CN117298345A - 脐带华通氏组织来源的可注射凝胶及其制备方法与应用 - Google Patents
脐带华通氏组织来源的可注射凝胶及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种脐带华通氏组织来源的可注射凝胶,通过以下方法制备得到:(1)将脐带组织切成小块,Triton‑X100处理6~12 h,加入DNase、RNase,37℃摇床酶解5~10 h;离心,冻干,得脱细胞华通氏胶;(2)将脱细胞华通氏胶与胃蛋白酶的盐酸溶液混合,形成预凝胶溶液;(3)将预凝胶溶液与京尼平溶液混合,加入核黄素,交联;(4)加入EDTA和氯已定,即得。所述脐带华通氏组织来源的可注射凝胶在制备组织修复支架材料、干细胞移植载体、药物载体中的应用。本发明的可注射凝胶富含结构蛋白和自然生长因子,适用于任何形状的伤口。本发明为伤口修复和实现医疗废物组织的再利用提供了新的优良选择。
Description
技术领域
本发明涉及一种脐带华通氏组织来源的可注射凝胶及其制备方法与应用,属于生物材料技术领域。
背景技术
难治性伤口如糖尿病足、烧伤和放射性溃疡,一旦病理炎症反应开始,使用常规药物很难在短时间内获得预期的治疗效果。组织损伤区域的大小、深度和炎症反应限制了其自我修复的能力。此外,随着年龄的增长,干细胞和祖细胞的活性及数量均呈下降趋势,会导致其再生能力下降,从而使组织修复更加困难。近年来脱细胞材料在组织工程领域得到越来越广泛的应用,为这种组织修复提供了新的思路。
细胞外基质(ECM)作为三维网络存在于所有的组织和器官中,它是许多细胞胞外大分子的集合。在显微镜下,ECM是一个复杂的三维大分子网络,由胶原原纤维主导,还包含蛋白多糖和糖蛋白等结构性生物分子,以及各种生长因子。ECM的三维网络与其内细胞的相互作用构成了一个高度动态的生态微环境,在这里细胞的生长、增殖、迁移、分化、自噬在ECM的“支持”下实现了可塑性。细胞在各种行为过程中也向ECM提供反馈,指导细胞外基质微环境的动态构建。ECM和细胞在这种动态平衡中维持着生物体的正常功能,并使组织具有高度的组织特异性。
脱细胞的细胞外基质(dECM)是一种细胞外基质,它从组织中去除所有细胞和细胞相关成分,并尽可能多地保留天然组织成分和结构。目前,脱细胞基质已被开发用于多种应用,包括基质粉末、贴片、类器官和水凝胶。
脱细胞的细胞外基质水凝胶(dECMH)最早由Fryetes等人报道。dECM片段可以被胃蛋白酶消化,当消化的pH和盐浓度调整回生理条件时,dECMH可以在37℃孵育30 min或体内注射自组装形成。虽然脱细胞化过程通常伴随着物理强度和微观结构解离的降低,并且不能自主恢复,但dECMH可以克服这一缺点,并在显微镜下重建细胞外基质的天然三维纤维网络结构。dECM预凝胶溶液是一种粘性液体,易于通过注射器注射到皮下,因此与微创治疗相匹配,它可以填补任何不规则的伤口,减少对患者的继发性损伤。目前,主要研究猪和牛动物组织来源的dECMH,但这些动物来源的dECMH在人类中的应用可能会由于异种移植原因引起不良免疫反应,并增加传染性疾病传播的风险。由于在非灵长类哺乳动物中存在α-gal表位,所有动物组织来源的材料都被人类抗gal阻止移植,形成了免疫屏障。然而,人类来源的dECMH可以作为同种移植物避免这种风险。因此,人体组织来源的dECMH变得更加可靠。例如,来自人类脂肪、肺、肝等来源的dECMH已被报道。然而,这些研究经常使用尸体作为其来源。众所周知,随着年龄的增长,脂肪沉积在人体组织中,ECM交联增加,因此与年轻的组织相比,其构建和组织重塑活性降低。不可避免的是,尸体供体的年龄是不可控的,不同批次的产品会有很大的差异,因此不可能从人体组织来源实现工业大规模生产dECMH。另外传统的细胞培养是基于细胞在二维平台上的生长,一般不能代表细胞在其原始身体组织中的真实状态。3D 交联网络在热凝胶中构建了一个多孔结构,赋予它类似支架的特性和类似 ECM的性质。具有先进特性的水凝胶能够模仿天然 ECM 的结构和生物学特性,这些支架提供机械、空间和生物信号来调节和指导细胞粘附、迁移、分化和增殖以促进组织再生。
胶原因其良好的组织相容性和可降解性被广泛用于组织工程研发领域。但由于其机械强度不足,在体内水解过程中不能保持空间构型,且降解过快,因此,有必要提高其稳定性。
发明内容
针对上述现有技术,本发明提供了一种脐带华通氏组织来源的可注射凝胶,及其制备方法与应用。
本发明是通过以下技术方案实现的:
脐带华通氏组织来源的可注射凝胶的制备方法,包括以下步骤:
(1)脱细胞华通氏胶(UC-dECM)的制备:将脐带组织切成小块,用PBS缓冲液和氯化钠溶液冲洗和浸泡,以去除血液;匀浆处理,加入Triton-X100处理6~12 h,离心去除上清;加入适量水,加入脱氧核糖核酸酶(DNase)、核糖核酸酶(RNase),37℃摇床酶解5~10 h;酶解液离心去除细胞碎片,用蒸馏水反复冲洗,冻干,得脱细胞华通氏胶;
(2)可溶性脱细胞华通氏胶的制备:将脱细胞华通氏胶与适量胃蛋白酶的盐酸溶液混合,在室温下搅拌直到完全溶解,形成预凝胶溶液;调整预凝胶溶液中脱细胞华通氏胶的浓度为0.6%~1.2%;
(3)交联:将预凝胶溶液与京尼平溶液混合,加入核黄素,在18~22℃交联20~28h;蒸馏水冲洗,置于饱和甘氨酸溶液中浸泡,换液至溶液不再变色,蒸馏水反复冲洗,冻干;置于铝箔上,紫外线照射1.5~2.5 h;
(4)向上述制备的预凝胶溶液中加入EDTA和氯已定,即得脐带华通氏组织来源的可注射凝胶,4℃冷藏保存。
进一步地,所述步骤(1)中,DNase的浓度为100 U/ml,RNase的浓度为100 U/ml。
进一步地,所述步骤(2)中,胃蛋白酶的盐酸溶液中胃蛋白酶的浓度为4 mg/ml。
进一步地,所述步骤(3)中,京尼平溶液的浓度为0.5%(g/ml);核黄素的终浓度为0.25%(g/ml)。
进一步地,所述步骤(4)中,EDTA的终浓度为17%(g/ml);氯已定的终浓度为0.1%(g/ml)。
利用上述方法制备得到的脐带华通氏组织来源的可注射凝胶,在作为或制备组织修复支架材料中的应用,在作为或制备干细胞移植载体中的应用,在作为或制备药物载体中的应用,在构建人工器官中的应用。
本发明的脐带华通氏组织来源的可注射凝胶,以脐带组织为来源,具有以下有益效果:
(1)脐带作为母体和新生儿组织之间的一种营养联系,富含多种生长因子,生物活性高;比成人组织具有更多的未成熟胶原蛋白,且ECM交联较少,对缺陷组织重塑的优势明显。
(2)全世界每年有多达1.5亿儿童出生,组织供应充足。
(3)年龄的负面影响可以被消除。
(4)实现了废物再利用:脐带作为一种生产过程中的废物,通常被用于分离人脐带间充质干细胞和血管内皮细胞。本发明利用其制备用于组织修复的水凝胶,实现了废物再利用。
本发明采用改良方法制备了基于人脐带组织源的dECMH,采用京尼平、核黄素和紫外线进行交联,并在凝胶中加入基质金属蛋白酶抑制剂EDTA和氯已定(抑制胶原降解,维持胶原结构的完整性,是提高再生材料稳定性的关键),并分析了其物理、化学和力学性能。人脐静脉内皮细胞(HUVECs)用来描述UC-dECMH的生物相容性。水凝胶在体外实验中显示出高迁移能力,这极大地恢复了真正的动态细胞外基质和细胞之间的微环境,以及显示微血管生成的潜力。本发明的脐带华通氏组织来源的可注射凝胶,富含结构蛋白和自然生长因子,适用于任何形状的伤口,同时由于交联剂和氯已定的加入,延长了可注射凝胶在体内的停留时间,为伤口修复和实现医疗废物组织的再利用提供了新的优良选择。本发明的研究有望促进伤口修复方面的进一步进展。
附图说明
图1:UC-dECMH的微观结构示意图,其中,IJKL分别表示浓度为0.6%、0.8%、1.0%、1.2%时的UC-dECMH;标尺标注为100μm。
图2:脐带组织、水凝胶的染色结果示意图,其中,A:脐带组织的H&E染色;B:水凝胶的H&E染色;C:脐带组织的Alisin蓝染色;D:水凝胶的Alisin蓝染色;E:脐带组织的天狼星红染色;F:水凝胶的天狼星红染色。
图3:水凝胶的降解能力及动力学测定结果示意图,其中,A:重量变化率的曲线;B:经典浊度曲线。
图4:不同浓度的预凝胶溶液的流变学特性测定结果示意图,其中,A:频率模量扫描,对不同浓度的水凝胶样品施加1%的固定应变,并在1-60 rad/s范围内进行频率扫描试验。B:时间模量扫描,开始时,温度设置为37℃,60 min后,温度迅速降低到4℃,继续扫描测试60 min;该程序采用的固定应变为0.5%,固定频率为1%。C:在37℃下水凝胶的平均模量。D:在时间模量扫描时,在4℃时的平均模量。
图5:HUVEC在水凝胶中生长(第1天、第3天、第5天)相关的图像及结果示意图,其中,A:有代表性的图像,比例尺标注为100μm;B:细胞在水凝胶中的存活;C:单个视场中的细胞计数;D:细胞的圆度指数;E:在三维环境中观察到的血管生成的代表性图像。
图6:在小鼠真皮和筋膜之间植入可注射凝胶后(第1天、第3天、第7天)相关的图像及结果示意图,其中,A:植入1天后的变化情况;B:植入3天后的变化情况;C:植入7天后的变化情况。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1 脐带华通氏组织来源的可注射凝胶的制备及性能分析
(一)脐带华通氏组织来源的可注射凝胶的制备,步骤如下:
(1)脱细胞华通氏胶(UC-dECM)的制备
新鲜的脐带组织(由医院产科提供)无菌冷藏保存运至实验室,-20℃冷冻保存。脐带组织切成约0.5 cm3的小块,用PBS缓冲液和1M氯化钠溶液冲洗和浸泡,以去除血液。在-80℃下冻融3次后,进行以下操作:用匀浆器匀浆处理,然后加入适量Triton-X100溶液(浓度为1%)处理10 h,3000 rpm离心去除上清;加入5倍量的蒸馏水,加入DNase和RNase(终浓度均为100 U/ml),在37℃摇床上酶解处理8 h。酶解液3000 rpm离心去除细胞碎片,用蒸馏水反复冲洗,冻干(LGJ-18,松原华兴,北京,中国),磨成碎片,供后续使用。
(2)可溶性脱细胞华通氏胶的制备
上述制备的UC-dECM 10 g,与20 ml胃蛋白酶的盐酸溶液(胃蛋白酶4 mg/ml,HCl0.01 mol/L)混合,在室温(26℃)下连续搅拌8小时,直到完全溶解,在37℃形成预凝胶溶液;通过添加PBS缓冲液调节预凝胶溶液中dECM的最终浓度(设置了4个实验组,dECM的浓度分别为0.6%,0.8%,1.0%和1.2%)。预凝胶溶液在37℃孵育30 min自组装形成3D网络结构的水凝胶。将水凝胶放入深低温冰箱中进行速冻过夜,然后在真空冷冻干燥机中冷冻干燥过夜。使用扫描电镜(JSM-IT200,JEOL,Japan)观察水凝胶支架的微观结构,如图1所示。
(3)可溶性华通氏胶的交联
将上述制备的预凝胶溶液10 ml,置于10 ml的0.5%京尼平水溶液中,加入终浓度为0.25%的核黄素,于20℃交联24 h;蒸馏水冲洗,置于饱和甘氨酸溶液中浸泡,换液至溶液不再变色,用蒸馏水反复冲洗,冻干。交联后的水凝胶放在铝箔上,距离254 nm紫外灯6寸高度,照射2 h。
(4)可注射水凝胶的抗降解性控制
上述制备的预凝胶溶液10 ml,加入EDTA使其终浓度为17%,加入氯已定使其终浓度为0.1%,4℃冷藏保存。
(二)性能分析
(1)组织学分析
将上述交联后的脐带组织、水凝胶块在4%多聚甲醛溶液中固定,并在分级乙醇系列中脱水。然后用石蜡包埋,用10μm厚的切片机进行切片,进行苏木精和伊红(H&E)染色,以确定剩余的细胞核。阿利新蓝染色(pH 2.5)和天狼星红分别染酸性粘多糖和胶原,倒置显微镜(TE2000-S尼康,日本)检查脱细胞化效率以及保留酸性粘多糖和胶原蛋白在组织和水凝胶块。
结果:H&E染色显示完整的细胞核均匀分布在脐带组织的基质中,如图2A所示;而脱细胞水凝胶没有显示完整的细胞核或细胞核片段,如图2B所示。阿利新蓝染色结果显示,脐带组织中存在大量的酸性粘多糖,如图2C所示,这些酸性粘多糖在去细胞化过程中被有效的再排列,在去细胞化水凝胶中均匀分布,形成一个没有紫红色核的网格结构,如图2D所示。通过天狼星红染色的结果也可以观察到胶原蛋白在脱细胞水凝胶中的保留,如图2E、F所示。所有结果表明,本发明的脱细胞过程是成功的,dECM的天然成分如胶原蛋白和蛋白聚糖等蛋白质结构,没有明显的丢失。
(2)UC-dECMH的降解
用浓度为0.1%的Ⅱ型胶原酶溶液检测水凝胶的降解性。上述制备的含有0.6%、0.8%、1.0%和1.2% dECM的交联水凝胶10 g(记为W0),然后置于10 ml胶原酶溶液中处理24小时,每2小时取出称重一次(记为Wi,共进行10次),计算重量变化率,如下式:重量变化率(%)。
结果:在Ⅱ型胶原酶溶液中观察到水凝胶的快速降解行为,如图3A所示,胶原酶降解2 h后,dECM含量分别为0.6%、0.8%、1.0%和1.2%的水凝胶块的质量分别降低到初始质量的91.16%、92.79%、93.95%和94.36%。胶原酶降解18 h后,其质量分别降低至初始质量的16.93%、24.55%、28.5%和29.42%。胶原酶降解24 h后,4个水凝胶块均完全降解。由于胶原酶对UC-dECMH的降解是水解胶原蛋白的过程,因此dECM浓度越高,水凝胶降解率越低。
(3)UC-dECMH的凝胶动力学
将不同浓度的预凝胶溶液(dECM的浓度分别为0.6%,0.8%,1.0%和1.2%)以每孔100μl加入96孔板,在4℃下放置过夜。将磁平板阅读器(680型,BIO-RAD,USA)在37℃下预热10min,然后每3 min以450 nm测试平板,共60 min。根据浊度的变化,测定水凝胶的凝胶化时间。
结果:经典浊度曲线如图3B所示,在37℃孵育10 min后,各浓度UC-dECMH的吸光度开始持续上升,直到30 min,各组的吸光度开始稳定,接近最大吸光度的95%。40 min后,所有水凝胶的吸光度均达到最大值。结果表明,随着UC-dECM浓度的增加,水凝胶的吸光度逐渐增加,说明高浓度的水凝胶可能具有较高的刚度。
(4)流变性能试验
采用旋转流变仪(MCR92,Anton Paar,奥地利)评价UC-dECMH的流变学性能。将dECM含量分别为0.6%、0.8%、1.0%和1.2%的预凝胶溶液加入流变仪的样品浴中,用硅油密封,以防止测试过程中水从凝胶中蒸发。将流变仪的起始温度设置为37℃,加入样品后孵育30 min。采用固定应变为1%、频率范围为0.1~60 rad/s的频率模量扫描,测试与频率相关的储能模量(G’)和损耗模量(G″)。在4~37℃下,凝胶化过程的第一个小时和反转过程的第二个小时,固定频率为1 rad/s,固定应变为0.5%进行时间模量扫描。
采用37℃频率模量扫描检测四种不同浓度的UC-dECM的存储和损失模量,结果如图4A所示。所有UC-dECM的存储模量(G’)均大于损失模量(G’),表明当dECM浓度大于0.6%时,可以形成稳定的水凝胶。模量与频率无显著相关性,但随着dECM浓度的增加而增大。在时间模量扫描试验中(结果如图4B所示),采用固定应变为0.5%,固定频率为1%。从37℃变化到4℃后,水凝胶的储存模量和损失模量在一定时间内下降,而储存模量的下降更为明显。当温度为37℃时,水凝胶的平均存储模量(G)远高于平均损失模量(G),如图4C所示,并表现为固态。当温度降低到4℃时,平均存储模量(G’)仅略大于平均损失模量(G’) ,如图4D所示。UC-dECMH的流变性能表明,它是一种温度敏感的可逆水凝胶(4℃⇆37℃),这对保持水凝胶的可注入性至关重要。
(5)细胞的体外增殖
将制备的dECM浓度为1.2%的预凝胶溶液与HUVEC细胞悬液混合,最终细胞浓度为2×105/ml,最终dECM浓度为1.0%。预凝胶在培养箱中孵育固化30 min,然后转移到24孔板中,培养基时间分别为1、3、5天。分别用Calcein-AM/PI活细胞/死细胞双染色试剂盒对细胞进行染色。使用激光扫描共聚焦显微镜(LSCM,LSM880,蔡司,德国)对水凝胶块进行拍照,并叠加来自相同视图的5张图像。使用Image J分析细胞活力,计数细胞并分析细胞圆度指数。细胞圆度指数采用下述公式计算:细胞圆度指数 ,其中,为细胞的面积,
结果如图5所示,HUVECs在UC-dECMH中显示出快速迁移、扩散和增殖的能力。如图5A所示,第一天,细胞在水凝胶中稀疏均匀分布,未扩散的细胞呈圆形。第三天,细胞数量明显增加,并开始表现出类似于二维平面生长的扩散模式,大部分细胞呈椭圆形,有触须。到第5天,UC-dECMH中充满了处于完全扩散状态的细胞。细胞表面积达到最大值,细胞之间明显相连。最后,随着细胞的扩散和增殖,细胞连接形成了一个密集的细胞网络。如图5B所示,第1天细胞活力约为85%,第3天和第5天达到95%左右。如图5C所示,特定的细胞数量(单个视野)从第1天的232±7显著增加到第5天的1472±54,细胞在第5天内增加了6.5倍。如图5D所示,细胞圆度指数也从第1天的约0.8下降到第5天的约0.3,进一步证实了高细胞扩散率。值得注意的是,UC-dECMH中的细胞并不局限于传统水凝胶中的圆形生长状态,而HUVECs和水凝胶的混合培养提供了更高的人体细胞真实生长环境的模拟。有趣的是,通过共聚焦激光扫描图像,在单层扫描图像中观察到UC-dECMH中HUVECs呈管状生长的趋势。这表明,UC-dECMH促进血管化的潜力在3D环境中仍然很明显,而不仅仅是在水凝胶表面(如图5E所示)。
(6)动物体内实验
在小鼠真皮和筋膜之间植入可注射凝胶。在第1天、第3天和第7天观察到,可注射凝胶均保持致密的结构,没有松动(图6-A)。在第3天观察到少量体内降解(图6-B),可注射凝胶在第7天仍没有出现明显的降解(图6-C)。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (7)
1.脐带华通氏组织来源的可注射凝胶的制备方法,其特征在于,包括以下步骤:
(1)脱细胞华通氏胶的制备:将脐带组织切成小块,用PBS缓冲液和氯化钠溶液冲洗和浸泡,以去除血液;匀浆处理,加入Triton-X100处理6~12 h,离心去除上清;加入水,加入DNase、RNase,37℃摇床酶解5~10 h;酶解液离心去除细胞碎片,用蒸馏水反复冲洗,冻干,得脱细胞华通氏胶;
(2)可溶性脱细胞华通氏胶的制备:将脱细胞华通氏胶与胃蛋白酶的盐酸溶液混合,在室温下搅拌直到完全溶解,形成预凝胶溶液;调整预凝胶溶液中脱细胞华通氏胶的浓度为0.6%~1.2%;
(3)交联:将预凝胶溶液与京尼平溶液混合,加入核黄素,在18~22℃交联20~28 h;蒸馏水冲洗,置于饱和甘氨酸溶液中浸泡,换液至溶液不再变色,蒸馏水反复冲洗,冻干;置于铝箔上,紫外线照射1.5~2.5 h;
(4)向预凝胶溶液中加入EDTA和氯已定,即得脐带华通氏组织来源的可注射凝胶。
2.根据权利要求1所述的脐带华通氏组织来源的可注射凝胶的制备方法,其特征在于:所述步骤(1)中,DNase的浓度为100 U/ml,RNase的浓度为100 U/ml。
3.根据权利要求1所述的脐带华通氏组织来源的可注射凝胶的制备方法,其特征在于:所述步骤(2)中,胃蛋白酶的盐酸溶液中胃蛋白酶的浓度为4 mg/ml。
4.根据权利要求1所述的脐带华通氏组织来源的可注射凝胶的制备方法,其特征在于:所述步骤(3)中,京尼平溶液的浓度为0.5%,核黄素的终浓度为0.25%。
5.根据权利要求1所述的脐带华通氏组织来源的可注射凝胶的制备方法,其特征在于:所述步骤(4)中,EDTA的终浓度为17%,氯已定的终浓度为0.1%。
6.利用权利要求1~5中任一项所述的制备方法制备得到的脐带华通氏组织来源的可注射凝胶。
7.权利要求6所述的脐带华通氏组织来源的可注射凝胶在制备组织修复支架材料中的应用,或在制备干细胞移植载体中的应用,或在制备药物载体中的应用,或在构建人工器官中的应用。
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