CN118240753A - 一种构建类骨组织的方法 - Google Patents
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Abstract
本发明公开了一种构建类骨组织的方法,提取自体骨髓或者脂肪间充质干细胞进行体外培养扩增;将间充质干细胞与纳米羟基磷灰石混合摇匀,制作细胞‑纳米羟基磷灰石混悬液,后滴加于低粘附培养板中离心以促进细胞间以及细胞‑材料间的接触,制作得到间充质干细胞微球;后将间充质干细胞微球置于成骨分化培养基中进行分化培养,获得通过间充质干细胞自组装和成骨分化形成的具备骨组织形态和功能的类骨组织,本申请在类骨组织中均匀添加具备成骨诱导活性的纳米材料能够在不影响其形态和结构的同时持续为其提供成骨诱导分化信号,同时纳米材料还可以作为药物载体进一步负载成骨诱导药物通过药物缓释来持续增强其成骨分化能力。
Description
技术领域
本发明涉及构建类骨组织的方法,用于骨缺损修复、药物筛选等领域。
背景技术
近年来,间充质干细胞作为一种器官再生和药物开发常用的研究工具得到了广泛的发展和应用;传统二维培养的间充质干细胞因为缺乏必要的细胞-细胞联系和细胞-基质联系会逐渐丧失干细胞特性,不能准确模拟组织和器官的发生发展过程;3D类器官或者类器官样结构作为一种新型的研究工具,能够保留细胞间以及细胞-基质间的联系,模拟器官组织的结构和功能,在再生医学、发育生物学以及药物筛选研究中得到了越来越多的重视。
先前研究已经利用间充质干细胞构建了3D类骨组织,发现3D类骨组织与2D单层细胞相比,在细胞增殖、成骨分化以及骨缺损修复能力等方面具备更大的优势;对于类骨组织的构建而言,快速、精准和功能化十分重要;但目前利用间充质干细胞构建类骨组织还存在诸多问题:例如间充质干细胞的不定向分化性、很难在体外分化成为成熟的骨细胞、类骨组织中组分的可控性差、以及建模周期长(诱导周期需要3-4周)。
发明内容
本发明所要解决的技术问题是提供一种构建类骨组织的方法,以解决现有技术中导致的上述多项缺陷。
为实现上述目的,本发明提供以下的技术方案:一种构建类骨组织的方法,包括以下步骤:
(1)提取自体骨髓或者脂肪间充质干细胞进行体外培养扩增,获得大量间充质干细胞;
(2)将间充质干细胞与纳米羟基磷灰石混合摇匀,制作细胞-纳米羟基磷灰石混悬液,后放入低粘附细胞培养板中离心,以促进细胞间以及细胞-材料间的接触,获得均匀掺杂纳米羟基磷灰石的间充质干细胞微球;
(3)将间充质干细胞微球置于成骨诱导分化培养基中进行分化培养,获得通过间充质干细胞自组装和成骨分化形成的具备骨组织形态和功能的类骨组织;
(4)在7天和14天,分别通过组织染色和成骨蛋白表达评估类骨组织的成骨分化能力和成骨分化阶段。
优选的:步骤(1)中间充质干细胞来源于自体骨髓或者脂肪组织,体外扩增培养的时间为7-14天。
优选的:步骤(2)中采用低粘附细胞培养板培养24小时,通过减少细胞在板底的附着来形成悬浮的间充质干细胞微球。
优选的:步骤(2)中离心速度为700-1500rpm。
优选的:步骤(2)中纳米羟基磷灰石的浓度为20-40μg/ml。
优选的:步骤(3)中成骨诱导分化时间为7-14天。
优选的:步骤(4)中Runx2、Osterix蛋白为早期成骨标记,OCN、Sclerostin蛋白为晚期成骨标记。
本申请的有益效果在于:
其一,制备方法相对简便,可以在短时间内大量获取不同尺寸的类骨组织,经济廉价;其二,该产品引入纳米羟基磷灰石这种批准用于临床治疗的材料,类骨组织构建效果更稳定,临床转化更为便捷;其三,该方法构建的类骨组织,与单纯间充质干细胞构建的类骨组织相比,干细胞成骨分化更为成熟,细胞外基质合成和矿化更符合软骨内成骨过程,结构和组分更接近于正常骨组织。
说明书附图
图1是本申请类骨组织构建流程图。
图2是本申请构建类骨组织的形态及分子特征的扫描电镜照片。
图3是本申请构建类骨组织的形态及分子特征的免疫荧光染色显示1周和2周成骨诱导分化后,早期成骨指标(Runx2蛋白、Osterix蛋白)和晚期成骨指标(OCN骨钙蛋白、Sclerostin骨硬化蛋白)等在类骨组织中的表达。
图4是本申请构建类骨组织的形态及分子特征的茜素红染色显示类骨组织中矿盐的沉积。
图5是本申请构建类骨组织的形态及分子特征的ALP染色显示类骨组织中碱性磷酸酶的表达。
图6是本申请构建类骨组织的形态及分子特征的番红固绿染色提示类骨组织中软骨内成骨分化过程。
具体实施方式
下面详细说明本发明的实施例一:
一种构建类骨组织的方法,包括以下步骤:
(1)间充质干细胞的提取和培养:通过骨髓穿刺的方式抽取骨髓血,采用贴壁培养的方式获取骨髓间充质干细胞,在完全培养基中培养扩增7-14天,获得大量间充质干细胞;
(2)间充质干细胞团的构建:将贴壁的间充质干细胞消化,制作浓度为5x105个细胞/mL的细胞悬液,加入浓度为30μg/ml的纳米羟基磷灰石混悬液,然后通过添加完全培养基将细胞-纳米羟基磷灰石混悬液中细胞浓度调整为4x105个细胞/mL;
然后将50μL间充质干细胞-纳米羟基磷灰石混悬液滴加于康宁384孔低粘附培养板中,后以1500rpm的速度在离心机中离心5分钟,最后放置于培养箱中培养。
(3)间充质干细胞团的成骨分化:24小时后,将低粘附培养板中形成的细胞团转移至低粘附培养皿中,将培养基更换为成骨诱导培养基,继续培养7-14天;
(4)成骨能力测定:在7天和14天时,分别通过组织染色(茜素红染色、ALP染色、番红固绿染色)和成骨蛋白表达(Runx2、OCN、Osterix、Sclerostin)评估类骨组织的成骨分化能力和分化阶段。
实施例二:
一种构建类骨组织的方法,包括以下步骤:
(1)脂肪间充质干细胞的提取和培养:收集患者脂肪组织,用胰蛋白酶消化,得到单细胞混悬液,通过贴壁培养的方式获取脂肪间充质干细胞,在完全培养基中培养扩增7-14天,获得大量间充质干细胞;
(2)间充质干细胞团的构建:将贴壁的脂肪间充质干细胞消化,制作浓度为4x106个细胞的细胞悬液,加入浓度为20μg/mL纳米羟基磷灰石混悬液,然后通过添加单纯培养基调整细胞-纳米羟基磷灰石混悬液中细胞浓度为3x106个细胞/mL;
最后将1ml间充质干细胞-纳米羟基磷灰石混悬液滴加于AggreWellTM800低粘附培养板中,后以1500rpm的速度离心5分钟,放置于培养箱中培养。
(3)间充质干细胞团的成骨分化:24小时后,将低粘附培养板中形成的细胞团转移至低粘附培养皿中,将培养基更换为成骨诱导分化培养基,继续培养7-14天;
(4)成骨能力测定:在7天和14天时,分别通过组织染色(茜素红染色、ALP染色、番红固绿染色)和成骨蛋白表达(Runx2、OCN、Osterix、Sclerostin)评估类骨组织的成骨分化能力和分化阶段。
纳米羟基磷灰石是骨组织中主要的无机成分,具有良好的成骨诱导活性,能够促进间充质干细胞向成骨方向分化;本发明基于以上特点,结合纳米羟基磷灰石生物相容性良好,已经被批准用于临床治疗,通过实现纳米羟基磷灰石在间充质干细胞球内均匀分布,在不影响间充质干细胞球结构和形态的前提下增强类骨组织的成骨分化能力。
以上所述的仅是本发明的优选实施方式,应当指出,对于本领域的普通技术人员来说,在不脱离本发明创造构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (7)
1.一种构建类骨组织的方法,其特征在于,包括以下步骤:
(1)提取自体骨髓或者脂肪间充质干细胞进行体外培养扩增,获得大量间充质干细胞;
(2)将间充质干细胞与纳米羟基磷灰石混合摇匀,制作细胞-纳米羟基磷灰石混悬液,后放入低粘附细胞培养板中离心,以促进细胞间以及细胞-材料间的接触,获得均匀掺杂纳米羟基磷灰石的间充质干细胞微球;
(3)将间充质干细胞微球置于成骨诱导分化培养基中进行分化培养,获得通过间充质干细胞自组装和成骨分化形成的具备骨组织形态和功能的类骨组织;
(4)在7天和14天,分别通过组织染色和成骨蛋白表达评估类骨组织的成骨分化能力和成骨分化阶段。
2.根据权利要求1所述的一种构建类骨组织的方法,其特征在于,步骤(1)中间充质干细胞来源于自体骨髓或者脂肪组织,体外扩增培养的时间为7-14天。
3.根据权利要求1所述的一种构建类骨组织的方法,其特征在于,步骤(2)中采用低粘附细胞培养板培养24小时,通过减少细胞在板底的附着来形成悬浮的间充质干细胞微球。
4.根据权利要求1所述的一种构建类骨组织的方法,其特征在于,步骤(2)中离心速度为700-1500rpm。
5.根据权利要求1所述的一种构建类骨组织的方法,其特征在于,步骤(2)中纳米羟基磷灰石的浓度为20-40μg/ml。
6.根据权利要求1所述的一种构建类骨组织的方法,其特征在于,步骤(3)中成骨诱导分化时间为7-14天。
7.根据权利要求1所述的一种构建类骨组织的方法,其特征在于,步骤(4)中Runx2、Osterix蛋白为早期成骨标记,OCN、Sclerostin蛋白为晚期成骨标记。
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