CN107823711B - 核壳结构复合材料的制备及利用其构建组织工程微组织的方法 - Google Patents
核壳结构复合材料的制备及利用其构建组织工程微组织的方法 Download PDFInfo
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Abstract
本发明提供一种核壳结构复合材料的制备及利用其构建组织工程微组织的方法。其具体是采购新鲜宰杀的大型哺乳动物的四肢骨依次进行脱钙、脱脂、脱蛋白后粉碎得到的骨颗粒再次进行脱脂、脱钙及脱蛋白处理得到脱钙骨DBM微颗粒;将明胶粉末添加到PBS缓冲液中配置成明胶溶液,然后明胶溶液涂抹到PDMS板上的微孔中,并在每个孔中加入2‑5粒DBM颗粒,再经过交联、冲洗和冻干后得到核壳结构复合材料;使用顶针将其顶出来,然后聚集成团并置于培养皿中,将配制的细胞悬液滴加到核壳结构复合材料上进行培养得到组织工程微组织。本发明制备的核壳结构材料既具有明胶的良好生物相容性,也具有脱钙骨基质的良好力学特性,在植入体内以后,有利于血管长入。
Description
技术领域
本发明涉及生物移植及构建,具体是一种核壳结构复合材料的制备方法,以及利用该核壳结构复合材料构建骨组织工程微组织的方法。
背景技术
基于创伤或其他原因所导致的大段骨缺损是临床上的一大难题,与传统修复方法(自体骨,生物材料填充等)相比,以间充质干细胞(mesenchymal stem cell,MSC)为核心的骨组织工程移植物(tissue engineering bone graft,TEBG)具有成骨活性好,创伤小,来源广,免疫原性低等优点,是一种较有潜力的治疗方式。但是,传统组织工程的构建过程存在两方面不足:(1)种子细胞首先需要在二维条件下进行平面扩增,这与体内三维生长环境不同,一定程度可能会影响细胞的代谢及再生潜能,此外传代中的反复胰酶消化也可能破坏细胞外基质影响干细胞的分化潜能;(2)构建方式是采用自上而下(Top-down)方式将种子细胞直接种植在三维多孔材料表面,由于材料孔径孔隙不均一、营养代谢交换不畅等原因,在构建大尺寸复杂微结构组织时种子细胞往往分布不均匀,进而导致体内修复效果参差不齐。
为了解决这一问题,人们研究出了组织工程微组织(tissue engineeredmicrotissue,TEMT)策略,其主要优势是:1)利用微流体、微孔阵列、微载体等微尺度技术,构建出模块化的三维结构组织工程微单元,使得种子细胞可在与体内细胞外基质相似的水凝胶等材料上进行三维扩增,或自组装形成细胞微球,从而模拟体内三维生长环境,保护细胞干性进而提高再生效率;2)可利用自下而上(Bottom-up)的方式将微单元利用模具约束法、磁力辅助组装、静电作用力组装、细胞打印等方法进行组装,构建出细胞均一分布的复杂微结构大块组织,模块间较均匀的孔隙也可能更有利于血管长入及营养交换,提高成骨效率。但无论是水凝胶或是细胞微球,在骨再生材料所需的力学弹性及骨诱导活性等方面均有所不足。
核壳结构材料是指通过化学键或其他方式包裹形成的复合材料,具有中心的核体及包裹的壳层。因此与单一材料相比,它可以整合内外两种材料的性质,并互相补充各自的不足。核壳结构材料组成繁多,可以用任意种类材料制备,金属、绝缘、半导体等均可作为核体,目前在新能源领域,生物医药等方面有广泛的应用前景,在骨再生领域也有部分研究报道,如Su Y等使用胶原/PLLACL制备了可缓释BMP2及地塞米松的组织工程用核壳结构纳米电纺丝;Yongxiang Luo报道使用藻酸盐/纳米羟基磷灰石颗粒构建了一种可用于体内生物矿化的核壳结构材料等等。但还未见相关文献报道将其应用在组织工程微组织的构建领域。
发明内容
本发明是针对现有技术的不足提供一种基于明胶-脱钙骨基质(demineralizedbone matrix,DBM)的核壳结构复合材料的制备及利用其构建组织工程微组织的方法,所述方法构建的明胶-DBM核壳结构组织工程微组织可利用DBM的良好力学特性及骨传导特性,提高微组织的骨再生性能。
本发明提供的技术方案:所述一种核壳结构复合材料的制备方法,其特征在于具体步骤如下:
(1)DBM内核材料的制备:
a.市场采购新鲜宰杀的大型哺乳动物的四肢骨,选取粗大股骨远端及股骨头部分锯下截成3-5cm3大小块状骨;
b.将步骤a中的块状骨置于浓度为0.6mol/L的稀盐酸中浸泡至大头针可扎入骨骺端为止,并剥去软骨层暴露松质骨,进行脱钙处理;
c.按甲醇:98%丙酮=1:1比例配制脱脂液,然后将步骤b中脱钙处理后的骨块置于配制好的脱脂液中浸泡48h以上,再将骨块取出放入浓度为3%的过氧化氢溶液中浸泡1小时以上,完成骨块的脱脂处理;
d.将浓度为2mol/L的CaCl2溶液、0.5mol/L的EDTA(乙二胺四乙酸)溶液和8mol/L的LiCl溶液按照按1:1:1比例配制脱蛋白液,将步骤c中脱脂处理后的骨块浸泡到配制好的脱蛋白液中,在温度为4℃的条件下浸泡16-24小时后,取出骨块用流水清洗多次洗掉骨块上的残留液体,之后置于-70-85℃的低温条件下进行预冻12-24h;
e.将步骤d中预冻后的骨块取出,对其进行冷冻干燥和灭菌处理后,再置于-80℃条件下冷冻40-60h,然后在液氮中进行冷冻粉碎,并确保粉碎温度在0℃以下,粉碎完成后通过筛分选取粒径为200-500μm之间的骨颗粒;
f.将步骤e中得到的200-500μm之间的颗粒重复步骤b至d进行脱脂、脱钙及脱蛋白处理得到脱钙骨DBM微颗粒,并对DBM微颗粒依次进行冷冻干燥、灭菌处理在4℃无菌条件下保存;
(2)明胶溶液的配制:将明胶粉末添加到PBS(磷酸缓冲盐溶液)中配置成明胶浓度为3%-10%的明胶溶液,并在4℃条件下放置3-6小时去除泡沫;
(3)核壳结构复合材料的制备:取0.5-0.8mm厚的PDMS(聚二甲基硅氧烷)板,采用激光打孔机于板上制备若干个直径为800-2000μm的孔,将步骤(2)中配制的明胶浓度为3%-10%明胶溶液利用盖玻片均匀涂抹到PDMS板上的孔中,然后将步骤(1)中制备的DBM颗粒均匀铺至每个填充有明胶溶液的孔中,每个孔中放置2-5粒DBM颗粒,将其预冻使明胶溶液变成固体后,将含有冻干明胶和DBM颗粒的PDMS板浸泡于0.3%的戊二醛溶液,交联0.5-2小时后,清洗去掉交联液后再次预冻凝固后,置于冷冻干燥机中在-60℃条件下冷冻干燥1-3h在每个孔中形成单颗核壳结构复合材料;
(4)使用顶针将PDMS板每个孔中的核壳结构复合材料顶出来,便采用去离子水对其进行冲洗后,冷冻干燥形成粒径为800-2000μm的颗粒状核壳结构复合材料。
本发明较优的技术方案:所述步骤(1)中的大型哺乳动物为牛或羊或猪。
本发明较优的技术方案:所述步骤(1)的e步骤中的冷冻干燥时间为12-24h,冷冻干燥后采用60Co辐射或者环氧乙烷灭菌,冷冻粉碎后分别使用200μm和500μm的筛网对粉碎颗粒进行筛选选取粒径为200-500um之间的骨颗粒。
本发明较优的技术方案:所述步骤(1)的f步骤中脱钙骨DBM微颗粒冷冻干燥时间为8-12h,之后采用环氧乙烷进行灭菌处理。
本发明较优的技术方案:所述步骤(3)中将加入DBM颗粒的明胶溶液在-20℃条件下预冻16-24h后,置于冷冻干燥机中在-60℃条件下冷冻干燥1-3h,随后将含有冻干明胶和DBM颗粒的PDMS板浸泡于0.3%的戊二醛溶液,交联0.5-2小时后,清洗去掉交联液后再次预冻凝固后,置于冷冻干燥机中在-60℃条件下冷冻干燥1-3h在每个孔中形成单颗核壳结构复合材料。
本发明较优的技术方案:所述步骤(4)中的顶针是用3D打印得到的750-1300μm的光敏树脂顶针,采用顶针从PDMS板每个孔中顶出的核壳结构复合材料采用去离子水冲洗后,先在-20℃条件下再次预冻16-24h后置于冷冻干燥机在-60℃条件下冷冻干燥1-3h形成颗粒状核壳结构复合材料。
本发明提供的一种利用核壳结构复合材料构建组织工程微组织的方法,其特征在于具体步骤如下:
(1)取50粒上述颗粒状核壳结构复合材料,聚集成团并置于培养皿中,然后将间充质干细胞的P3-P5代细胞配制成(1~5)×106/ml的细胞悬液,并将配制的细胞悬液按照(50~70)μl/50粒核壳结构复合材料的量滴加到聚集成团的颗粒状核壳结构复合材料上;
(2)将步骤(1)中滴入接种细胞悬液的核壳结构复合材料转入37℃、5%二氧化碳的细胞培养箱中2h后,在培养皿中加入培养液,并将滴入接种细胞悬液的核壳结构复合材料完全淹没,在上述培养条件下培养七天,其培养过程中每2天更换一次培养液;
(3)在步骤(2)中培养完成后,将培养液更换为成骨诱导液,在同样的条件下成骨诱导培养14天后得到核壳结构的组织工程微组织,其培养过程中每3天更换一次成骨诱导液。
本发明较优的技术方案:所述步骤(1)中的间充质干细胞为骨髓干细胞、胚胎干细胞、脐带干细胞、脂肪干细胞中的任意一种;将间充质干细胞放置培养皿中普通培养至P3代后,吸去培养皿中的培养液后向培养皿中按照(1.5~2)ml/106个细胞的添加量加入胰酶,并拍打培养皿,将细胞消化下来后再按照与胰酶等体积的量加入培养液对胰酶进行中和,避免对胰酶太多会对细胞有损伤,然后将含细胞的液体转移到离心管中,在1500rbpm的条件下离心5min后吸去上清液,再采用培养液将其配制成浓度为(1~5)×106/ml细胞悬液。
本发明步骤(2)中的培养液是按照以下配方配制而成:
L-DMEM培养液 500ml Hyclone
FBS(胎牛血清) 10ml Gibco
青/链霉素 1ml Hyclone
本发明步骤(3)中的成骨诱导液是按照以下配方配制而成:
所述步骤(1)的f步骤中脱钙骨DBM微颗粒冷冻干燥时间为8-12h,之后采用环氧乙烷进行灭菌处理。(et hylene oxide,EO);
本发明的有益效果:1、本发明制备的核壳结构材料既具有明胶的良好生物相容性,也具有脱钙骨基质的良好力学特性,而且其力学特性强弱可通过调整两种成分的比例来调节;
2、本发明制备的骨组织工程微组织相比于传统的大块骨移植物而言,由于每个微组织上都载有种子细胞,最终形成的骨移植物上细胞分布更均匀;
3、本发明制备的骨组织工程微组织间有较大空隙,在植入体内以后,相比于传统的大块骨移植物更有利于血管长入。
附图说明
图1是本发明的脱钙骨基质微颗粒;
图2是本发明的核壳结构微凝胶示意图(虚线表示的是DBM内核);
图3是核壳结构微凝胶与单纯明胶微凝胶的弹性模量;
图4是种植细胞于材料上进行MTT的实验结果;
图5是种植细胞14天活死细胞染色并在共聚焦显微镜下观察的结果(绿色为活细胞,红色为死细胞)。
图6是成骨诱导14天后采用Von Kossa法检测微组织上种子细胞成骨分化活性。
具体实施方式
下面结合附图和实例对本发明进行详细的说明。
以下实施例中DBM内核材料的制备过程如下:
a.市场采购新鲜宰杀的牛四肢骨,选取粗大股骨远端及股骨头部分锯下截成3-5cm3大小块状;
b.将步骤a中的块状骨置于浓度为0.6mol/L的稀盐酸中浸泡至大头针可扎入骨骺端为止,并剥去软骨层暴露松质骨,进行脱钙处理;
c.按甲醇:98%丙酮=1:1比例配制脱脂液,然后将步骤b中脱钙处理后的骨块置于配制好的脱脂液中浸泡48h以上,再将骨块取出放入浓度为3%的过氧化氢溶液中浸泡1小时以上,完成骨块的脱脂处理;
d.将浓度为2mol/L的CaCl2溶液、0.5mol/L的EDTA溶液和8mol/L的LiCl溶液按照按1:1:1比例配制脱蛋白液,将步骤c中脱脂处理后的骨块浸泡到配制好的脱蛋白液中,在温度为4℃的条件下浸泡24小时后,取出骨块用流水清洗三次洗掉骨块上的残留液体,之后置于-80℃的低温条件下进行预冻12h;
e.将步骤d中预冻后的骨块取出,置入冻干机中冷冻干燥8h,之后采用环氧乙烷灭菌,再置于-80℃条件下冷冻48h,然后在液氮中进行冷冻粉碎12h,并确保粉碎温度在0℃以下,随后分别使用200um及500um筛网对粉碎颗粒进行筛选,选取其中颗粒直径在200-500um之间的颗粒;
f.将步骤e中得到的200-500μm之间的颗粒重复步骤b至d进行脱脂、脱钙及脱蛋白处理得到脱钙骨DBM微颗粒,并对DBM微颗粒依次进行冷冻干燥、灭菌处理在4℃无菌条件下保存;所述骨颗粒的脱脂、脱钙、脱蛋白的处理过程大致与骨块的处理过程相同,为了避免骨颗粒在处理过程中流失,其脱脂液、脱钙液和脱蛋白液的去除可以采用吸附的方式,然后将骨颗粒晾干便可。
实施例1.一种核壳结构复合材料的制备方法,其特征在于具体步骤如下:
(1)按照上述方法制备内核材料DBM颗粒;
(2)明胶溶液的配制:将明胶粉末添加到PBS缓冲液中配置成明胶浓度为6%的明胶溶液,并在4℃条件下放置3小时去除泡沫;
(3)核壳结构复合材料的制备:取0.5厚的PDMS板,采用激光打孔机于板上制备若干个直径为1500μm的孔,将步骤(2)中配制的明胶浓度为6%明胶溶液用盖玻片均匀涂布到PDMS板上的孔中,然后将步骤(1)中制备的DBM颗粒均匀铺至每个填充有明胶溶液的孔中,每个孔中放置4-5粒DBM颗粒,所述步骤(3)中将加入DBM颗粒的明胶溶液在-20℃条件下预冻24h后,置于冷冻干燥机中在-60℃条件下冷冻干燥3h,随后将含有冻干明胶和DBM颗粒的PDMS板浸泡于0.3%的戊二醛溶液,交联2小时后,清洗去掉交联液后再次预冻凝固后,置于冷冻干燥机中在-60℃条件下冷冻干燥3h在每个孔中形成单颗核壳结构复合材料;
(4)采用3D打印得到的1300μm的光敏树脂顶针从PDMS板每个孔中顶出的核壳结构复合材料,并采用去离子水冲洗后,先在-20℃条件下再次预冻16h后置于冷冻干燥机在-60℃条件下冷冻干燥2h形成直径为1500μm的颗粒状核壳结构复合材料。
实施例2.一种核壳结构复合材料的制备方法,其特征在于具体步骤如下:
(1)按照上述方法制备内核材料DBM颗粒;
(2)明胶溶液的配制:将明胶粉末添加到PBS缓冲液中配置成明胶浓度为7%的明胶溶液,并在4℃条件下放置5小时去除泡沫;
(3)核壳结构复合材料的制备:取0.7mm厚的PDMS板,采用激光打孔机于板上制备若干个直径为800μm的孔,将步骤(2)中配制的明胶浓度为7%明胶溶液用盖玻片均匀涂布到PDMS板上的孔中,然后将步骤(1)中制备的DBM颗粒均匀铺至每个填充有明胶溶液的孔中,每个孔中放置2-3粒DBM颗粒,所述步骤(3)中将加入DBM颗粒的明胶溶液在-20℃条件下预冻16h后,置于冷冻干燥机中在-60℃条件下冷冻干燥1h,随后将含有冻干明胶和DBM颗粒的PDMS板浸泡于0.3%的戊二醛溶液,交联1小时后,清洗去掉交联液后再次预冻凝固后,置于冷冻干燥机中在-60℃条件下冷冻干燥2h在每个孔中形成单颗核壳结构复合材料;
(4)采用3D打印得到的750μm的光敏树脂顶针从PDMS板每个孔中顶出的核壳结构复合材料,并采用去离子水冲洗后,先在-20℃条件下再次预冻22h后置于冷冻干燥机在-60℃条件下冷冻干燥2h形成直径为800μm的颗粒状核壳结构复合材料。
实施例3.一种核壳结构复合材料的制备方法,其特征在于具体步骤如下:
(1)按照上述方法制备内核材料DBM颗粒;
(2)明胶溶液的配制:将明胶粉末添加到PBS缓冲液中配置成明胶浓度为4%的明胶溶液,并在4℃条件下放置6小时去除泡沫;
(3)核壳结构复合材料的制备:取0.5mm厚的PDMS板,采用激光打孔机于板上制备若干个直径为1200μm的孔,将步骤(2)中配制的明胶浓度为8%明胶溶液用盖玻片均匀涂布到PDMS板上的孔中,然后将步骤(1)中制备的DBM颗粒均匀铺至每个填充有明胶溶液的孔中,每个孔中放置3-4粒DBM颗粒,所述步骤(3)中将加入DBM颗粒的明胶溶液在-20℃条件下预冻20h后,置于冷冻干燥机中在-60℃条件下冷冻干燥2h,随后将含有冻干明胶和DBM颗粒的PDMS板浸泡于0.3%的戊二醛溶液,交联2小时后,清洗去掉交联液后再次预冻凝固后,置于冷冻干燥机中在-60℃条件下冷冻干燥3h在每个孔中形成单颗核壳结构复合材料;
(4)采用3D打印得到的1000μm的光敏树脂顶针从PDMS板每个孔中顶出的核壳结构复合材料,并采用去离子水冲洗后,先在-20℃条件下再次预冻16h后置于冷冻干燥机在-60℃条件下冷冻干燥2h形成直径为1200μm的颗粒状核壳结构复合材料。
采用扫描电镜对实施例1、实施例2和实施例3的步骤(1)中制备的DBM颗粒以及三个实施例中制备的颗粒状核壳结构复合材料的表面,剖面形态,以及微凝胶弹性进行表征;其中DBM颗粒的电镜图如图1所示,可以看到在电镜下DBM颗粒是直径在200um左右的均匀块状固体。
本发明三个实施例中颗粒状核壳结构复合材料的微凝胶示意图如图2所示,电镜下可见此复合材料是以DBM为内核、明胶的微凝胶为外壳的3D复合物,虚线标示的部分即为DBM内核;
三个实施例中制备的颗粒状核壳结构复合材料的微凝胶与单纯明胶微凝胶的弹性模量的对比图如图3所示,可以看到核壳结构材料的弹性模量数值大于单纯明胶,这说明核壳结构材料的力学特性优于单纯明胶。
实施例4.利用实施例1中制备的核壳结构复合材料构建组织工程微组织,其特征在于具体步骤如下:
(1)大鼠BMSC分离培养:新生1-3天SD大鼠颈椎脱臼法处死,并置于75%酒精中浸泡10分钟,作皮肤消毒处理,消毒后戴无菌手套去皮,去皮后更换无菌手套,剔除四肢长骨,仔细剥离去除四肢长骨附着肌肉,保留完整软骨生长面,将其置于盛有培养液的培养皿中保持湿润,用消毒后的眼科剪去除长骨(股骨或胫骨)两端软骨,暴露骨髓腔孔,使用1mL注射器抽取10%FBS低糖DMEM培养液后,插入骨髓腔内冲洗骨髓腔,并收集冲洗液于培养皿中,反复重复冲洗过程3-5次后,长骨颜色转白,摇晃培养皿使皿中所得骨髓细胞分布均匀,将培养皿转入普通培养箱中静置五天,无须换液,待原代贴壁细胞长满后换液传代;
(2)当步骤(1)中的原代细胞传至P3代后,吸去培养皿中的培养液,去培养皿中的培养液后向培养皿中按照(1.5~2)ml/106细胞的添加量加入胰酶,并拍打培养皿,将细胞消化下来后再按照与胰酶等体积的量加入培养液对胰酶进行中和,然后将含细胞的液体转移到离心管中,在1500rbpm的条件下离心5min后吸去上清液,再采用生理盐水将其配制成浓度为(1~5)×106/ml细胞悬液;
(3)取50粒上述颗粒状核壳结构复合材料,聚集成团并置于培养皿中,然后将间充质干细胞的P3-P5代细胞配制成(1~5)×106/ml的细胞悬液,并将配制的细胞悬液按照(50~70)μl/50粒核壳结构复合材料的量滴加到聚集成团的颗粒状核壳结构复合材料上;
(4)将步骤(3)中滴入接种细胞悬液的核壳结构复合材料转入37℃、5%二氧化碳的细胞培养箱中2h后,在培养皿中加入培养液,并将滴入接种细胞悬液的核壳结构复合材料完全淹没,在上述培养条件下培养七天,其培养过程中每2天更换一次培养液;
(5)在步骤(4)中培养完成后,将培养液更换为成骨诱导液,在同样的条件下成骨诱导培养14天后得到核壳结构的组织工程微组织,其培养过程中每3天更换一次成骨诱导液。
其中,所述步骤(2)中的培养液是按照以下配方配制而成:
L-DMEM培养液 500ml
胎牛血清 10ml
青/链霉素 1ml
步骤(3)中的成骨诱导液是按照以下配方配制而成:
在实施例4步骤(5)的培养过程中,分别于0,3,7,10,14天取部分微室分散到96孔板,20个/孔(根据结果后面选择其中一个),每孔加培养液100ul,设置5个复孔,5个对照孔(完全培养基无细胞),然后参照试剂盒说明书进行MTT检测,其检测结果如图4所示,横坐标代表天数,纵坐标代表在酶标仪上的吸光度,吸光度越高代表材料上细胞活性越好,可以见到核壳结构材料上吸光度基本都是高于单纯明胶的,这说明核壳结构材料上细胞活性优于单纯明胶。
实验1、在接种细胞14天后,对核壳结构材料进行活死细胞染色并于共聚焦显微镜下观察以检测实施例2中构建的组织工程微组织上细胞生长状况,具体步骤如下:
(1)配置FDA(荧光素双醋酸酯)染液:5mgFDA粉末溶于1ml丙酮,用PBS(磷酸盐缓冲液)稀释至1mg/ml后避光保存,使用前用PBS缓冲液稀释500倍后方可使用;
(2)配备PI(聚酰亚胺)染液:10mg的PI粉末溶于1ml丙酮,用PBS(磷酸盐缓冲液)稀释至1mg/ml储存,使用前用PBS稀释10倍后方可使用;
(3)首先用PBS(磷酸盐缓冲液)漂洗样本三次,将其在37℃漂浮于FDA(荧光素双醋酸酯)染液30min,然后吸去FDA染液,再用PBS漂洗3次;然后将样品在20℃漂浮于PI(聚酰亚胺)染液10min,吸去PI染液,并用PBS漂洗一次;最后将样品漂浮于PBS中,用锡纸包住,放至共聚焦显微镜下观察,如图5所示;绿色荧光代表的是活细胞,红色荧光代表的是死细胞,可以观察到材料上活细胞很多而死细胞很少,而且可以观察到细胞伸张良好,这说明材料上种子细胞生长状况良好,进一步表明了核壳结构材料非常适合于种子细胞生长。
实验2:采用Von Kossa法检测成骨诱导14天后微组织上种子细胞成骨分化活性。
Von kossa:自培养箱内取出待染微组织,用PBS(磷酸盐缓冲液)洗两次,然后采用4%多聚甲醛溶液室温固定1小时,去掉固定液,并用双蒸水清洗培养皿,之后加入1mL,2%AgNO3(溶解在双蒸水中,现配现用)室温闭光,染色10分钟,曝光15分钟(不超过1小时),再用双蒸水洗两次。在空气中晾干后,显微镜下拍照,具体如图6所示,黑色部分代表的是矿化结节,可以观察到核壳结构材料上矿化结节很多,这说明核壳结构材料有助于矿化结节的沉积,也表明核壳结构材料能诱导种子细胞成骨分化。
Claims (8)
1.一种核壳结构复合材料的制备方法,其特征在于具体步骤如下:
(1)DBM内核材料的制备:
a.市场采购新鲜宰杀的大型哺乳动物的四肢骨, 选取粗大股骨远端及股骨头部分锯下截成 3-5cm3大小块状骨;
b.将步骤a中的块状骨置于浓度为0.6mol/L的稀盐酸中浸泡至大头针可扎入骨骺端为止,并剥去软骨层暴露松质骨,进行脱钙处理;
c.按甲醇:98%丙酮=1:1 比例配制脱脂液,然后将步骤b中脱钙处理后的骨块置于配制好的脱脂液中浸泡48h 以上,再将骨块取出放入浓度为3%的过氧化氢溶液中浸泡1 小时以上,完成骨块的脱脂处理;
d.将浓度为2mol/L的 CaCl2溶液、0.5mol/L 的EDTA溶液和8mol/L 的LiCl溶液按照按1:1:1 比例配制脱蛋白液,将步骤c中脱脂处理后的骨块浸泡到配制好的脱蛋白液中,在温度为4℃的条件下浸泡16-24小时后,取出骨块用流水清洗多次洗掉骨块上的残留液体,之后置于-70~-85 ℃的低温条件下进行预冻12~24h;
e.将步骤d中预冻后的骨块取出,对其进行冷冻干燥和灭菌处理后,再置于-80℃条件下冷冻40-60h,然后在液氮中进行冷冻粉碎,并确保粉碎温度在0℃以下,粉碎完成后通过筛分选取粒径为200-500μm之间的骨颗粒;
f.将步骤e中得到的200-500μm之间的颗粒重复步骤b至d进行脱钙、脱脂及脱蛋白处理得到脱钙骨 DBM 微颗粒,并对DBM 微颗粒依次进行冷冻干燥、灭菌处理在4 ℃无菌条件下保存;
(2)明胶溶液的配制:将明胶粉末添加到 PBS缓冲液中配置成明胶浓度为3%-10%的明胶溶液,并在4℃条件下放置3-6小时去除泡沫;
(3)核壳结构复合材料的制备:取0.5-0.8 mm厚的PDMS板,采用激光打孔机于板上制备若干个直径为800-2000μm 的孔,将步骤(2)中配制的明胶浓度为3%-10%明胶溶液利用盖玻片均匀涂抹到PDMS板上的孔中,然后将步骤(1)中制备的DBM颗粒均匀铺至每个填充有明胶溶液的孔中,每个孔中放置2-5粒DBM颗粒,将其在-20 ℃条件下预冻 16-24h,再置于冷冻干燥机中在-60 ℃条件下冷冻干燥1-3h使明胶溶液变成固体后,将含有冻干明胶和DBM颗粒的PDMS板浸泡于0.3%的戊二醛溶液,交联 0.5-2小时后,采用PBS 缓冲液清洗 2-4次,然后在-20 ℃条件下再次预冻 16-24h后,置于冷冻干燥机中在-60 ℃条件下冷冻干燥1-3h在每个孔中形成单颗核壳结构复合材料;
(4)使用顶针将PDMS板每个孔中的核壳结构复合材料顶出来,采用去离子水对其进行冲洗后,先在-20 ℃条件下再次预冻 16-24h后,再置于冷冻干燥机在-60 ℃条件下冷冻干燥1-3h形成粒径为800-2000μm的颗粒状核壳结构复合材料。
2.根据权利要求1所述的一种核壳结构复合材料的制备方法,其特征在于:所述步骤(1)中的大型哺乳动物为牛或羊或猪。
3.根据权利要求1所述的一种核壳结构复合材料的制备方法,其特征在于:所述步骤(1)的e步骤中的冷冻粉碎时间为12-24h,冷冻粉碎后采用60Co 辐射或者环氧乙烷灭菌,冷冻粉碎后分别使用200μm 和 500μm的筛网对粉碎颗粒进行筛选选取粒径为 200-500μm之间的骨颗粒。
4.根据权利要求1所述的一种核壳结构复合材料的制备方法,其特征在于:所述步骤(1)的f步骤中脱钙骨 DBM 微颗粒冷冻干燥时间为8-12h, 之后采用环氧乙烷进行灭菌处理。
5.一种利用权利要求1中的方法制备的核壳结构复合材料构建组织工程微组织的方法,其特征在于具体步骤如下:
(1)取50粒上述颗粒状核壳结构复合材料,聚集成团并置于培养皿中,然后将间充质干细胞的P3-P5代细胞配制成(1~5)×106个/mL的细胞悬液,并将配制的细胞悬液按照(50~70)μL /50粒核壳结构复合材料的量滴加到聚集成团的颗粒状核壳结构复合材料上;
(2)将步骤(1)中滴入接种细胞悬液的核壳结构复合材料转入37 ℃、5%二氧化碳的细胞培养箱中2h后,在培养皿中加入培养液,并将滴入接种细胞悬液的核壳结构复合材料完全淹没,在上述培养条件下培养七天,其培养过程中每2天更换一次培养液;
(3)在步骤(2)中培养完成后,将培养液更换为成骨诱导液,在同样的条件下成骨诱导培养14天后得到核壳结构的组织工程微组织,其培养过程中每3天更换一次成骨诱导液。
6.根据权利要求5所述的一种利用核壳结构复合材料构建组织工程微组织的方法,其特征在于:所述步骤(1)中的间充质干细胞为骨髓干细胞、胚胎干细胞、脐带干细胞、脂肪干细胞中的任意一种;将间充质干细胞放置培养皿中普通培养至P3代后,吸去培养皿中的培养液后向培养皿中按照(1.5~2)×106个细胞/mL的添加量加入胰酶,并拍打培养皿,将细胞消化下来后再按照与胰酶等体积的量加入培养液对胰酶进行中和,然后将含细胞的液体转移到离心管中,在1500rbpm的条件下离心5min后吸去上清液,再采用培养液将其配制成浓度为(1~5)×106个/mL细胞悬液。
7.根据权利要求5所述的一种利用核壳结构复合材料构建组织工程微组织的方法,其特征在于所述步骤(2)中的培养液是按照以下配方配制而成:
L-DMEM培养液 500mL
胎牛血清 10mL
青/链霉素 1mL。
8.根据权利要求5所述的一种利用核壳结构复合材料构建组织工程微组织的方法,其特征在于所述步骤(3)中的成骨诱导液是按照以下配方配制而成:L-DMEM培养液500mL,胎牛血清10mL,青/链霉素1mL,地塞米松0.022mg,β-甘油磷酸钠1.188g,L-抗坏血酸-2-磷酸酯镁盐7.96mg。
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