CN113509596A - 一种3d打印支架及制备方法和应用 - Google Patents

一种3d打印支架及制备方法和应用 Download PDF

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CN113509596A
CN113509596A CN202110518519.9A CN202110518519A CN113509596A CN 113509596 A CN113509596 A CN 113509596A CN 202110518519 A CN202110518519 A CN 202110518519A CN 113509596 A CN113509596 A CN 113509596A
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林贤丰
顾辰辉
陈鹏飞
范顺武
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Hangzhou Yuanbao Biotechnology Co ltd
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Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
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Abstract

本发明公开了一种3D打印支架及制备方法和应用，本发明将间充质干细胞外泌体搭载于细胞外基质凝胶并进行3D打印，得到特定构象的组织修复支架，使其具有更强的生物降解能力和外泌体释放能力；搭载间充质干细胞外泌体的细胞外基质3D打印支架具有高生物活性、高支撑性和高定制性，可更好地针对特定缺损进行修复。

Description

一种3D打印支架及制备方法和应用
技术领域
本发明属于生物材料组织修复及免疫调控领域，具体涉及搭载间充质干细胞外泌体的细胞外基质3D打印支架及制备方法和其在组织修复免疫调控中的应用。
背景技术
外泌体相关材料已在组织修复中取得较大关注，它是由特定细胞分泌的一类小型细胞外囊泡，其粒径约60-150nm，通常携带多种生物活性分子，通过融入靶细胞而实现细胞交互。其中间充质干细胞外泌体，已被认为具有广大的生物治疗前景，并已开始向临床转化。
细胞外基质凝胶是指由细胞外基质及常用凝胶如GelMA混合形成的凝胶。其中，细胞外基质具有细胞分泌的多糖、蛋白、蛋白聚糖等生物大分子，组成复杂的结构网络，支持细胞生长并调节细胞的生物行为，GelMA提供了更加有保证的支撑性及成胶性，使得细胞外基质凝胶可以快速凝固成形。
3D打印技术是一种快速数字化成型技术，在计算机软件控制下实现个性化、精准化、效率高、利用率高的成形过程。已被广泛利用于特定形貌的物件成形，并且已在临床得到初步应用。
目前对于细胞外基质凝胶3D打印支架和外泌体的应用较为分离，缺少同时具有外泌体高生物活性分子释放能力和细胞外基质凝胶3D打印支架高个体化和成型性的复合结构。申请号为CN202010084165.7的专利公布了一种修复软骨缺损的生物3D打印复合墨水的制备方法，将软骨细胞外基质与混合材料按比例打印，得到了具有快速交联和高生物相容性的支架，但是未搭载高生物活性成分如外泌体。申请号为CN201710797883.7的专利公布了一种定向释放功能的外泌体复合胶原生物支架及其制备方法和用途，将外泌体分散至纳米胶原小球，实现外泌体释放，但并未实现3D打印成形，个体化定制及支撑性具有限制。同时，上述专利并未研究支架对于组织修复免疫的调控作用，其作用机制相对不明晰。
因此，需要一种搭载间充质干细胞外泌体的细胞外基质3D打印支架及其在组织修复免疫调控中的应用，达到高生物活性、高支撑性和高定制性。
发明内容
本发明针对现有技术的不足，提供一种搭载间充质干细胞外泌体的细胞外基质3D打印支架及制备方法和其在组织修复免疫调控中的应用。将间充质干细胞外泌体搭载于细胞外基质凝胶并进行3D打印，得到特定构象的组织修复支架，使其具有更强的生物降解能力和外泌体释放能力。
一种3D打印支架，将间充质干细胞外泌体与细胞外基质凝胶混合，并由3D打印平台光固化成形。
作为优选，所述3D打印平台光固化成形时采用的3D打印支架构型包括径向构型、同心圆构型和实心构型。
作为优选，所述的细胞外基质凝胶包括细胞外基质和甲基丙烯酰化凝胶。
作为优选，所述的细胞外基质包括软骨细胞外基质、骨膜细胞外基质、肌腱细胞外基质。
作为优选，细胞外基质凝胶混合浓度为细胞外基质1-10％，单位为w/v，甲基丙烯酰化凝胶8-15％，单位为w/v。
作为优选，所述的细胞外基质采用软骨细胞外基质；所述的细胞外基质浓度为1-3％，单位为w/v，甲基丙烯酰化凝胶浓度为10％，单位为w/v。
一种3D打印支架的制备方法，该方法具体包括以下步骤：
步骤一：采用差速离心+超滤法提取间充质干细胞外泌体，
步骤二：混合1-10％细胞外基质及8-15％甲基丙烯酰化凝胶并搭载外泌体蛋白的浓度为0.1％-2.0％的间充质干细胞外泌体；
步骤三：设计支架构型，并由3D打印平台光固化成形。
作为优选，所述的细胞外基质包括软骨细胞外基质、骨膜细胞外基质、肌腱细胞外基质。
作为优选，所述的软骨细胞外基质的制备方法，具体包括以下步骤：
步骤一：将软骨、骨膜或肌腱进行三次反复冻融，并使用1％Triton X-100洗脱24小时；
步骤二：将洗脱后软骨、骨膜或肌腱经1％SDS洗脱24小时，并使用DNAse I处理12小时；
步骤三：将步骤二处理后的产物冻干后磨粉，并经蛋白酶消化至透亮。
一种搭载间充质干细胞外泌体的细胞外基质3D打印支架在组织修复免疫调控中的应用。
本发明的有益效果：
一种搭载间充质干细胞外泌体的细胞外基质3D打印支架及其在组织修复免疫调控中的应用。将间充质干细胞外泌体搭载于细胞外基质凝胶并进行3D打印，得到特定构象的组织修复支架，使其具有更强的生物降解能力和外泌体释放能力。
相比现有外泌体及细胞外基质凝胶3D打印修复材料，本发明显著的进步在于：
1)使用细胞外基质凝胶搭载间充质干细胞外泌体，同时具有生物活性及细胞支撑性。
2)使用3D打印技术构建特定支架构型，具有高度个体性及空间相容性。
因此，搭载间充质干细胞外泌体的细胞外基质3D打印支架具有高生物活性、高支撑性和高定制性，可更好地针对特定缺损进行修复。
附图说明
图1搭载间充质干细胞外泌体的细胞外基质3D打印支架的制备及作用机制示意图；
图2搭载间充质干细胞外泌体的细胞外基质3D打印支架的CAD模型及构型表征；
图3搭载间充质干细胞外泌体的细胞外基质3D打印支架对损伤部位的免疫调控作用，显示可促进组织损伤部位巨噬细胞由促炎M1细胞转向促愈M2细胞。
图4搭载间充质干细胞外泌体的细胞外基质3D打印支架对组织损伤的修复作用的标本大体图片。
具体实施方式
下面结合实施例对本发明提供的一种搭载间充质干细胞外泌体的细胞外基质3D打印支架及其在组织修复免疫调控中的应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
本发明中的搭载间充质干细胞外泌体的细胞外基质3D打印支架的优选案例为为采用差速离心+超滤法提取间充质干细胞外泌体，混合2％(w/v)软骨细胞外基质及10％(w/v)GelMA并搭载0.2％(w/v)间充质干细胞外泌体，设计径向支架，并由3D打印平台光固化成形。具体步骤如实施例1。
本发明还可以使用其他上述的外泌体制备方案、其他上述的细胞外基质、其他上述的细胞外基质、GelMA及外泌体的混合比例，其他上述的3D打印支架构形，均可获得相同的技术效果。
实施例1、间充质干细胞外泌体与软骨细胞外基质凝胶混合光固化3D打印径向支架
1、采用差速离心法获得外泌体粗产物，并进行超滤，进行外泌体蛋白定量。
2、将软骨进行3次反复冻融，并使用1％Triton X-100洗脱24小时。
3、将洗脱后软骨经1％SDS洗脱24小时，并使用DNAse I处理12h小时。
4、将脱细胞软骨冻干后磨粉，并经蛋白酶消化至透亮。
5、将软骨细胞外基质、GelMA、外泌体蛋白按2％：10％：0.2％(w/v)混合，加入0.25％LAP后经3D打印平台光固化为径向支架，外泌体蛋白来源于间充质干细胞外泌体，实际加入为间充质干细胞外泌体。
实施例2、间充质干细胞外泌体与骨膜细胞外基质凝胶混合光固化3D打印同心圆支架
1、采用差速离心法获得外泌体粗产物，并进行超滤，进行外泌体蛋白定量。
2、将骨膜进行3次反复冻融，并使用1％Triton X-100洗脱24小时。
3、将洗脱后骨膜经1％SDS洗脱24小时，并使用DNAse I处理12h小时。
4、将脱细胞骨膜冻干后磨粉，并经蛋白酶消化至透亮。
5、将骨膜细胞外基质、GelMA、外泌体蛋白按2％：10％：0.2％(w/v)混合，加入0.25％LAP后经3D打印平台光固化为同心圆支架，外泌体蛋白来源于间充质干细胞外泌体，实际加入为间充质干细胞外泌体。
实施例3、间充质干细胞外泌体与肌腱细胞外基质凝胶混合光固化3D打印实心支架
1、采用差速离心法获得外泌体粗产物，并进行超滤，进行外泌体蛋白定量。
2、将骨膜进行3次反复冻融，并使用1％Triton X-100洗脱24小时。
3、将洗脱后肌腱经1％SDS洗脱24小时，并使用DNAse I处理12h小时。
4、将脱细胞肌腱冻干后磨粉，并经蛋白酶消化至透亮。
5、将肌腱细胞外基质、GelMA、外泌体蛋白按2％：10％：0.2％(w/v)混合，加入0.25％LAP后经3D打印平台光固化为实心支架。外泌体蛋白来源于间充质干细胞外泌体，实际加入为间充质干细胞外泌体。
实施例4、间充质干细胞外泌体与肌腱细胞外基质凝胶混合光固化3D打印实心支架
1、采用差速离心法获得外泌体粗产物，并进行超滤，进行外泌体蛋白定量。
2、将骨膜进行3次反复冻融，并使用1％Triton X-100洗脱24小时。
3、将洗脱后肌腱经1％SDS洗脱24小时，并使用DNAse I处理12h小时。
4、将脱细胞肌腱冻干后磨粉，并经蛋白酶消化至透亮。
5、将肌腱细胞外基质、GelMA、外泌体蛋白按10％：15％：2％(w/v)混合，加入0.25％LAP后经3D打印平台光固化为实心支架。外泌体蛋白来源于间充质干细胞外泌体，实际加入为间充质干细胞外泌体。
实施例5、间充质干细胞外泌体与肌腱细胞外基质凝胶混合光固化3D打印实心支架
1、采用差速离心法获得外泌体粗产物，并进行超滤，进行外泌体蛋白定量。
2、将骨膜进行3次反复冻融，并使用1％Triton X-100洗脱24小时。
3、将洗脱后肌腱经1％SDS洗脱24小时，并使用DNAse I处理12h小时。
4、将脱细胞肌腱冻干后磨粉，并经蛋白酶消化至透亮。
5、将肌腱细胞外基质、GelMA、外泌体蛋白按1％：8％：0.1％(w/v)混合，加入0.25％LAP后经3D打印平台光固化为实心支架。外泌体蛋白来源于间充质干细胞外泌体，实际加入为间充质干细胞外泌体。
实施例1中搭载间充质干细胞外泌体的细胞外基质3D打印支架的制备及作用机制示意图
如图1所示，将间充质干细胞外泌体、细胞外基质和GelMA混合后经3D打印成形为径向支架，并填充入兔膝软骨缺损模型中，以促进兔膝关节骨软骨修复。
实施例1中搭载间充质干细胞外泌体的细胞外基质3D打印支架的CAD模型及构型表征
1、该支架设计为径向放射状结构，并含有中空通道，打印后成体构型符合设计。
2、对支架进行扫描电镜发现支架的径向放射状结构存在，且形态较为规整。如图2.
实施例1中搭载间充质干细胞外泌体的细胞外基质3D打印支架对损伤部位的免疫调控作用
1、将实验动物分为1组(不做治疗)、2组(GelMA支架治疗)、3组(细胞外基质/GelMA凝胶支架治疗)、4组(搭载间充质干细胞外泌体的细胞外基质/GelMA凝胶支架治疗)
2、术后6周、12周可见3组、4组促愈型M2巨噬细胞marker CD163含量较多，促炎型M1巨噬细胞marker iNOS含量较少，证明细胞外基质/GelMA凝胶支架及搭载间充质干细胞外泌体的细胞外基质/GelMA凝胶支架可调控骨软骨修复免疫，将M1促炎巨噬细胞转为M2促愈巨噬细胞。如图3；
实施例1中搭载间充质干细胞外泌体的细胞外基质3D打印支架对组织损伤的修复作用的标本宏观图片及ICRS评分
1、将实验动物分为1组(不做治疗)、2组(GelMA支架治疗)、3组(细胞外基质/GelMA凝胶支架治疗)、4组(搭载间充质干细胞外泌体的细胞外基质/GelMA凝胶支架治疗)
2、术后6周、12周可见4组软骨关节面显著较1、2、3组修复完善、关节面光滑。通过ICRS评分可知，搭载间充质干细胞外泌体的细胞外基质/GelMA凝胶支架治疗效果优于其他三组，说明搭载间充质干细胞外泌体的细胞外基质/GelMA凝胶支架具有较好的骨软骨组织修复实际效果。如图4.
对实施例2、3所得的搭载间充质干细胞外泌体的细胞外基质3D打印支架分别进行CAD建模、打印及构型表征、对损伤部位免疫调控作用验证、对组织损伤的修复作用的标本宏观图片及ICRS评分，与实施例1中间充质干细胞外泌体与软骨细胞外基质凝胶混合光固化3D打印径向支架结果相似，这表明可通过其他上述的外泌体制备方案、其他上述的细胞外基质、其他上述的细胞外基质、GelMA及外泌体的混合比例，其他上述的3D打印支架构形，实现效果类似的搭载间充质干细胞外泌体的细胞外基质3D打印支架制备及其在组织修复免疫调控中的应用
由上述实施例可知，本发明提供的搭载间充质干细胞外泌体的细胞外基质3D打印支架及其在组织修复免疫调控中的应用。将间充质干细胞外泌体搭载于细胞外基质凝胶并进行3D打印，得到特定构象的组织修复支架，使其具有更强的生物降解能力和外泌体释放能力。
以上所述仅是本发明的优选实施方式，应当指出，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本技术领域的技术人员来应当理解，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围，不偏离本发明权利要求书所限定的范围。

Claims (9)

1.一种3D打印支架，其特征在于：将间充质干细胞外泌体与细胞外基质凝胶混合，并由3D打印平台光固化成形。
2.根据权利要求1所述的一种3D打印支架，其特征在于：所述3D打印平台光固化成形时采用的3D打印支架构型包括径向构型、同心圆构型和实心构型。
3.根据权利要求1所述的一种3D打印支架，其特征在于：所述的细胞外基质凝胶包括细胞外基质和甲基丙烯酰化凝胶。
4.根据权利要求1所述的一种3D打印支架，其特征在于：所述的细胞外基质包括软骨细胞外基质、骨膜细胞外基质、肌腱细胞外基质。
5.根据权利要求1所述的一种3D打印支架，其特征在于：细胞外基质凝胶混合浓度为细胞外基质1-10％，单位为w/v，甲基丙烯酰化凝胶8-15％，单位为w/v。
6.根据权利要求1所述的一种3D打印支架，其特征在于：所述的细胞外基质采用软骨细胞外基质；所述的细胞外基质浓度为1-3％，单位为w/v，甲基丙烯酰化凝胶浓度为10％，单位为w/v。
7.根据权利要求1所述的一种3D打印支架的制备方法，其特征在于，方法具体包括以下步骤：
步骤一：采用差速离心+超滤法提取间充质干细胞外泌体，
步骤二：混合1-10％细胞外基质及8-15％甲基丙烯酰化凝胶并搭载外泌体蛋白的浓度为0.1％-2.0％的间充质干细胞外泌体；三者单位皆为w/v；
步骤三：设计径向构型、同心圆构型或实心构型支架，并由3D打印平台光固化成形。
8.根据权利要求1所述的一种3D打印支架的制备方法，其特征在于：所述的细胞外基质包括软骨细胞外基质、骨膜细胞外基质、肌腱细胞外基质。
9.根据权利要求1所述的一种3D打印支架的应用，其特征在于：一种搭载间充质干细胞外泌体的细胞外基质3D打印支架在组织修复免疫调控中的应用。
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