds as crosslinking agents is widely used in the preparation of a hyaluronic acid gel. 它可以与透明质酸分子中的羟基和羧基发生反应形成醚键和酯键,本发明在反应体系中混合入胶原蛋白后,环氧化合物也可以与胶原蛋白中的羟基和羧基反应,并且也可以与其中的氨基反应形成仲氨基团,因此,使得环氧化合物反应更彻底,所形成的三维结构更加致密,可以有效的提升产物水凝胶的机械性能。 It may form an ether bond and an ester bond reacts with the hyaluronic acid molecule hydroxyl and carboxyl groups, the reaction system in the present invention is mixed into the collagen, the epoxy compound may be reacted with the collagen hydroxyl and carboxyl groups, and also an amino group which can react with secondary amino groups are formed, and therefore, such an epoxy compound is more thorough, the formed three-dimensional structure more compact, can effectively improve the mechanical properties of the product hydrogels. 发明内容 SUMMARY

[0007] 本发明的目的是提供一种使用更安全、缓释作用更好的注射用透明质酸-胶原蛋白复合水凝胶及其制备方法。 [0007] The object of the present invention is to provide a safer, better sustained release injectable hyaluronic acid - hydrogel and method for preparing collagen composite.

[0008] 本发明所采用的技术方案是: [0008] The technical proposal of the present invention is:

一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: An injection of hyaluronic acid - Preparation collagen composite hydrogel, wherein:

由以下步骤实现: Implemented by the following steps:

步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为6%-20% ; Step a: The sodium hyaluronate is dissolved in alkaline solution, stirred to complete dissolution, the mixed solution of hyaluronic acid concentration of 6% to 20%;

步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为1_6%,搅拌均匀; 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为1_10%,反应温度30-60°C,反应时间1-14小时; Step two: collagen was added, the mixed solution so that the concentration of collagen 1_6%, stir; Step three: adding a crosslinking agent, the mixed solution so that the concentration of crosslinking agent 1_10%, the reaction temperature is 30-60 ° C, the reaction time is 1-14 hours;

步骤四:除去残留交联剂; Step Four: remove residual crosslinking agent;

步骤五:得到的产物在生理盐水中平衡3-6小时; Step Five: The product obtained equilibrate in physiological saline for 3-6 hours;

步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 Step Six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.

[0009] 步骤一中,透明质酸钠的分子量为50-300万。 [0009] Step a, the molecular weight of sodium hyaluronate 50-300 million.

[0010] 步骤一中,碱性溶液选自氢氧化钠、氢氧化钾、碳酸钠溶液,摩尔体积分数为 [0010] Step one, the alkaline solution is selected from sodium hydroxide, potassium hydroxide, sodium carbonate solution, molar volume fraction

0.02-2 mol/Lo 0.02-2 mol / Lo

[0011] 步骤二中,胶原蛋白选自从动物组织提取的全长胶原蛋白、胶原蛋白多肽、明胶;或采用基因工程方法生产的重组胶原蛋白、类人胶原蛋白。 [0011] Step II, collagen extracted from animal tissue selected from the full length collagen, collagen peptide, gelatin; or a genetic engineering method for producing recombinant collagen, human-like collagen.

[0012] 步骤三中,交联剂选自乙二醇二缩水甘油醚、I,4- 丁二醇二缩水甘油醚、二甘醇二缩水甘油醚、聚乙二醇二缩水甘油醚、聚丙二醇二缩水甘油醚、1,6-己二醇二缩水甘油醚、1,2,7,8- 二环氧羊烧、二乙稀基讽。 In [0012] Step three, the crosslinking agent selected from ethylene glycol diglycidyl ether, I, 4- butanediol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyethylene propylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, 1,2,7,8-diepoxy burning sheep, di ethylene group ridicule.

[0013] 步骤四中,除去残留交联剂的方法选自水洗、有机溶剂清洗、高温高压蒸馏。 In [0013] Step 4 of the method for removing residual cross-linking agent is selected from water, organic solvent cleaning, high temperature high pressure distillation.

[0014] 步骤六中,粉碎处理选自挤压、研磨、切割方式。 [0014] Step six, the crushing treatment is selected from extrusion, grinding, cutting mode.

[0015] 一种如所述的制备方法制得的注射用透明质酸-胶原蛋白复合水凝胶。 [0015] A method of preparing the injectable hyaluronic acid obtained - collagen composite hydrogel.

[0016] 本发明具有以下优点: [0016] The present invention has the following advantages:

本发明采用透明质酸钠和胶原蛋白为原料,缩水甘油醚类为交联剂制备水凝胶。 The present invention employs sodium hyaluronate and collagen as raw material, glycidyl ethers as crosslinking agent a hydrogel. 单纯使用透明质酸钠制备水凝胶时存在降解速度过快、机械强度低的问题,不能完全满足临床应用。 Simple use of the presence of sodium preparing the hydrogel degradation rate is too fast, low mechanical strength of hyaluronic acid, does not fully meet the clinical application. 因此我们引入胶原蛋白制备得到更加理想的水凝胶。 Thus obtained prepared collagen we introduce more desirable hydrogels. 得到的产品中交联剂残留量低,对生物体损害较小,且胶原蛋白可提高水凝胶的机械强度,并对产品的细胞粘附性和促细胞生长性产生积极的影响,从而使该水凝胶作为软组织填充材料表现出更大的优势。 The resulting product is low crosslinking agent residual amount, less damage to the living body, and can improve the mechanical strength of collagen hydrogel, and a positive impact on the growth of the product and cell adhesion promoting cell, thereby the hydrogel as soft tissue filler material exhibits a greater advantage.

附图说明 BRIEF DESCRIPTION

[0017] 图1为水凝胶分别在体外培养1、3、5天后的相对细胞存活率。 [0017] FIG. 1 relative cell viability were cultured in vitro 1,3,5 days hydrogel.

[0018] 图2为水凝胶的压缩位移与压缩载荷之间的关系曲线。 [0018] FIG 2 is a plot of compressive deformation and the compression load between the hydrogel.

具体实施方式 detailed description

[0019] 下面结合具体实施方式对本发明进行详细的说明。 [0019] The present invention will be described in detail with reference to specific embodiments.

[0020] 本发明的目的是提供一种注射用透明质酸-胶原蛋白复合水凝胶及制备方法,将透明质酸钠与胶原蛋白以一定比例混合溶于碱性溶液中,搅拌均匀后向反应体系中加入缩水甘油醚,混合均匀于一定温度下反应,得到块状凝胶,经过清洗或蒸馏除去残留交联剂,均质化使其成为颗粒状水凝胶。 [0020] The object of the present invention is to provide an injection of hyaluronic acid - collagen composite hydrogel and method for preparing the collagen and sodium hyaluronate at a certain mixing ratio was dissolved in alkaline solution, stirred to uniformity ether was added to the reaction system, the reaction mixed at a certain temperature, to obtain a massive gel, washing or distillation residue after removal of a crosslinking agent, to become homogenized the particulate hydrogel.

[0021] 本发明所涉及的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: [0021] The present invention relates to an injection of hyaluronic acid - collagen composite hydrogel preparation, which is characterized in that:

由以下步骤实现: Implemented by the following steps:

步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为6%-20%。 Step a: Sodium hyaluronate was dissolved in alkaline solution, stirred to complete dissolution, the mixed solution of hyaluronic acid concentration was 6-20%.

[0022] 透明质酸钠的分子量为50-300万;碱性溶液选自氢氧化钠、氢氧化钾、碳酸钠溶液,摩尔体积分数为0.02-2 mol/L。 [0022] The molecular weight of sodium hyaluronate 50-300 million; the basic solution is selected from sodium hydroxide, potassium hydroxide, sodium carbonate solution, molar volume fraction of 0.02-2 mol / L.

[0023] 步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为1_6%,搅拌均匀。 [0023] Step Two: collagen was added, the mixed solution so that the concentration of collagen 1_6%, stir.

[0024] 胶原蛋白选自从动物组织提取的全长胶原蛋白、胶原蛋白多肽、明胶;或采用基因工程方法生产的重组胶原蛋白、类人胶原蛋白。 [0024] collagen extracted from animal tissue selected from the full length collagen, collagen peptide, gelatin; or a genetic engineering method for producing recombinant collagen, human-like collagen.

[0025] 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为1_10%,反应温度30-60°C,反应时间1-14小时。 [0025] Step Three: adding a crosslinking agent, the mixed solution so that the concentration of crosslinking agent 1_10%, the reaction temperature is 30-60 ° C, the reaction time is 1-14 hours.

[0026] 交联剂选自乙二醇二缩水甘油醚、1,4- 丁二醇二缩水甘油醚、二甘醇二缩水甘油醚、聚乙二醇二缩水甘油醚、聚丙二醇二缩水甘油醚、1,6-己二醇二缩水甘油醚、1,2,7,8- 二环氧羊烧、二乙稀基讽。 [0026] The crosslinking agent is selected from ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, 1,2,7,8-diepoxy burning sheep, di ethylene group ridicule.

[0027] 步骤四:除去残留交联剂。 [0027] Step Four: remove residual crosslinking agent.

[0028] 除去残留交联剂的方法选自水洗、有机溶剂清洗(乙醇、丙酮等)、高温高压蒸馏,也可多种方法配合使用。 [0028] A method for removing residual cross-linking agent is selected from water, organic solvent cleaning (ethanol, acetone, etc.), high temperature and high pressure distillation, it can also be used with a variety of methods.

[0029] 步骤五:得到的产物在生理盐水中平衡3-6小时。 [0029] Step Five: The product obtained in 3-6 hours equilibrate in physiological saline.

[0030] 步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 [0030] Step Six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.

[0031 ] 粉碎处理选自挤压、研磨、切割方式。 [0031] The pulverization treatment is selected from extrusion, grinding, cutting mode.

[0032] 实施例1: [0032] Example 1:

步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为6%。 Step a: Sodium hyaluronate was dissolved in alkaline solution, stirred to complete dissolution, the mixed solution of hyaluronic acid concentration was 6%.

[0033] 透明质酸钠的分子量为50-300万;碱性溶液选取氢氧化钠溶液,摩尔体积分数为 [0033] The molecular weight of sodium hyaluronate 50-300 million; the alkaline solution a sodium hydroxide solution selected, molar volume fraction

0.02mol/L。 0.02mol / L.

[0034] 步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为1%,搅拌均匀。 [0034] Step Two: collagen was added, the mixed solution so that the concentration of collagen is 1%, and stirred uniformly.

[0035] 胶原蛋白选取从动物组织提取的全长胶原蛋白。 [0035] The collagen extracted from animal tissues selected full-length collagen.

[0036] 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为1%,反应温度30°C,反应时间I小时。 [0036] Step Three: adding a crosslinking agent, the mixed solution so that the concentration of crosslinker is 1%, the reaction temperature of 30 ° C, the reaction time of I hour.

[0037] 交联剂选自乙二醇二缩水甘油醚、1,4_ 丁二醇二缩水甘油醚。 [0037] The crosslinking agent is selected from ethylene glycol diglycidyl ether, butanediol diglycidyl ether 1,4_.

[0038] 步骤四:除去残留交联剂。 [0038] Step Four: remove residual crosslinking agent.

[0039] 除去残留交联剂的方法选取水洗。 [0039] A method for removing residual cross-linking agent selected washing.

[0040] 步骤五:得到的产物在生理盐水中平衡3小时。 [0040] Step Five: The product obtained equilibrate in physiological saline for 3 hours.

[0041] 步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 [0041] Step Six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.

[0042] 粉碎处理选取挤压方式。 [0042] The extrusion pulverization select mode. [0043] 实施例2: [0043] Example 2:

步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为10%。 Step a: Sodium hyaluronate was dissolved in alkaline solution, stirred to complete dissolution, the concentration of hyaluronic acid in the mixed solution was 10%.

[0044] 透明质酸钠的分子量为50-300万;碱性溶液选取氢氧化钾溶液,摩尔体积分数为0.5 mol/L。 [0044] sodium hyaluronate molecular weight of 50 to 300 million; the selected basic solution of potassium hydroxide solution, molar volume fraction of 0.5 mol / L.

[0045] 步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为2%,搅拌均匀。 [0045] Step Two: collagen was added, the mixed solution so that the concentration of collagen is 2%, stir.

[0046] 胶原蛋白选取从动物组织提取的胶原蛋白多肽。 [0046] collagen polypeptide selected from collagen extracted from animal tissues.

[0047] 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为4%,反应温度40°C,反应时间5小时。 [0047] Step Three: adding a crosslinking agent, the mixed solution so that the concentration of crosslinker is 4%, the reaction temperature of 40 ° C, the reaction time of 5 hours.

[0048] 交联剂选自二甘醇二缩水甘油醚、聚乙二醇二缩水甘油醚。 [0048] The crosslinking agent is selected from diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether.

[0049] 步骤四:除去残留交联剂。 [0049] Step Four: remove residual crosslinking agent.

[0050] 除去残留交联剂的方法选取有机溶剂清洗(乙醇、丙酮等)。 [0050] The method of removing residual organic solvent selected crosslinker washed (ethanol, acetone, etc.).

[0051] 步骤五:得到的产物在生理盐水中平衡4小时。 [0051] Step Five: The product obtained equilibrate in physiological saline for 4 hours.

[0052] 步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 [0052] Step Six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.

[0053] 粉碎处理选取研磨。 [0053] grinding the pulverization treatment selected.

[0054] 实施例3: 步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为15%。 [0054] Example 3: Step 1: Sodium hyaluronate was dissolved in alkaline solution, stirred to complete dissolution, the concentration of hyaluronic acid in the mixed solution was 15%.

[0055] 透明质酸钠的分子量为50-300万;碱性溶液选取碳酸钠溶液,摩尔体积分数为Imol/L。 [0055] The molecular weight of sodium hyaluronate 50-300 million; the selected basic solution of sodium carbonate solution, molar volume fraction Imol / L.

[0056] 步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为4%,搅拌均匀。 [0056] Step Two: collagen in the mass concentration in the mixed solution of 4% collagen, stir.

[0057] 胶原蛋白选取从动物组织提取的明胶。 [0057] Collagen select gelatin extracted from animal tissues.

[0058] 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为7%,反应温度50°C,反应时间10小时。 [0058] Step Three: adding a crosslinking agent, so that the concentration of the crosslinking agent in the mixed solution of 7%, the reaction temperature of 50 ° C, the reaction time of 10 hours.

[0059] 交联剂选自聚丙二醇二缩水甘油醚、I,6-己二醇二缩水甘油醚。 [0059] The crosslinking agent is selected from polyethylene glycol diglycidyl ether, I, 6- hexanediol diglycidyl ether.

[0060] 步骤四:除去残留交联剂。 [0060] Step Four: remove residual crosslinking agent.

[0061] 除去残留交联剂的方法选取高温高压蒸馏。 [0061] A method for removing residual cross-linking agent selected high temperature and pressure distillation.

[0062] 步骤五:得到的产物在生理盐水中平衡5小时。 [0062] Step Five: The product obtained equilibrate in physiological saline for 5 hours.

[0063] 步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 [0063] Step Six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.

[0064] 粉碎处理选取切割方式。 [0064] pulverization selected cutting mode.

[0065] 实施例4: [0065] Example 4:

步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为20%。 Step a: Sodium hyaluronate was dissolved in alkaline solution, stirred to complete dissolution, the concentration of the mixed solution of hyaluronic acid was 20%.

[0066] 透明质酸钠的分子量为50-300万;碱性溶液选取碳酸钠溶液,摩尔体积分数为2mol/L。 [0066] The molecular weight of sodium hyaluronate 50-300 million; the selected basic solution of sodium carbonate solution, molar volume fraction of 2mol / L.

[0067] 步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为6%,搅拌均匀。 [0067] Step Two: collagen in the mixed solution concentration 6% collagen, stir.

[0068] 胶原蛋白选取采用基因工程方法生产的重组胶原蛋白、类人胶原蛋白。 [0068] Collagen selected using recombinant collagen produced by genetic engineering methods, human-like collagen.

[0069] 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为10%,反应温度60°C,反应时间14小时。 [0069] Step Three: adding a crosslinking agent, so that the concentration of the crosslinking agent in the mixed solution is 10%, reaction temperature 60 ° C, the reaction time is 14 hours. [0070] 交联剂选自1,2,7,8-二环氧辛烷、二乙烯基砜。 [0070] The crosslinker is selected from 1,2,7,8-diepoxy octane, divinyl sulfone.

[0071] 步骤四:除去残留交联剂。 [0071] Step Four: remove residual crosslinking agent.

[0072] 除去残留交联剂的方法采用水洗、有机溶剂清洗(乙醇、丙酮等)、高温高压蒸馏中多种方法的配合。 [0072] A method for removing residual cross-linking agent employed washed with water, washed with an organic solvent, a variety of high temperature and pressure distillation method (ethanol, acetone, etc.).

[0073] 步骤五:得到的产物在生理盐水中平衡6小时。 [0073] Step Five: The product obtained equilibrate in physiological saline for 6 hours.

[0074] 步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 [0074] Step Six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.

[0075] 粉碎处理采用挤压、研磨、切割方式中多种方法的配合。 [0075] pulverizing treatment using extrusion, grinding, cutting method with a variety of ways.

[0076] 以下为本发明所涉及的注射水凝胶的相关性能测试: [0076] The injectable hydrogel of the present invention is related to performance test:

1、材料的细胞毒性检测 1, material Cytotoxicity Detection

BHK细胞为仓鼠肾细胞,材料对该细胞的生长和增殖的影响通过新型细胞增殖及细胞毒性检测试剂盒(cck-8)来分析。 Hamster kidney cells BHK cells, the material affect cell growth and proliferation was analyzed by novel cell proliferation and cytotoxicity detection kit (cck-8). 它的基本原理是活细胞线粒体中的琥珀酸脱氢酶可以将2- (2-甲氧基-4-硝基苯基)-3- (4-硝基苯基)-5- (2,4- 二磺酸苯)-2H-四唑单钠盐(WST-8)还原为水溶性的黄色的甲臢产物,细胞增殖越快越多,颜色越深,细胞毒性越大,颜色越浅,颜色的深浅与活细胞的数量成正比,从而可以得到细胞的增殖情况。 Its basic principle is the mitochondria of living cells can succinate dehydrogenase 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, acid 4-phenyl) -2H- tetrazolium monosodium salt (WST-8) is reduced to a yellow water-soluble formazan product, the more faster the cell proliferation, the darker the color, the higher the cytotoxicity, the lighter the color , color depth proportional to the number of living cells, so that cell proliferation can be obtained.

[0077] 将贴壁细胞用胰蛋白酶消化后,用培养液制成细胞悬浮液,按照每孔IO3-1O4个细胞接种于96孔板中,每孔100 μ L,并将孔板置于细胞培养箱,培养Id使细胞贴壁。 [0077] The adherent cells were trypsinized, a cell suspension culture was prepared, IO3-1O4 cells per well were seeded in 96-well plates, each well 100 μ L, was placed and the plate cells incubator, the cells were cultured adherent Id.

[0078] 将材料浸泡在培养液中于37°C下浸提72h后,用浸提液培养贴壁细胞,以加入正常培养液的孔作为对照。 [0078] The material was immersed in the culture solution after leaching 72h, extract with adherent cells cultured at 37 ° C, a normal culture medium was added to the hole as a control. 分别于1,3,5d后,每孔加入10 μ L cck-8,继续培养4小时后,使用酶标仪在450nm处测定吸光度。 Respectively, after 1,3,5d, each well was added 10 μ L cck-8, after culture for four hours, and the absorbance was measured at 450nm using a microplate reader.

[0079] 各组OD值取均值后,计算细胞相对存活率如下: [0079] The OD values ​​of each group averaging, calculation Relative survival following:

Figure CN103333349AD00071

根据IS010993-1.1997及国家标准GB/T16886.5-2003,透明质酸凝胶与透明质酸-胶原蛋白复合凝胶的细胞毒性等级为O级,并且在培养5天后,复合水凝胶的促细胞生长作用明显比透明质酸凝胶良好。 The IS010993-1.1997 and national standards GB / T16886.5-2003, hyaluronic acid gel and the hyaluronic acid - a cytotoxic level of collagen gel composite is O stage and after 5 days in culture, pro-composite hydrogel cELL gROWTH excellent than hyaluronic acid gel. 通过对比实验结果,我们可以得出:在透明质酸凝胶基础上复合了胶原蛋白以后,可以有效促进细胞的生长,从而证明了该材料的对细胞生长及增殖的促进作用。 By comparing the experimental results, we can conclude: hyaluronic acid complexed with the collagen gel on the basis of the future, can effectively promote the growth of the cells, thus demonstrating that material promoting cell growth and proliferation. 因此,实验证明两种水凝胶的浸提液对细胞的生长都有促进作用,使得细胞的相对存活率随培养时间而增加,说明该水凝胶无明显的细胞毒性,并且具有良好的生物相容性,符合生物材料应用的要求。 Thus, two kinds of experiments show that the extract hydrogel on cell growth have promoted, so that the relative survival rate of the cells increases over the culture time, indicating that the hydrogel without significant cytotoxicity, and have good biological compatibility, meet the requirements of biomaterials applications.

[0080] 2、材料的可注射性 [0080] 2, injectable material

将样品灌装入ImL注射器中,实验时,安装上27G注射针,模拟实际使用情况。 ImL samples were filled into the syringe, the experiment, the 27G needle mounted to simulate actual usage. 以恒定推动速度30mm/min推动推进杆,注射器中的样品经由针头被推挤出。 Promote a constant speed 30mm / min push the push rod, sample injector needle is pushed through the extruder. 得到推挤力曲线。 Pushing force curve obtained.

[0081] 由图可知,在一定的压缩位移内,可注射透明质酸一类人胶原蛋白复合水凝胶的压缩载荷变化范围在在5 — 8N之间,压缩载荷的变化幅度很小,说明推挤力的高低落差较小,表明样品分散均匀,注射较为容易和方便,并且说明可注射透明质酸-类人胶原蛋白复合水凝胶的颗粒粒径较为均一。 [0081] The figure shows, in a certain compression displacement, a class of injectable hyaluronic acid compressive load variation range of human collagen in the composite hydrogel 5 - small change in amplitude between 8N, compressive load described pushing force is smaller gap height, the sample showed a uniform dispersion, injection easier and convenient, and can be described injectable hyaluronic acid - like collagen composite hydrogel particle size more uniform.

[0082] 3、凝胶的粒径分布 [0082] 3, the gel particle size distribution

取凝胶产物0.5g,加入0.2%甲苯胺蓝-乙醇溶液染色lmin,而后离心取沉淀,加入ImL蒸馏水静置lmin,离心后弃上清液,加入95%的乙醇5mL分色5min,再离心弃乙醇,加入ImL生理盐水稀释,在100倍光学显微镜下观察计数、测定粒径。 Take gel product 0.5g, 0.2% toluidine blue - Lmin stained ethanol solution, followed by centrifugation the precipitate, distilled water was added ImL Lmin standing, the supernatant was discarded after centrifugation, was added 5mL of 95% ethanol dichroic 5min, centrifuged again ethanol was discarded, diluted with ImL saline was added, the count was observed at 100 times optical microscope to measure the particle size.

[0083] 4、凝胶中透明质酸含量测定 [0083] 4. Determination of the hyaluronic acid gel

取产物0.5g,加入4.6M HCl于70°C水浴中水解2h,溶液定容到IOOmL容量瓶中,采用咔唑法测定其中糖醛酸含量,从而可以得到透明质酸含量(A)。 The product take 0.5g, was added 4.6M HCl in 70 ° C water bath hydrolysis 2h, the solution was brought IOOmL flask, wherein the uronic acid content was measured using the carbazole method, which can obtain a hyaluronic acid content of (A).

[0084] 5、凝胶的抗酶解性能测定 [0084] 5. Determination of the anti-enzymatic properties of the gel

取凝胶0.5g,加入3mg/mL透明质酸酶溶液0.5mL, 37°C下水解15h,0.22 μ m滤膜过滤后取0.5mL滤液,定容至50mL,咔唑法测定其中糖醛酸浓度,从而得到透明质酸含量(B),凝胶的抗酶解性能用B/A值表示。 Take gel 0.5g, was added 3mg / mL of hyaluronidase solution 0.5mL, hydrolysis 15h at 37 ° C, after the 0.22 μ m filter membrane to take 0.5mL filtrate volume to 50mL, wherein the measured uronic acid carbazole concentration, whereby the hyaluronic acid content (B), anti-gel digestion performance represented by B / a values. 结果见表I。 The results are shown in Table I.

[0085] 表I凝胶测定结果 [0085] The measurement results in Table I Gel

Figure CN103333349AD00081

由表中数据可以看出,在同样的工艺条件下,制备所得到的透明质酸凝胶和透明质酸-胶原蛋白复合凝胶粒径分布相差不大,但产物中透明质酸含量有所不同,并且透明质酸凝胶的抗酶解性能明显比透明质酸-胶原蛋白复合凝胶差,说明透明质酸-胶原蛋白复合凝胶在体内的存留时间可明显延长。 Can be seen from the data in the table, under the same conditions, the resulting hyaluronic acid gel prepared and hyaluronic acid - Compound collagen gel or less the particle size distribution, but the acid content in the product has different and enzymatic stability of a hyaluronic acid gel properties significantly to hyaluronic acid - collagen composite gel difference described hyaluronic acid - collagen composite gel can be significantly longer retention time in the body.

[0086] 6、凝胶产物中交联剂残留的测定 [0086] 6, as determined by gel crosslinking agent remaining in the product

采用荧光分光光度法测定凝胶中交联剂的残留量,结果见表2。 Residues using fluorescence spectrophotometry gel crosslinking agent, the results shown in Table 2.

[0087] [0087]

表2凝胶中交联剂的残留量 TABLE 2 Residues crosslinker gel

Figure CN103333349AD00082

由于交联剂具有较低毒性,因此产物中交联剂残留量必须达标。 Since the crosslinking agent has low toxicity, and therefore the amount of product remaining in the crosslinking agent must be compliance. 规定凝胶中交联剂残留量不能高于2μ g/g,由表中数据可知,交联剂浓度较低时,可直接通过水洗使交联剂残留量降到标准以下,但当提高交联剂浓度时,水洗效果不能达标,因此我们与高温高压蒸馏法相结合,可以明显地去除凝胶中残留交联剂,从而得到生物相容性较好,毒性较低,适用于软组织填充或组织修复的水凝胶材料。 Gel predetermined crosslinker residues not exceed 2μ g / g, seen from the data in the table, lower concentrations of the crosslinking agent, the crosslinking agent can be obtained by the direct-washed residual amount falls less standard, but increasing the cross when the concentration of the crosslinking agent, the effect of washing is not standard, we combined with high temperature and high pressure distillation wears, the gel can be clearly remove residual crosslinking agent, to obtain a good biocompatibility, low toxicity, is suitable for filling soft tissue or tissue repair hydrogel material.

[0088] 本发明的内容不限于实施例所列举,本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换,均为本发明的权利要求所涵盖。 [0088] The present invention is not limited to the embodiments exemplified embodiment, any equivalent converted to those of ordinary skill in the art to take a reading of the description of the invention of the present invention, the claims are encompassed by the present invention.

Claims (8)

  1. 1.一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 由以下步骤实现: 步骤一:将透明质酸钠溶解于碱性溶液中,搅拌使其完全溶解,使混合溶液中透明质酸的质量浓度为6%-20% ; 步骤二:加入胶原蛋白,使混合溶液中胶原蛋白的质量浓度为1_6%,搅拌均匀; 步骤三:加入交联剂,使混合溶液中交联剂的质量浓度为1_10%,反应温度30-60°C,反应时间1-14小时; 步骤四:除去残留交联剂; 步骤五:得到的产物在生理盐水中平衡3-6小时; 步骤六:离心除去生理盐水,所得产物进行粉碎形成颗粒。 An injectable hyaluronic acid - Preparation collagen composite hydrogel, wherein: implemented by the following steps: Step 1: Sodium hyaluronate was dissolved in alkaline solution, stirred and completely dissolved, mixed solution of hyaluronic acid concentration of 6-20%; two steps: addition of collagen, so that the concentration of collagen in the mixed solution of 1_6%, stir; step three: adding a crosslinking agent, mixing mass concentration in the solution 1_10% crosslinking agent, the reaction temperature is 30-60 ° C, the reaction time is 1-14 hours; step four: remove residual crosslinking agent; step five: the product obtained in physiological saline balance 3-6 h; step six: centrifugal physiological saline, the resulting product is pulverized to form particles removed.
  2. 2.根据权利要求1所述的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 步骤一中,透明质酸钠的分子量为50-300万。 2. An injection according to claim 1 of hyaluronic acid - Preparation collagen composite hydrogel, wherein: in step a, a molecular weight sodium hyaluronate 50-300 million.
  3. 3.根据权利要求1或2所述的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 步骤一中,碱性溶液选自氢氧化钠、氢氧化钾、碳酸钠溶液,摩尔体积分数为0.02-2mol/L0 3. An injection of hyaluronic acid according to claim 1 or claim 2 - Preparation collagen composite hydrogel, characterized by: a step, the alkaline solution is selected from sodium hydroxide, potassium hydroxide, sodium carbonate solution, molar volume fraction 0.02-2mol / L0
  4. 4.根据权利要求3所述的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 步骤二中,胶原蛋白选自从动物组织提取的全长胶原蛋白、胶原蛋白多肽、明胶;或采用基因工程方法生产的重组胶原蛋白、类人胶原蛋白。 An injection according to claim 3, wherein said hyaluronic acid - Preparation collagen composite hydrogel, wherein: the step II, collagen extracted from animal tissue selected from the full length collagen, Collagen polypeptide, gelatin; or by recombinant collagen produced by genetic engineering methods, human-like collagen.
  5. 5.根据权利要求4所述的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 步骤三中,交联剂选自乙二醇二缩水甘油醚、I,4- 丁二醇二缩水甘油醚、二甘醇二缩水甘油醚、聚乙二醇二缩水甘油醚、聚丙二醇二缩水甘油醚、1,6-己二醇二缩水甘油醚、1,2,7,8_ 二环氧羊烧、二乙稀基讽。 An injection according to claim 4, wherein the hyaluronic acid - Preparation collagen composite hydrogel, wherein: in step three, the crosslinking agent selected from ethylene glycol diglycidyl ether, I, 1,4-butanediol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, 1,2, 7,8_ diepoxy burning sheep, di ethylene group ridicule.
  6. 6.根据权利要求5中所述的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 步骤四中,除去残留交联剂的方法选自水洗、有机溶剂清洗、高温高压蒸馏。 An injection according to claim 5, wherein hyaluronic acid - Preparation collagen composite hydrogel, wherein: in step four, a method for removing residual cross-linking agent is selected from water, organic solvent cleaning , high temperature and pressure distillation.
  7. 7.根据权利要求6所述的一种注射用透明质酸-胶原蛋白复合水凝胶的制备方法,其特征在于: 步骤六中,粉碎处理选自挤压、研磨、切割方式。 An injection according to claim 6, wherein said hyaluronic acid - Preparation collagen composite hydrogel, wherein: step six, the pulverization treatment is selected from extrusion, grinding, cutting mode.
  8. 8.—种如权利要求1所述的制备方法制得的注射用透明质酸-胶原蛋白复合水凝胶。 Injection of hyaluronic acid preparation method of the kind as claimed in claim 1 8.- obtained - collagen composite hydrogel.
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