CN106479965A - 一种增强聚乙二醇水凝胶生物相容性和降解性的方法 - Google Patents
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Abstract
本发明公开了一种增强聚乙二醇水凝胶生物相容性和降解性的方法,包括以下步骤:(1)酰基化聚乙二醇的制备、(2)酰基化多肽的制备、(3)人间充质干细胞的分离、(4)间充质干细胞诱导分化成软骨细胞。本发明的优点在于:本发明具有工艺先进、步骤简单、操作简便、可以提升生物材料支架的生物相容性和可降解性、促进细胞繁殖和组织再生。
Description
技术领域
本发明涉及生物材料技术领域,具体涉及一种增强聚乙二醇水凝胶生物相容性和降解性的方法。
背景技术
聚乙二醇具有安全、可自由改性、优良的生物物理性能等优点,已经广泛应用于多个医药领域,并且是通过美国FDA安全认证的可外用内服的生物材料。基于聚乙二醇这些优良的性能,通过多种交联技术而构建的生物材料支架可以广泛应用于组织工程领域。但是该生物材料目前在运用中也存在一些问题,首先聚乙二醇对较为敏感的细胞生物相容性不太理想,其次聚乙二醇生物材料无法被生物体降解,这些缺点限制了它更为广泛的应用,而对其缺点的改进方法研究较少,目前仍然没有很好的方法能够解决此问题。
多肽链GRGDS和MMP-sensitive可以促进细胞生长,并且能为细胞提供可降解的位点。将多肽酰基化,并且与酰基化的聚乙二醇同时交联,可以将这些多肽链化学结合至聚乙二醇骨架,由此提升该生物材料支架的生物相容性和可降解性,促进细胞繁殖和组织再生。
发明内容
本发明的目的在于解决现有聚乙二醇生物材料存在的不足,现提供一种增强聚乙二醇水凝胶生物相容性和降解性的方法。
为解决上述技术问题,本发明采用的技术方案为:一种增强聚乙二醇水凝胶生物相容性和降解性的方法,其创新点在于:包括以下步骤:
(1)酰基化聚乙二醇的制备:在氮气条件下,取一定量聚乙二醇与甲基丙烯酰氯过夜反应,反应完成后纯化合成的酰基化聚乙二醇,然后通过核磁共振检验,检验其纯度是否大于95%;
(2)酰基化多肽的制备:在氮气条件下,取一定量的GRGDS、MMP-sensitive多肽溶解在无水甲醇中,再加入甲基丙烯酰氯和二甲基氨基吡啶,室温下反应2小时,反应完成后加入乙醇旋转蒸发得酰基化多肽,将其溶解在去离子水中备用,所述蒸发温度为50℃,旋转蒸发至乙醇消失为止;
(3)人间充质干细胞的分离:无菌条件下取健康成人骨髓液,用硫化铅漂洗并计数,将漂洗后的细胞培养在α-MEM基础培养基中,置于细胞培养箱培养;
(4)间充质干细胞诱导分化成软骨细胞:取生长状态良好的第二代或第三代间充质干细胞传代,调整密度至5×103个/cm2,待细胞贴壁将基础培养基更换为DMEM诱导培养基,置于细胞培养箱培养。
进一步的,所述步骤(1)中每克聚乙二醇加0.2毫升甲基丙烯酰氯反应,纯化方法为乙醚析出和过夜冻干,纯化后的酰基化聚乙二醇纯度大于95%。
进一步的,所述步骤(2)中无水甲醇、甲基丙烯酰氯的量对于GRGDS、MMP-sensitive多肽为过量反应;二甲基氨基吡啶的量占总体积比的5%,旋转蒸发次数为5次,每次加入5mL甲醇以去除丙烯酸甲酯副产物。
进一步的,所述步骤(3)中α-MEM基础培养基含体积浓度为20%的胎牛血清和体积浓度为1%的PSG,细胞培养箱内温度为37℃,含体积浓度为5%的CO2,培养48小时后更换培养液,之后每3天更换一次培养液。
进一步的,所述步骤(4)中DMEM诱导培养基包括1×胰岛素-转铁蛋白-硒-丙酮酸钠,0.1mmol/L抗坏血酸磷酸盐, 1.25mg/mL人血清白蛋白, 10-7mol/L地塞米松, 1%体积浓度的青霉素-链霉素-庆大霉素,10 ng/mL TGF-b1,细胞培养箱内温度为37℃,含体积浓度为5%的CO2,培养48小时后更换培养液,之后每3天更换一次培养液,培养时间2-6周。
本发明的有益效果如下:本发明具有工艺先进、步骤简单、操作简便、可以提升生物材料支架的生物相容性和可降解性、促进细胞繁殖和组织再生。
附图说明
图1为本发明制备的酰基化聚乙二醇核磁共振谱图;
图2为间充质干细胞在该生物材料中的成骨分化结果;
图3为间充质干细胞在该生物材料中的成软骨分化的结果。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。
一种增强聚乙二醇水凝胶生物相容性和降解性的方法,包括以下步骤:
(1)酰基化聚乙二醇的制备:在氮气条件下,取一定量聚乙二醇与甲基丙烯酰氯过夜反应,反应完成后纯化合成的酰基化聚乙二醇,每克聚乙二醇加0.2毫升甲基丙烯酰氯反应,纯化方法为乙醚析出和过夜冻干,然后通过核磁共振检验,如图1所示,其检验纯度大于95%,酰基化程度接近100%;
(2)酰基化多肽的制备:在氮气条件下,取一定量的GRGDS、MMP-sensitive多肽溶解在无水甲醇中,再加入甲基丙烯酰氯和二甲基氨基吡啶,室温下反应2小时,反应完成后加入乙醇旋转蒸发得酰基化多肽,将其溶解在去离子水中备用,蒸发温度为50摄氏度,旋蒸至乙醇基本消失为止,无水甲醇、甲基丙烯酰氯的量对于GRGDS、MMP-sensitive多肽为过量反应;二甲基氨基吡啶的量占总体积比的5%,旋转蒸发次数为5次,每次加入5mL甲醇以去除丙烯酸甲酯副产物;
(3)人间充质干细胞的分离:无菌条件下取健康成人骨髓液,用硫化铅漂洗并计数,将漂洗后的细胞培养在α-MEM基础培养基中,置于细胞培养箱培养,α-MEM基础培养基含体积浓度为20%的胎牛血清和体积浓度为1%的PSG,细胞培养箱内温度为37℃,含体积浓度为5%的CO2,培养48小时后更换培养液,之后每3天更换一次培养液;
(4)间充质干细胞诱导分化成软骨细胞:取生长状态良好的第二代或第三代间充质干细胞传代,调整密度至5×103个/cm2,待细胞贴壁将基础培养基更换为DMEM诱导培养基,置于细胞培养箱培养,DMEM诱导培养基包括1×胰岛素-转铁蛋白-硒-丙酮酸钠,0.1mmol/L抗坏血酸磷酸盐,1.25mg/mL人血清白蛋白,10-7mol/L地塞米松,1%体积浓度的青霉素-链霉素-庆大霉素,10 ng/mL TGF-b1,细胞培养箱内温度为37℃,含体积浓度为5%的CO2,培养48小时后更换培养液,之后每3天更换一次培养液,培养时间2-6周,如图2和图3所示。
本发明具有工艺先进、步骤简单、操作简便、可以提升生物材料支架的生物相容性和可降解性、促进细胞繁殖和组织再生。
上述实施例只是本发明的较佳实施例,并不是对本发明技术方案的限制,只要是不经过创造性劳动即可在上述实施例的基础上实现的技术方案,均应视为落入本发明专利的权利保护范围内。
Claims (5)
1.一种增强聚乙二醇水凝胶生物相容性和降解性的方法,其特征在于:包括以下步骤:
(1)酰基化聚乙二醇的制备:在氮气条件下,取一定量聚乙二醇与甲基丙烯酰氯过夜反应,反应完成后纯化合成的酰基化聚乙二醇,然后通过核磁共振检验,检验其纯度是否大于95%;
(2)酰基化多肽的制备:在氮气条件下,取一定量的GRGDS、MMP-sensitive多肽溶解在无水甲醇中,再加入甲基丙烯酰氯和二甲基氨基吡啶,室温下反应2小时,反应完成后加入乙醇旋转蒸发得酰基化多肽,将其溶解在去离子水中备用,所述蒸发温度为50℃,旋转蒸发至乙醇消失为止;
(3)人间充质干细胞的分离:无菌条件下取健康成人骨髓液,用硫化铅漂洗并计数,将漂洗后的细胞培养在α-MEM基础培养基中,置于细胞培养箱培养;
(4)间充质干细胞诱导分化成软骨细胞:取生长状态良好的第二代或第三代间充质干细胞传代,调整密度至5×103个/cm2,待细胞贴壁将基础培养基更换为DMEM诱导培养基,置于细胞培养箱培养。
2.根据权利要求1所述的一种增强聚乙二醇水凝胶生物相容性和降解性的方法,其特征在于:所述步骤(1)中每克聚乙二醇加0.2毫升甲基丙烯酰氯反应,纯化方法为乙醚析出和过夜冻干,纯化后的酰基化聚乙二醇纯度大于95%。
3.根据权利要求1所述的一种增强聚乙二醇水凝胶生物相容性和降解性的方法,其特征在于:所述步骤(2)中无水甲醇、甲基丙烯酰氯的量对于GRGDS、MMP-sensitive多肽为过量反应;二甲基氨基吡啶的量占总体积比的5%,旋转蒸发次数为5次,每次加入5mL甲醇以去除丙烯酸甲酯副产物。
4.根据权利要求1所述的一种增强聚乙二醇水凝胶生物相容性和降解性的方法,其特征在于:所述步骤(3)中α-MEM基础培养基含体积浓度为20%的胎牛血清和体积浓度为1%的PSG,细胞培养箱内温度为37℃,含体积浓度为5%的CO2,培养48小时后更换培养液,之后每3天更换一次培养液。
5.根据权利要求1所述的一种增强聚乙二醇水凝胶生物相容性和降解性的方法,其特征在于:所述步骤(4)中DMEM诱导培养基包括1×胰岛素-转铁蛋白-硒-丙酮酸钠,0.1mmol/L抗坏血酸磷酸盐, 1.25mg/mL人血清白蛋白, 10-7mol/L地塞米松, 1%体积浓度的青霉素-链霉素-庆大霉素, 10 ng/mL TGF-b1,细胞培养箱内温度为37℃,含体积浓度为5%的CO2,培养48小时后更换培养液,之后每3天更换一次培养液,培养时间2-6周。
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