CN114870088B - 一种Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架的制备方法与应用 - Google Patents
一种Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架的制备方法与应用 Download PDFInfo
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Abstract
本发明涉及骨科移植物医用材料技术领域,采用冻融循环和脱氧核糖核酸酶消化的脱细胞方法获得Wnt信号激活的骨细胞脱细胞基质及其表衬的硬材料支架。该技术去除了所有细胞器和95%的DNA,保留胶原、糖胺多糖等细胞基质成分,获得的脱细胞基质保持了完整的细胞基膜,以及密集的椭圆形和蜂窝状结构。该脱细胞基质可以诱导应力纤维产生,利于细胞粘附、铺展、成骨分化和矿化;并增加BMSC中RANKL和MCSF、Vegfa和Angpt1以及Ngf的表达,植入后生成大量与宿主骨相似并整合良好的新骨和致密排列的I型胶原,诱导破骨细胞、血管和神经生成,实现骨缺损的快速修复,有望达到自体骨移植的效果,适合转化应用。
Description
技术领域
本发明涉及骨科移植物医用材料技术领域,尤其是涉及一种Wnt信号激活骨细胞脱细胞基质材料以及该脱细胞基质表衬的骨修复支架的制备方法与应用。
背景技术
临界骨缺损的再生修复是骨科临床面临的主要挑战,自体骨移植因其良好的成骨性能被认为是临床治疗骨缺损的金标准,但存在来源有限、二次手术和引起供体部位感染的问题。冻干骨和异体骨被用于代替自体骨治疗骨缺损,但其价格昂贵,并存在交叉传染、传播疾病的风险。因此,金属、生物陶瓷和合成材料被开发应用于骨缺损修复,但仍存在生物活性不足、翻修等困扰。3D生物打印能够仿生模拟在体骨发育微环境,构建骨修复支架可显著提高移植物的生物活性和促成骨性能,但活细胞会引起一系列伦理问题,向临床转化仍极具挑战。因此,从仿生成骨微环境角度研发具备良好促成骨生物活性的无细胞新型骨科移植物是当前骨组织工程的研究热点。
脱细胞基质(decellularized matrix,DM)最大去除细胞中DNA等具有免疫原性的成分,同时保留原细胞基质成分蛋白质和多糖组成的精确有序的网络结构,其三维微结构可仿生模拟机体内最接近细胞生长的微环境,且富含的各种活性分子为各种细胞活动提供基础,因此是一种理想的组织工程材料。组织源性脱细胞基质如脱细胞异体骨具有天然骨组织的结构和生物活性,有效促进骨再生,已应用于组织工程和再生医学,但其来源有限、成本昂贵,仍具有潜在的病原体感染的可能性,并可发生退化,引起炎症、免疫反应等,有一定的局限性[Biotechnol Adv,2014,32,462]。与此相反,细胞来源的脱细胞基质是将特定细胞在无菌条件下体外培养、使其分泌大量细胞基质,再脱去细胞而获得,它几乎不存在潜在的病原体感染,并且可以构建高度特异性的细胞基质。不同组织细胞的脱细胞基质具有该组织特异的微环境,对该组织的再生起着重要作用[Bioactive Materials,2022,10,15]。Kohn等人发现牛原代软骨细胞和成骨细胞的脱细胞基质分别诱导人间充质干细胞(hMSCs)向软骨细胞和成骨细胞分化[J Mater Sci:Mater Med,2017,28,100]。Pati等人发现成骨诱导培养的MSCs矿化后的脱细胞基质修饰的PCL/PLGA/β-磷酸三钙支架显著增强MSCs的粘附、增殖、分化与矿化,在植入大鼠顶骨临界骨缺损模型8周后形成的骨为对照组支架的两倍[Biomaterials,2015,37,230]。但是,干细胞用于临床还存在亟待解决的问题,比如来源就限制了其转化应用[Transplantation,2007,83,1019],而且,其在体外扩增最大24-40次后进入老化或生长停滞期,不利于应用[Tissue Eng,2007,13,1059]。此外,干细胞需要外界诱导产生成骨分化后才具备成骨微环境,而骨类细胞(前成骨细胞、成骨细胞、骨细胞等)的脱细胞基质可能更具备成骨特异性诱导性能[J Mater Sci:Mater Med,2017,28,100]。Aldemir Dikici等人采用骨发育终端的骨细胞(MLO-A5)的脱细胞基质修饰3D打印的PCL支架,发现其具有促进骨形成和血管生成的功能[ACS Appl Mater Interfaces,2020,12,12510]。但也需要评定其植入后的功能。综上所述,骨组织中细胞的特异性脱细胞基质可以指导骨组织再生,特别是小鼠骨细胞样细胞脱细胞基质仿生构建的天然骨组织微环境具备成骨和成血管性能,从而提示我们从骨发育的角度考虑筛选合适的细胞类型进行脱细胞处理以获得保留骨组织特异性生化成分和超微结构的脱细胞基质,能够更好的模拟体内骨组织发育生物微环境,进而高效高质量的促进骨再生完成骨修复。
我们团队的前期研究发现激活小鼠骨细胞的经典Wnt信号,小鼠骨量增加,骨形成速度加快,还有意想不到的骨吸收[Proc Natl Acad Sci U S A.,2015,112,E478]。基于这项创新的研究成果,我们构建了基于Wnt信号激活骨细胞生物微环境骨修复功能模块,体外培养结果提示该微环境显著促进功能模块中BMSCs的增殖、成骨分化和矿化,体内骨缺损修复实验显示负载生物微环境的功能模块能够快速修复骨缺损,并促进血管及神经形成。这些发现证明骨细胞是骨组织中经典Wnt/β-catenin信号合成代谢作用的中心靶细胞,并提示Wnt信号激活骨细胞可能是制备具有促进成骨的生物微环境的脱细胞基质的合适细胞来源。
鉴于此,特提出本发明。
发明内容
本发明立足于扎实的前期研究基础,目的在于提供一种Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架的制备方法及应用,Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架有效去除DNA,保留细胞基质成分,营造了无细胞的仿生成骨微环境,具有良好的生物相容性,利于细胞黏附、生长、增殖、成骨分化及生物矿化;植入后,该支架促进骨缺损部位的成骨分化、破骨生成、血管和神经形成,实现骨缺损的快速修复,特别适于制备硬组织替换或修复材料产品。
本发明提供的技术方案如下:
在一个方面,本发明提供了一种基于Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架的制备方法,采用三次冻融循环和脱氧核糖核酸酶消化联用的方式对接种了Wnt信号激活骨细胞的3D打印硬材料支架进行脱细胞处理,得到Wnt信号激活骨细胞脱细胞基质表衬的骨修复支架;包括在3D打印的硬材料支架上接种培养Wnt信号激活骨细胞,之后通过冻融循环和DNA酶处理进行脱细胞处理,得到Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架。
本发明脱细胞基质为细胞来源的脱细胞基质,去除了细胞及DNA等引起免疫排斥的成分,保留细胞基质中胶原、糖胺多糖等成分。所述的脱细胞基质保留原细胞基质的胶原及糖胺多糖70%以上,DNA去除90%以上。
在一个实施方案中,所述细胞为骨细胞Wnt信号激活小鼠(daβcatOt小鼠)的骨细胞(daβc-Ot)。经典Wnt信号在小鼠骨细胞中激活后诱导骨合成代谢而不影响小鼠存活。在一个对比方案中,所述细胞选自C57BL/6小鼠的骨髓基质细胞(BMSCs)、daβcatOt小鼠同笼对照野生型小鼠的骨细胞(WT-Ot)的任一种。
在一个实施方案中,所述的3D打印硬材料支架为天然或合成的生物相容性良好的硬材料通过3D打印的方式加工制备的三维多孔支架;支架的材料束直径为300μm~400μm,孔径为300μm~600μm,孔隙率为20%~80%,且孔隙结构均匀。
在一个实施方案中,所述3D打印硬材料支架的材料选自天然或合成的生物相容性良好的硬材料,包括壳聚糖、纤维素、聚己内酯、聚乳酸、聚乳酸-羟基乙酸共聚物、聚乙醇酸中的一种或多种或其共聚物;优选地,所述材料为聚己内酯,聚己内酯降解速度较其他材料低,适合骨移植物这种长期植入物。
在一个实施方案中,所述聚己内酯的分子量在40000~100000之间。
在一个实施方案中,3D打印聚己内酯支架孔径约300μm,这个尺寸利于骨和血管形成。
在一个实施方案中,所述Wnt信号激活骨细胞的Wnt信号激活方式包括基因编辑、生物因子、多肽、小分子药物激活中的任一种。
在一个实施方案中,向所述支架上接种10μL-50μL细胞密度为2×106~2×108个/mL的骨细胞悬液;优选地,接种10μL的细胞密度为2×107个/mL的细胞悬液。
在一个实施方案中,在所述支架上接种细胞后培养1~16天进行脱细胞;优选地,接种细胞后培养14天进行脱细胞。
在一个实施方案中,所述脱氧核糖核酸酶(DNA酶)包括重组DNA酶;优选DNA酶I;优选地,所述DNA酶的浓度为0.05~0.8mg/mL;优选0.2mg/mL。
在一个实施方案中,所述冻融循环包括在液氮中冷冻5~60min,然后于35℃~37℃水浴中5~60min,之后用无菌PBS浸洗去除细胞碎片;优选地,所述冻融循环进行3~4次。
在一个具体的实施方案中,本发明的制备方法的步骤如下:
(1)3D打印聚己内酯支架:通过3D Bio-maker软件设置打印聚己内酯支架,并将支架进行灭菌处理、浸洗以及晾干;打印的聚己内酯支架材料束宽约300μm,孔径约400μm,规格15mm×15mm×1mm,将其修剪为5mm×5mm×1mm的规格备用;
(2)在聚己内酯支架上接种细胞:用血清浸润支架并晾干,将细胞消化后配制成2×107个/mL的细胞悬液,每个支架接种10μL细胞悬液;将支架放入孵箱2h让细胞黏附;加入完全培养基(含10%胎牛血清和1%青霉素-链霉素的α-MEM),隔天换液;
(3)冻融循环-DNA酶消化联用进行脱细胞处理:
a.弃去培养基,无菌PBS浸洗2次;
b.支架转移至15mL离心管,加入5mL无菌PBS;
c.将离心管放入液氮10min,之后放入37℃水浴锅10min,无菌PBS浸洗3次(3min/次)去除细胞碎片;该步骤重复3次;
d.加入DNA酶I于37℃孵育1h;
e.无菌PBS浸洗3次(3min/次);置于4℃PBS中保存。
在另一个方面,本发明还提供了所述的制备方法制备得到的Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架,在体外能够促进骨髓基质细胞的成骨分化及生物矿化。
在另一个方面,本发明还提供了Wnt信号激活骨细胞脱细胞基质表衬的骨修复支架制备骨组织替换或修复材料产品中应用。在所述骨修复支架上体外培养细胞时具有高存活率和增殖活性,成功实现成骨分化和矿化;植入小鼠临界原位骨缺损部位后具有骨再生、骨吸收、血管及神经生成功能。
有益效果:
(1)本发明在3D打印的硬材料支架上接种骨细胞后进行冻融循环和DNA酶消化联用的脱细胞处理,去除了90%以上DNA,降低其抗原性,并且保留70%以上细胞基质成分,具有良好的生物相容性;特别地,Wnt信号激活骨细胞脱细胞基质支持骨髓间充质干细胞(BMSCs)黏附和增殖,能够诱导BMSCs成骨分化,而且3D打印支架的孔隙利于骨和血管形成,促进体内骨缺损的快速再生修复;
(2)本发明的制备方法,取材简单,操作方便,成本低,不含活细胞,可用于骨缺损修复治疗,具有良好的临床应用前景;特别地,Wnt信号激活骨细胞脱细胞基质表衬的骨修复支架保留骨细胞基质成分,具有良好的生物相容性,利于细胞黏附、生长、增殖、成骨分化和生物矿化,能够促进原位骨缺损的修复即成骨分化、破骨生成、血管及神经形成,加速骨修复,具有良好的临床应用前景。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明3D打印的PCL支架以及脱细胞前后扫描电镜图;
图2为实施例1、对比例1和对比例2脱细胞前后细胞核染色及DNA、胶原和糖胺多糖定量;
图3为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架细胞接种BMSCs后细胞存活情况(活死染色)、细胞铺展情况(细胞骨架染色)及增殖活性(CCK-8测定);
图4为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架接种BMSCs后成骨分化情况(碱性磷酸酶染色及生化定量)、成骨标志基因表达情况(实时荧光定量PCR)及生物矿化情况(茜素红染色);
图5为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入C57BL/6小鼠顶骨临界骨缺损的修复情况(PET-CT检测);
图6为不同脱细胞方法DNA残留情况的对比结果图;
图7为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入小鼠顶骨临界骨缺损的骨修复情况(HE染色);
图8为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入小鼠顶骨临界骨缺损的血管生成情况(HE染色)及BMSCs血管相关基因表达情况;
图9为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入小鼠顶骨临界骨缺损的胶原形成情况(天狼星红染色);
图10为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入小鼠顶骨临界骨缺损的破骨细胞形成情况(TRAP染色);
图11为实施例1-5细胞在支架上培养后蛋白总量对比(BCA总蛋白测定);
图12为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入小鼠顶骨临界骨缺损的H型血管形成情况(H型血管标志物CD31、Endomucin免疫荧光染色);
图13为实施例1、对比例1和对比例2脱细胞基质表衬的3D打印PCL支架植入小鼠顶骨临界骨缺损的神经形成情况(神经标志物β3-Tubulin、NeuN免疫荧光染色)及BMSCs神经基因表达情况。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种Wnt信号激活骨细胞脱细胞基质表衬的骨修复支架的制备方法,包括以下步骤:
a.3D打印PCL支架:打开3D Bio-maker软件,根据程序设置打印PCL支架,将其修剪为5mm×5mm×1mm的规格;之后将支架放于75%酒精灭菌1h,无菌PBS浸洗5次(3min/次),将支架放于超净台晾干,紫外照射支架正反面各20min灭菌。
b.提取骨细胞Wnt激活小鼠的骨细胞(daβc-Ot):小鼠乙醚麻醉后脱颈处死,无菌条件下解剖取下双侧股骨、胫骨,去除软组织后取骨干,将骨干剪为1~2mm大小的碎片,加入1mg/mL I型胶原酶溶液于37℃摇床消化15min,之后于冰上完全培养基中静置5min,此过程重复3次;I型胶原酶(15min)-4mM EDTA(5min)于37℃交替处理3次,冲洗骨碎片后进行培养,每两天换液,待骨细胞长满培养皿70%左右使用并移走骨碎片继续培养。
c.3D打印PCL支架上接种Wnt激活骨细胞(daβc-Ot):在支架上滴加10μL血清浸润支架并晾干;之后消化骨细胞并计数,配制成2×107/mL的细胞悬液,每个支架接种10μL细胞悬液,将支架放入孵箱2h让细胞黏附,之后加入完全培养基(10%胎牛血清和1%青霉素-链霉素α-MEM)进行培养,隔天换液。
d.冻融循环/DNA酶脱细胞:培养14天后弃去培养基,无菌PBS浸洗2次;将支架转移至15mL离心管,加入5mL无菌PBS。进行冻融循环脱细胞处理,具体步骤如下,将离心管放入液氮10min,之后放入37℃水浴锅10min,无菌PBS浸洗支架3次(3min/次)去除细胞碎片(重复3次),加入0.2mg/mL的DNA酶于37℃孵育1h;无菌PBS浸洗3次(3min/次),置于4℃无菌PBS中保存。
e.使用时,将c步骤制得的支架放于超净台晾干,紫外照射正反面各10min。
实施例2
本实施例与实施例1不同在于,本实施例中细胞在支架上培养1天。
实施例3
本实施例与实施例1不同在于,本实施例中细胞在支架上培养4天。
实施例4
本实施例与实施例1不同在于,本实施例中细胞在支架上培养7天。
实施例5
本实施例与实施例1不同在于,本实施例中细胞在支架上培养10天。
对比例1
一种小鼠骨髓基质细胞(BMSCs)来源的脱细胞基质表衬的3D打印PCL支架的制备方法,包括以下步骤:
a.3D打印PCL支架:打开3D Bio-maker软件,根据程序设置打印PCL支架,将其修剪为5mm×5mm×1mm的规格;之后将支架放于75%酒精灭菌1h,无菌PBS浸洗5次(3min/次),将支架放于超净台晾干,紫外照射支架正反面各20min灭菌。
b.提取小鼠BMSCs:小鼠乙醚麻醉后脱颈处死,无菌条件下解剖取下双侧股骨、胫骨,去除软组织后取骨干,冲洗骨髓腔,吸取所冲出的骨髓于15mL离心管,吹打均匀后1000r/min离心5min,弃去上清进行培养,获得的细胞每两天换液,待细胞长满培养皿80%-90%后传代,培养至第3代的细胞用于后续实验。
c.在3D打印PCL支架上接种BMSCs:在支架上滴加10μL血清浸润支架并晾干;之后消化BMSCs并计数,配制成2×107/mL的细胞悬液,每个支架接种10μL细胞悬液,将支架放入孵箱2h让细胞黏附,之后加入完全培养基进行培养,隔天换液。
d.冻融循环/DNA酶脱细胞:培养14天后弃去培养基,无菌PBS浸洗2次;将支架转移至15mL离心管,加入5mL无菌PBS。进行冻融循环脱细胞处理,具体步骤如下:将离心管放入液氮10min,之后放入37℃水浴锅10min,无菌PBS浸洗支架3次(3min/次)去除细胞碎片(重复3次),加入0.2mg/mL的DNA酶于37℃孵育1h;无菌PBS浸洗3次(3min/次),置于4℃无菌PBS中保存。
e.使用时,将c步骤制得的支架放于超净台晾干,紫外照射正反面各10min。
对比例2
一种骨细胞来源的脱细胞基质表衬的3D打印PCL支架的制备方法,包括以下步骤:
a.3D打印PCL支架:打开3D Bio-maker软件,根据程序设置打印PCL支架,将其修剪为5mm×5mm×1mm的规格;之后将支架放于75%酒精灭菌1h,无菌PBS浸洗5次(3min/次),将支架放于超净台晾干,紫外照射支架正反面各20min灭菌。
b.提取同笼对照小鼠骨细胞(WT-Ot):小鼠乙醚麻醉后脱颈处死,无菌条件下解剖取下双侧股骨、胫骨,去除软组织后取骨干,反复冲洗去除骨髓,将骨干剪为1~2mm大小的碎片,加入1mg/mL I型胶原酶溶液于37℃摇床消化15min,之后于冰上完全培养基中静置5min,此过程重复3次;I型胶原酶(15min)-4mM EDTA(5min)于37℃交替处理3次,冲洗骨碎片后进行培养,每两天换液,待骨细胞长满培养皿70%左右使用并移走骨碎片继续培养。
c.在3D打印PCL支架上接种骨细胞(WT-Ot):在支架上滴加10μL血清浸润支架并晾干;之后消化骨细胞并计数,配制成2×107/mL的细胞悬液,每个支架接种10μL细胞悬液,将支架放入孵箱2h让细胞黏附,之后加入完全培养基(10%胎牛血清和1%青霉素-链霉素α-MEM)进行培养,隔天换液。
d.冻融循环/DNA酶脱细胞:培养14天后弃去培养基,无菌PBS浸洗2次;将支架转移至15mL离心管,加入5mL无菌PBS。进行冻融循环脱细胞处理,具体步骤如下:将离心管放入液氮10min,之后放入37℃水浴锅10min,无菌PBS浸洗支架3次(3min/次)去除细胞碎片(重复3次),加入0.2mg/mL的DNA酶于37℃孵育1h;无菌PBS浸洗3次(3min/次),置于4℃无菌PBS中保存。
e.使用时,将c步骤制得的支架放于超净台晾干,紫外照射正反面各10min。
性能检测及功能测试
1.3D打印支架接种细胞后培养时间的确定
在3D打印的PCL支架接种细胞1、4、7、10、14天分别收样进行BCA总蛋白测定。根据碧云天BCA总蛋白定量检测试剂盒进行检测。图11蛋白定量结果所示,第14天支架上积累的蛋白量显著高于1、4、7天(P<0.05),比第10天稍高但无显著性差异,故选择第14天进行脱细胞处理。
2.细胞来源的脱细胞基质表衬的3D打印PCL支架表征
对脱细胞处理前后的脱细胞基质表衬的PCL支架进行扫描电镜(SEM)表征:将支架样品放入4%多聚甲醛4℃固定过夜,PBS浸洗3次后进行梯度脱水,乙醇浓度依次为30%、50%、70%、80%、90%和100%,每个梯度浸泡15min;放入冷冻干燥机内冷冻干燥12h;将冻干后样品通过溅射镀膜在样品表面喷金60s,在电子加速电压为10KV下,扫描电镜观察。图1中a所示,3D打印PCL支架规格为5mm×5mm×1mm。图1中b和图1中c分别为脱细胞处理前后支架的SEM图像。图1中b脱细胞前后扫描电镜图显示,脱细胞前细胞及其分泌的细胞基质基本完全覆盖支架,图1中c显示脱细胞后支架上基本未见细胞形态,有大量脱细胞基质黏附。
3.脱细胞基质表衬的3D打印PCL支架的脱细胞效果评估
3.1 Hochest 33258细胞核染色
脱细胞处理前后的支架用PBS浸洗3次,加入200μL染色液,15min后弃去染色液,PBS浸洗3次后荧光显微镜下观察拍照。图2中a的上图和下图分别为脱细胞前和脱细胞后细胞核的染色的情况。
3.2 DNA提取、纯化及检测
每个支架用PBS轻柔浸洗3次,吸弃PBS,尽量吸干;加入300μL胰酶消化,置于37℃水浴10min,加入300μL PBS终止消化,吹打支架,将细胞吹打下来;800r/min离心5min,弃去上清和支架;每个样本加入200μL消化缓冲液和1μL蛋白酶K,56℃水浴3h,加入200μL 100%异丙醇,快速拨弹管底,室温12000r/min离心5min,吸弃上清。加入150μL 70%乙醇轻柔颠倒润洗,室温13000r/min离心2min,吸弃上清。超净工作台中风干5~10min,加入100μL TE缓冲液。轻弹管底沉淀充分溶解后置-20℃保存。
消化缓冲液的配制:2.5mL 2M Tris/HCl(pH8.8)+0.5mL 0.5M EDTA+1mL 10%SDS+2mL 5M NaCl+44mL ddH2O;
蛋白酶K的配制:储存浓度为10mg/mL,100mg+10mL MiniQ分装1mL/EP管-20℃保存;
EB缓冲液的配制:10mM Tris/HCl pH 8(母液为1M Tris/HCl pH 8高压后室温保存)。
DNA定量选用Invitrogen的Quant-iTdsDNA Assay Kit。在试管中,用1×TE缓冲液(试剂盒所带,保存DNA)将实验DNA溶液稀释至100μL的最终体积。
向每个样品中添加100μL Quant-iT 试剂的水溶液(2×PicoGreen试剂的工作溶液)。在避光条件下,于室温下孵育2~5分钟。孵育后,96孔板每孔加200μL溶液。使用分光荧光计或荧光酶标仪测定标准荧光素波长(激发光480nm,发射光520nm)。所有样品保持恒定的荧光测量时间。
标准曲线的制备:在TE中准备2μg/mL dsDNA储备溶液。根据光程为1cm的比色杯中260nm(A260)的吸光度确定DNA浓度;A260为0.04对应于2μg/mL dsDNA溶液。在Quant-iT试剂盒中以100μg/mL提供的lambda DNA标准品可以在TE中简单地稀释50倍,制成2μg/mL的工作溶液。创建从1ng/mL到1μg/mL的五点(0ng/mL、1ng/mL、10ng/mL、100ng/mL、1μg/mL)标准曲线,充分混合并在避光条件下于室温下孵育2~5分钟。96孔板每孔加200μL溶液。使用分光荧光计或荧光酶标仪测定标准荧光素波长(激发光480nm,发射光520nm)。
3.3糖胺多糖(GAGs)检测
根据GENMED细胞糖胺多糖(GAG)总含量阿尔新蓝(alcian blue)比色法定量检测试剂盒进行检测。将培养支架的孔板中的培养液小心抽去,无菌PBS浸洗1次,加入400μL胰酶于37℃消化10min。反复吹打支架30次,涡旋振荡30s。800r/min离心5min,小心抽去上清液,转移到一个1.5mL离心管,加入200μL GENMED萃取液,强力涡旋震荡1分钟,充分混匀。放进4℃冰箱里孵育16小时,期间涡旋震荡1分钟五次。放进微型台式离心机离心10分钟,速度为15000g,小心移取上清液到新的1.5毫升离心管,置于冰槽里备用。
加入20μL上述制备的待测样品到1.5毫升离心管。加入20μL GENMED高盐液,混匀。室温下孵育15分钟。加入20μL GENMED酸性液,混匀。室温下孵育15分钟。加入300μL GENMED染色工作液,涡旋震荡15秒。室温下孵育15分钟,避免光照。即刻放进微型台式离心机离心15分钟,速度为15000g。小心抽去上清液(注意:确保没有水滴残留)。加入400μL GENMED清理液(Reagent E),涡旋震荡15秒(注意:确保重悬颗粒)。放进微型台式离心机离心15分钟,速度为15000g。小心抽去上清液(注意:确保没有水滴残留),加入400μL GENMED解离液,涡旋震荡15秒。室温下孵育15分钟,避免光照(注意:确保充分溶解)。每个样本在96孔板加样200μL,即刻放进分光光度仪检测(波长600nm):获得样品吸光读数,根据上述标准曲线获得样品对应糖胺多糖含量(微克)。
标准样品配制:准备好5个1.5μL离心管,标记为1至5号管,分别加入20μL GENMED萃取液到1至5号管。移取20μL GENMED标准液到1号管,混匀。小心移取20μL 1号管稀释的GENMED标准液到2号管,混匀。小心移取20μL 2号管稀释的GENMED标准液(Reagent G)到3号管,混匀。小心移取20μL 3号管稀释的GENMED标准液到4号管,混匀。将1至5号管放进冰槽里备用;标准管浓度见下表。
浓度计算:【根据上述标准曲线样品对应糖胺多糖含量(微克)X样品稀释倍数】÷0.050(样品容量;毫升)=微克糖胺多糖/毫升。
3.4胶原检测
根据南京建成的羟脯氨酸测定试剂盒进行检测。将支架中培养液轻柔吸弃,PBS浸洗1次。将支架轻柔转移至1.5mL EP管中。样本水解:每样本加水解液400μL,混匀,37℃水浴3h;调pH值至6.0~6.8左右。将各试管流水冷却后各管加指示剂4μL,摇匀;各管准确加入调pH甲液0.4mL,混匀(此时溶液应为红色);用200μL的加样器吸取调pH的乙液向各管内逐滴小心加入调pH的乙液,每滴加入后均要混匀,直至液体中指示剂的颜色变成黄绿色(亦即红色消失时)。此时pH值在6.0~6.8左右(约加入调pH乙液40μL~200μL左右)。注意:加调pH乙液时,每加一滴均要混匀,为防止液体外溢,如果没有带盖的玻璃磨口试管,可用普通玻璃试管代替,每次混匀时可用塑料薄膜或冰箱保鲜膜压住试管口,充分旋涡混匀即可。
然后加双蒸水至1mL,混匀;水解液中加适量活性炭(约2~3mg左右,以上清液离心后澄清无色为准),混匀,3500转/分离心10分钟,小心取上清100μL作检测。
操作表:
(5)计算公式:羟脯氨酸含量(μg/mL)=(测定OD值-空白OD值)/(标准OD值-空白OD值)×标准品含量(5μg/mL)×水解液总体积(mL)/取样量(mL)。
图2中a脱细胞前后Hochest 33258细胞核染色显示脱细胞后基本没有DNA的蓝色荧光。图2中b-d脱细胞前后DNA、胶原和糖胺多糖定量显示冻融循环/DNA酶脱细胞处理去除90%以上DNA,细胞基质主要成分胶原和糖胺多糖保留70%以上。
4.脱细胞基质表衬的3D打印PCL支架的生物相容性
4.1活死染色
根据Invitrogen的Live/Dead试剂盒进行染色。
脱细胞基质表衬的3D打印PCL支架接种BMSCs培养24小时后,加入200μL染色液,室温染色30min后弃去染液,PBS浸洗3次终止染色,于荧光显微镜下摄取照片。
4.2细胞骨架染色
根据Cytoskeleton Inc的罗丹明-鬼笔环肽染色液进行染色。
脱细胞基质表衬的3D打印PCL支架接种BMSCs培养24小时后,加入200μL染色液,室温染色30min后弃去染液,PBS浸洗3次终止染色,再加入200μL DAPI染色室温染色15min,于荧光显微镜下摄取照片。
4.3 CCK-8细胞增殖活性检测
根据同仁CCK-8试剂盒进行测定。
脱细胞基质表衬的3D打印PCL支架接种BMSCs培养至第1天、4天和7天时,吸弃培养基,向样本中加入无血清培养基稀释的10%(v/v)的CCK-8工作液没过支架,于细胞培养箱内孵育1小时后,吸取上清液,用酶标仪测定在450nm处的OD值。
图3中a、b活死染色显示脱细胞基质表衬的3D打印PCL支架接种BMSCs 24h后细胞存活良好。图3中c-d F-actin细胞骨架染色显示BMSCs在daβc-Ot-DM表衬的3D打印PCL支架上铺展最好,应力纤维最长。图3中e的CCK-8测定显示BMSCs在脱细胞基质表衬的3D打印PCL支架上增殖良好,相比于纯PCL支架,脱细胞基质促进细胞的增殖。
5.细胞来源脱细胞基质表衬的3D打印PCL支架的成骨及矿化性能
5.1碱性磷酸酶染色
(1)脱细胞基质表衬的3D打印PCL接种BMSCs培养7和14天后,吸弃培养基,PBS缓冲液浸洗2次。
(2)加入4%多聚甲醛溶液,4℃固定20分钟。吸弃多聚甲醛溶液,PBS缓冲液浸洗2次。
(3)加入碱性磷酸酶显色混合液200μL,避光显色20min。
(4)吸弃显色液,用PBS洗涤2~3次终止显色,采集图片。
显色混合液配制:根据需要量按如下比例配制:
图4中a的ALP染色显示在7天和14天接种BMSCs后,daβc-Ot-DM表衬的3D打印PCL支架促进成骨分化显著强于其他两组。
5.2 ALP活性测定
脱细胞基质表衬的3D打印PCL接种BMSCs培养7天和14天后,用碧云天裂解液(P0013J,无抑制剂)裂解支架上的细胞,用碱性磷酸酶活性定量检测试剂盒(碧云天P0321S)对细胞裂解后的碱性磷酸酶活性进行定量检测,方法参见文献“Tu X et al(2007)Noncanonical Wnt signaling through G protein-linked PKCδactivation promotesbone formation.Dev Cell 12(1):113–27”。图4中b的ALP生化定量显示在脱细胞基质表衬的支架接种BMSCs7天和14天后,daβc-Ot-DM表衬的3D打印PCL支架ALP活性显著强于其他两组。
5.3 RNA提取及基因表达水平的测定
脱细胞基质表衬的3D打印PCL接种BMSCs培养7天和14天后,弃去培养基,加入4℃预冷的无菌PBS液体浸洗3次。将支架转移至1.5mL无菌无酶EP管中,消化得到的培养细胞,对细胞中表达的成骨细胞标志蛋白的编码基因的表达水平进行检测,具体的检测方法参见文献“Tu X et al(2015)Osteocytes mediate the anabolic actions of canonicalWnt/β-catenin signaling in bone.Proc Natl Acad Sci USA.112(5):E478-86”。
5.4茜素红染色
(1)脱细胞基质表衬的3D打印PCL接种BMSCs成骨诱导14天后,吸弃培养基,PBS缓冲液浸洗2次。
(2)加入4%多聚甲醛溶液,4℃固定20分钟。吸弃多聚甲醛溶液,PBS缓冲液浸洗2次。
(3)加入0.1%茜素红染液染色10min。
(4)吸弃染液,用PBS洗涤5-10次,采集图片。
(5)将染色的样品风干后浸泡在10%的氯化十六烷基吡啶中20min,转移洗脱液至96孔板中,用酶标仪在562nm处测定吸光度。
图4中a和b碱性磷酸酶染色及Alp生化定量结果显示在脱细胞基质表衬的支架上接种BMSCs 7天和14天后,daβc-Ot-DM表衬的3D打印PCL支架显色最深,说明其促进BMSCs成骨分化能力最强。图4中c实时荧光定量PCR显示daβc-Ot-DM表衬的3D打印PCL支架上BMSCs成骨基因表达上调显著强于其他两组(P<0.05),表明daβc-Ot-DM表衬的3D打印PCL支架具有很好的成骨分化效果。图4中d和e茜素红染色及定量分析显示在脱细胞基质表衬的支架上接种BMSCs成骨诱导14天后,daβc-Ot-DM表衬的3D打印PCL支架染色最深,矿化结节最多,其促进BMSCs矿化显著强于其他两组(P<0.05)。
6.细胞来源脱细胞基质表衬的3D打印PCL支架的骨修复性能
6.1 C57小鼠顶骨4.5mm骨缺损模型建立
本实验均按照重庆医科大学实验动物部的规范标准程序,进行动物手术过程。选取24只8周龄雄性C57小鼠进行实验,在小鼠腹腔中注射阿佛丁(0.2mL/10g)以麻醉,剂量为每100g体重0.1mL,待小鼠失去反射反应后,剔除头部毛发,用碘伏消毒,剪开头部皮肤暴露顶骨位置,钝性分离筋膜,使用4.5mm的环形钻头在顶骨左右两边各钻一个缺损,每只小鼠缺损处各植入1种脱细胞基质表衬的3D打印PCL支架。
植入支架4周和8周后,乙醚麻醉处死相应的小鼠,将小鼠顶骨取出,放入4%多聚甲醛4℃固定12h,随后将样品进行PET-CT检测,观察骨修复效果。
6.2 PET-CT检测
对4周和8周的顶骨样本用micro-CT扫描仪进行扫描分析。采用70kV、100mA的CT成像技术,以50μm分辨率对样品进行CT成像,并利用计算机软件(Pmod)进行三维重建。使用Image J从三维重建图像中计算出缺损部位的再生骨面积和缺损面积。图5中a、b的PET-CT检测及分析显示将脱细胞基质表衬的支架植入小鼠顶骨临界骨缺损4周和8周后,daβc-Ot-DM表衬的3D打印PCL支架4周修复约40%,8周约54%,显著高于其他三组。
6.3组织切片形态学分析
对4周和8周的顶骨样本脱钙、脱水后石蜡包埋,切片后进行H&E(图7、8)、天狼星红(图9)及TRAP染色(图10)。
图7为H&E染色显示,将脱细胞基质表衬的支架植入小鼠顶骨临界骨缺损4周后,daβc-Ot-DM表衬的3D打印PCL支架中长入的组织较为致密,而其他几组为疏松的组织;8周后,daβc-Ot-DM表衬的3D打印PCL支架中有大量新骨生成,WT-Ot-DM表衬的3D打印PCL支架中也有少量骨生成,而其他几组为一些类骨组织。H&E分析显示4周和8周daβc-Ot-DM表衬的3D打印PCL支架BV/TV(骨体积/总组织体积)、N.Ob/T.Ar(成骨细胞数量/总组织面积)、N.Ob/B.S(成骨细胞数量/骨面积)显著高于其他几组。
图8的H&E染色及分析显示,将脱细胞基质表衬的支架植入小鼠顶骨临界骨缺损8周时,daβc-Ot-DM表衬的3D打印PCL支架中长入的血管最多,而且体外在daβc-Ot-DM表衬的3D打印PCL支架模块上生长的BMSCs血管生成相关因子Vegfa和Angpt1的表达最高,而血管在骨形成过程中具有运输养分的作用,对骨形成极为重要。
图9的天狼星红染色显示,将脱细胞基质表衬的支架植入小鼠顶骨临界骨缺损4周时,daβc-Ot-DM表衬的3D打印PCL支架中胶原纤维较为致密排列整齐,WT-Ot-DM表衬的3D打印PCL支架中胶原纤维较少排列稍疏松,而其他几组疏松组织排列紊乱;8周后,daβc-Ot-DM表衬的3D打印PCL支架中有大量胶原纤维生成排列致密规则,与天然骨组织中胶原极为相似,而其他几组胶原较少排列无序。结果证明daβc-Ot-DM表衬的3D打印PCL支架新骨的成熟度较高,接近天然骨胶原。
图10的TRAP染色显示,将脱细胞基质表衬的支架植入小鼠顶骨临界骨缺损4周和8周时,daβc-Ot-DM表衬的3D打印PCL支架中破骨细胞围绕骨组织周围,数量较其他几组多,而破骨细胞行使骨吸收的功能,与成骨细胞协同在骨骼的发育和形成过程中发挥重要作用。结果证明daβc-Ot-DM表衬的3D打印PCL支架骨再生活跃。
图12血管标志物CD31和Endomucin免疫荧光染色进一步验证血管的生成,支架植入8周后的组织切片染色结果显示,daβc-Ot-DM组中红色荧光(CD31阳性)绿色荧光(Endomucin阳性)显著多于其他三组。这些结果表明daβc-Ot-DM具有促进体内血管生成的功能,从而保证支架内部的细胞的新陈代谢,有利于骨缺损的再生修复。
图13神经标志物β3-Tubulin和NeuN免疫荧光染色(β3-Tubulin阳性为绿色荧光,NeuN阳性为红色荧光)8周染色结果显示,daβc-Ot-DM组中β3-Tubulin和NeuN表达显著多于其他三组,而β3-Tubulin和NeuN在none-DM组无表达,BMSC-DM和WT-Ot-DM组两组有少量表达。这些结果表明daβc-Ot-DM可以促进体内神经生成。
对比例1:不同脱细胞方法的比较
与实施例1类似,不同之处在于仅采用冻融循环脱细胞。具体为:弃去培养基,无菌PBS浸洗2次;将支架转移至15mL离心管,加入5mL无菌PBS;冻融循环,具体步骤如下,将离心管放入液氮10min,之后放入37℃水浴锅10min,无菌PBS浸洗3次(3min/次)去除细胞碎片,重复此步骤3次。检测实施例3和对比例1的DNA残留情况,结果如图6所示。图6中a为冻融循环脱细胞,DNA残留22%左右,图6中b为冻融循环-DNA酶脱细胞,DNA残留4%左右。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种Wnt信号激活骨细胞脱细胞基质表衬的骨修复支架的制备方法,其特征在于,包括以下步骤:
a. 3D打印PCL支架:打开3D Bio-maker软件,根据程序设置打印PCL支架,将其修剪为5mm×5 mm×1 mm的规格;之后将支架放于75%酒精灭菌1 h,无菌PBS浸洗5次,每次清洗3min,将支架放于超净台晾干,紫外照射支架正反面各20 min灭菌;
b. 提取骨细胞Wnt激活小鼠的骨细胞:小鼠乙醚麻醉后脱颈处死,无菌条件下解剖取下双侧股骨、胫骨,去除软组织后取骨干,将骨干剪为1~2 mm大小的碎片,加入1 mg/mL I型胶原酶溶液于37℃摇床消化15 min,之后于冰上完全培养基中静置5 min,此过程重复3次;I型胶原酶于37℃处理15 min,4 mM EDTA于37℃处理5 min,交替处理3次,冲洗骨碎片后进行培养,每两天换液,待骨细胞长满培养皿70%使用并移走骨碎片继续培养;
c. 3D打印PCL支架上接种Wnt激活骨细胞:在支架上滴加10 μL血清浸润支架并晾干;之后消化骨细胞并计数,配制成2×107/mL的细胞悬液,每个支架接种10 µL细胞悬液,将支架放入孵箱2 h让细胞黏附,之后加入完全培养基进行培养,隔天换液;
d. 冻融循环和DNA酶脱细胞:培养14天后弃去培养基,无菌PBS浸洗2次;将支架转移至15 mL离心管,加入5 mL无菌PBS;进行冻融循环脱细胞处理,将离心管放入液氮中10 min,之后放入37℃水浴锅10 min,无菌PBS浸洗支架3次去除细胞碎片,每次清洗3 min,加入0.2mg/mL的DNA酶于37℃孵育1 h;无菌PBS浸洗3次,每次清洗3 min,置于4℃无菌PBS中保存;
e.使用时,将c步骤制得的支架放于超净台晾干,紫外照射正反面各10 min;
所述完全培养基包括:10%胎牛血清和1%青霉素-链霉素α-MEM。
2.根据权利要求1所述制备方法制得的Wnt信号激活骨细胞脱细胞基质表衬的骨修复硬材料支架。
3.权利要求2所述的Wnt信号激活骨细胞脱细胞基质表衬的骨修复硬材料支架在制备骨组织替换或修复材料产品中应用;所述骨组织为骨及软骨组织。
4.根据权利要求3所述的应用,其特征在于,所述骨修复硬材料支架植入体内修复长骨、短骨、扁骨、不规则骨的缺损或病变,通过促进破骨细胞生成,产生成骨、成血管和神经标志物表达的生物医学功能。
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