CN117138123A - 一种具有类骨结构的微米生物材料及其制备方法和应用 - Google Patents
一种具有类骨结构的微米生物材料及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及生物医学材料技术领域，尤其涉及一种具有类骨结构的微米生物材料及其制备方法和应用。所述制备方法包括：将间充质干细胞进行矿化培养，培养过程中使用改良矿化培养基，所述改良矿化培养基中包括万古霉素，所述万古霉素的浓度为0.1~1.0mM。本发明研究发现在制备以间充质干细胞为基础的微米生物材料的过程中加入万古霉素可以有效促进自组装过程中间充质干细胞的分化，形成发育程度更高，胶原纤维结构更完整的具有类骨结构的微米生物材料。本发明提供的制备方法制备得到的微米生物材料能够应用于修复感染性骨缺损，对于颅颌面感染性骨缺损的安全有效的治疗，具有较高的应用价值。
Description
技术领域
本发明涉及生物医学材料技术领域，尤其涉及一种具有类骨结构的微米生物材料及其制备方法和应用。
背景技术
感染、炎症、外伤、肿瘤等多种原因可引起颅颌面骨缺损，需要植骨手术恢复骨形态及功能，而植骨手术费用往往较高。影响骨缺损修复预后的重要原因是植骨区的继发感染，而口腔的有菌环境使创口更易感染，过程更加复杂。目前预防和治疗植骨手术相关感染的方法主要是局部清创并配合足量足疗程的抗生素全身给药，而这又可能带来包括细菌耐药和肝肾功能损伤在内的严重问题。同时，慢性感染及抗生素的全身使用均可能引起菌群失调，影响缺损区域骨免疫稳态，干扰局部间充质细胞功能，难以实现感染性骨缺损的修复/再生。因此，亟待研发一种对颅颌面感染性骨缺损安全、有效的治疗新策略，为广大患者解决病痛，为社会民生减轻负担。
抗生素成本低廉、性质明确、毒性可控，但全身使用会引起局部有效浓度低、肝肾毒性和耐药性等问题。同时，由于大部分抗生素半衰期较短，局部高浓度给药可能影响pH值和机体细胞活性，因此需要用生物活性材料负载抗生素以实现有效抗菌浓度内的抗生素缓释。有研究显示水凝胶可以缓释抗生素从而诱导牙周骨缺损修复，但其机械强度差，难以单独作为骨替代材料应用；还有研究选择抗生素-骨水泥链珠植于缺损局部，但骨水泥等材料在体内代谢速率极慢，释放抗生素的效率较低且不利于骨再生。现有外源性支架材料的降解速率难以控制，不易匹配缺损区抗菌需求。同时，材料降解过程有可能引发免疫原性反应和局部炎症反应等问题，无法同时满足抗菌和诱导骨再生的要求。
发明内容
为了解决现有技术存在的问题，本发明提供一种具有类骨结构的微米生物材料及其制备方法和应用。
万古霉素是一种大环内酯类抗生素，其主要通过抑制细菌的蛋白质合成来杀死或抑制细菌的生长，现有技术有研究万古霉素对于兔骨髓间充质干细胞的增殖和分化的影响，得到的结论是在高浓度的条件下（临床用药浓度的数百倍），万古霉素不影响兔骨髓间充质干细胞的增殖和分化，而当超过一定用量时，才会抑制兔骨髓间充质干细胞的增殖和分化过程。这也符合本领域对于抗生素功能的一般预期，但是本发明在研究的过程中意外发现，在矿化条件下，万古霉素对于牙髓间充质干细胞向着成骨的分化表现出了明显的促进作用，并且可以有效提高牙髓间充质干细胞的胶原分泌能力。这就使得制备得到的生物材料不仅拥有更完整的胶原纤维结构（整体生物材料也就相应地成为成熟度更高的类骨结构），还具备了一定的抗感染能力，在应用于感染性骨缺损时具有极优的技术效果
第一方面，本发明提供一种微米生物材料的制备方法，包括：
将间充质干细胞进行矿化培养，培养过程中使用改良矿化培养基，所述改良矿化培养基中包括万古霉素，所述万古霉素的浓度为0.1~1.0mM。
现有技术中，常采用间充质干细胞、Ca2+和PO4 3-进行矿化培养制备微米生物材料用于骨缺损的修复/再生，本发明在研究中发现在制备相关材料的过程中加入万古霉素可以促进间充质干细胞向着成骨分化，并同时促进其中胶原的形成和成熟程度，形成的胶原纤维也可见到明显的周期性结构。
进一步地，所述改良矿化培养基包括Ca2+、PO4 3-、聚天冬胺酸和万古霉素。
进一步地，所述Ca2+和所述PO4 3-的摩尔比为（6~15）：（2~6）；和/或，所述聚天冬胺酸的浓度为0.1~1mg/ml。
进一步地，所述Ca2+来源于CaCl2；和/或，所述PO4 3-来源于K2HPO4。
进一步地，所述改良矿化培养基还包括α-MEM培养基。
α-MEM培养基为微米生物材料的形成和生长提供了充分的营养条件。本发明所用α-MEM培养基可市售购得，实际上也可采用其他包含细胞增殖和分化所需营养成分的培养基。
进一步地，所述培养的培养条件包括：
在5~10%的CO2浓度、35~38℃的条件下进行培养。
进一步地，所述间充质干细胞为牙髓间充质干细胞或骨髓间充质干细胞。
第二方面，本发明提供一种微米生物材料，所述微米生物材料由所述的制备方法制备得到。
本发明进一步提供一种微米生物材料，包括：
间充质干细胞、细胞外基质、万古霉素、聚天冬胺酸和矿化物质；
所述细胞外基质在所述微米生物材料中呈现D-周期条纹型的成熟胶原纤维结构。
第三方面，本发明提供所述的微米生物材料在骨折、骨关节炎或骨缺损中的应用。
进一步地，所述骨缺损优选为感染性骨缺损。
本发明进一步提供所述的微米生物材料在制备治疗骨折、骨关节炎或骨缺损的药物或试剂盒中的应用。
本发明进一步提供万古霉素在促进间充质干细胞向成骨分化或提高间充质干细胞的胶原分泌能力中的应用。
本发明进一步提供万古霉素在促进间充质干细胞向成骨分化或提高间充质干细胞的胶原分泌能力的药物或试剂盒中的应用。
进一步地，在矿化条件下促进间充质干细胞向成骨分化或提高间充质干细胞的胶原分泌能力。
进一步地，所述矿化条件为含有Ca2+和PO4 3-的条件下。
本发明具备如下有益效果：
本发明采用含有万古霉素的矿化培养基进行微米生物材料的制备，发现这一过程制备得到的微米生物材料具备发育程度更高的周期性的胶原纤维结构。
本发明提供的制备方法制备得到的微米生物材料中胶原纤维结构的成熟程度更高，整体生物材料表现为类骨结构，更加适用于骨缺损的修复，而且还能够缓释抗生素，对于感染性骨缺损的修复具有重要意义。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，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是本发明实施例1提供的MSC@M-V和MSC@M两种微球在光镜和透射电子显微镜下的形貌结构图。
图2是本发明实施例2提供的MSC@M-V和MSC@M两种微球的抑菌效果示意图。
图3是本发明实施例3提供的MSC@M-V和MSC@M两种微球中成骨相关基因的表达水平示意图。
图4是本发明实施例4提供的动物模型示意图。
图5是本发明实施例4提供的micro-CT检查缺损区成骨情况的结果示意图。
图6是本发明实施例4提供的4周时用HE、Masson染色缺损组织的结果示意图。
图7是本发明实施例4提供的12周时用HE、Masson染色缺损组织的结果示意图。
图8是本发明实施例4提供的12周时免疫组织化学染色缺损组织的结果示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明中的附图，对本发明中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作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
本实施例提供一种微米生物材料（自组装的干细胞-基质-微球）的制备方法，具体如下：
获取12-18岁之间患者因正畸拔除的、牙体牙周均健康的前磨牙，口内新洁尔灭充分消毒，拔除牙齿后立即放入常规细胞培养基中储存。超净台中暴露牙髓，取出并充分切碎至泥浆状。加入胶原酶与分散酶消化后，使用α-MEM培养基重悬，置于37℃含5% CO2（体积百分含量）的孵箱中培养，每隔2-3天更换新鲜培养基。待牙髓间充质干细胞（以下均称DPSCs）至80-90%融合后消化，传代至第4代（P4），将P4 DPSCs接种于三维细胞微球培养六孔板（市售商品，杭州睿凯生物科技有限公司），使用实验培养基1进行MSC@M-V的制备，使用实验培养基2进行MSC@M的制备。培养期间每2天换液一次，培养7天。
其中，实验培养基1是在α-MEM培养基的基础上加入9 mM CaCl2、4.2 mM K2HPO4、0.2 mg/ml PASP和1mM万古霉素得到。
实验培养基2是在α-MEM培养基的基础上加入9 mM CaCl2、4.2 mM K2HPO4和0.2mg/ml PASP得到。
本发明进一步通过光镜和透射电镜对制备得到的MSC@M-V和MSC@M两种微球进行观察，具体采用2.5%戊二醛溶液固定，树脂包埋，切片，醋酸铀复染之后置于光镜或电镜下观察。
光镜观察结果和透射电镜观察结果如图1所示，从图中可以看出两种微球内部都可见到新合成胶原纤维，其中MSC@M-V中的胶原纤维可见到明显的周期性结构。以上结果表明本发明中提供的培养环境中，MSC@M-V相较于MSC@M而言，其中胶原形成和成熟的程度更高，表现为类骨结构。
实施例2
本实施例验证实施例1制备得到的微米生物材料MSC@M-V微球的抗菌性能，具体流程如下：
实验微生物采用金黄色葡萄球菌，在培养皿中划线培养后，分别加入50个微球，共培养24小时，通过肉眼和透射电镜对培养皿中的金黄色葡萄球菌进行观察，并统计结果。
结果如图2所示，在共培养24小时之后，MSC@M-V组几乎所有金黄色葡萄球菌均被杀灭，这说明了本申请实施例1制备得到的MSC@M-V微球具备优异的抑菌效果。
实施例3
本实施例验证实施例1制备得到的微米生物材料MSC@M-V微球促进成骨的能力，具体流程如下：
本申请通过荧光实时定量PCR检测COL1A1、BMP2和OCN三种成骨相关基因在两种细胞微球中表达水平的差异，具体在两种微球培养第3天时进行如下流程的检测：
在Trizol提取总RNA后用SuperScript® III One-Step RT-PCR系统和Platinum® Taq High Fidelity（Invitrogen）合成cDNA。以SYBR Green（Invitrogen LifeTechnologies）标记，在7900HT Fast Real Time PCR机器（Applied Biosystems）上检测。
结果如图3所示，图3显示在MSC@M-V微球和MSC@M微球培养3天时，COL1A1、BMP2和OCN三种成骨相关基因在MSC@M-V微球中的表达水平显著高于未添加万古霉素的MSC@M组，这说明1 mM万古霉素可以在含钙磷的矿化培养基环境下进一步促进DPSCs的成骨向分化。
实施例4
本实施例进一步验证实施例1制备得到的MSC@M-V微球和MSC@M微球促进感染性骨缺损修复的能力，具体流程如下：
1、本发明采用如下方法构建大鼠颅部感染性骨缺损模型：
将6-8周龄SD大鼠用1%戊巴比妥钠腹腔注射麻醉，剪毛，切开头部皮肤，分离肌层，暴露颅骨骨面；使用种植机1200转/min配合环形骨钻（外径5 mm）磨除全层骨组织，无菌生理盐水冷却，压迫止血，缺损区涂抹3×105CFU/mL金黄色葡萄球菌悬液，植入材料后缝合。构建的颅骨缺损如图4所示。
之后分别将实施例1制备得到的MSC@M微球或MSC@M-V微球植入缺损区，在4周、8周和12周后取材，通过micro-CT分析骨缺损的修复效果，结果如图5（图中4w为4周，8w为8周，12w为12周）所示：三个时间点MSC@M-V组新生骨的数量和质量均显著优于MSC@M组。
2、本发明进一步对4周和12周得到的材料进行HE和Masson染色观察，具体步骤如下：
HE染色步骤：
组织10%福尔马林固定后，10% EDTA脱钙，常规脱水，包埋，制作连续石蜡切片，层厚5 μm。烘干切片后，二甲苯及梯度酒精脱蜡水化。石蜡切片入苏木素染0.5-1分钟，自来水漂洗，1%盐酸酒精分化数秒，自来水漂洗，然后1%氨水水溶液返蓝1分钟，流水冲洗数秒，放入伊红染液中染色数秒，流水漂洗。石蜡切片依次放入70%乙醇10秒 -80%乙醇10秒 -90%乙醇10秒 -95%乙醇10秒 -无水乙醇5分钟 -无水乙醇5分钟 -二甲苯5分钟透明，将切片从二甲苯拿出来中性树胶封片。
Masson染色步骤：
组织10%福尔马林固定后，10% EDTA脱钙，常规脱水，包埋，制作连续石蜡切片，层厚5 μm。烘干切片后，二甲苯及梯度酒精脱蜡水化。用Weigert铁苏木素染色液染色5-10分钟。酸性乙醇分化液分化5-15秒，水洗。Masson蓝化液返蓝3-5分钟，水洗。蒸留水洗1分钟。丽春红品红染色液染色5-10分钟。在上述操作过程中按蒸馏水: 弱酸溶液=2:1 比配置弱酸工作液，用弱酸工作液洗1分钟磷钜酸溶液洗 1-2分钟。用配置好的弱酸工作液洗l分钟。直接放入苯胺蓝染色液中染色1-2分钟。用配置好的弱酸工作液洗分钟。95%乙醇快速脱水2-3秒，无水乙醇脱水 3次，每次 5-10秒。二甲苯透明 3 次，每次 1-2分钟，中性树胶封固。
之后进行观察，结果如图6所示：4周时MSC@M-V组骨量显著高于MSC@M组和对照组。如图7所示：12周时MSC@M-V组感染性颅骨缺损完全修复，新骨充满缺损区，而MSC@M组仅有少量新骨形成，对照组几乎没有新骨形成。
本发明进一步采用免疫组化染色，具体步骤如下：
织10%福尔马林固定后，10% EDTA脱钙，常规脱水，包埋，制作连续石蜡切片，层厚5μm。烘干切片后，二甲苯及梯度酒精脱蜡水化。采用0.125%胰酶+10 μg/mL蛋白酶K混合酶消化液，室温孵育30分钟进行抗原修复；3% H2O2溶液避光室温孵育30分钟；5%山羊血清室温封闭30分钟；用抗体稀释液进行稀释并滴加适量体积一抗，4°C过夜；应用一抗种属对应的超敏二步法试剂盒，滴辣根过氧化物酶标记二抗，37°C水浴30分钟；DAB显色；苏木素复染细胞核，盐酸酒精分化，流水冲洗返蓝；梯度酒精脱水，二甲苯透明，中性树胶封片。
得到如图8所示的结果：12周时MSC@M-V组成骨向标记物RUNX2和OCN的表达量显著高于MSC@M组和对照组。这些均表示万古霉素促进了成骨相关基因的表达，即促进了微米生物材料修复感染性骨缺损的能力。
最后应说明的是：以上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精神和范围。
Claims (10)
1.一种微米生物材料的制备方法，其特征在于，包括：
将间充质干细胞进行矿化培养，培养过程中使用改良矿化培养基，所述改良矿化培养基中包括万古霉素，所述万古霉素的浓度为0.1~1.0mM。
2.根据权利要求1所述的制备方法，其特征在于，所述改良矿化培养基包括Ca2+、PO4 3-、聚天冬胺酸和万古霉素。
3.根据权利要求2所述的制备方法，其特征在于，所述Ca2+和所述PO4 3-的摩尔比为（6~15）：（2~6）；和/或，所述聚天冬胺酸的浓度为0.1~1mg/ml。
4.根据权利要求3所述的制备方法，其特征在于，所述Ca2+来源于CaCl2；和/或，所述PO4 3-来源于K2HPO4。
5.根据权利要求2所述的制备方法，其特征在于，所述改良矿化培养基还包括α-MEM培养基。
6.根据权利要求2-5任一项所述的制备方法，其特征在于，所述培养的培养条件包括：
在5~10%的CO2浓度、35~38℃的条件下进行培养。
7.一种微米生物材料，其特征在于，所述微米生物材料由权利要求1-6任一项所述的制备方法制备得到。
8.一种微米生物材料，其特征在于，所述微米生物材料包括：间充质干细胞、细胞外基质、万古霉素、聚天冬胺酸和矿化物质；所述细胞外基质在所述微米生物材料中呈现D-周期条纹型的成熟胶原纤维结构。
9.权利要求7或8所述的微米生物材料在骨折、骨关节炎或骨缺损中的应用。
10.万古霉素在促进间充质干细胞向成骨分化或提高间充质干细胞的胶原分泌能力中的应用。
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