CN111518755A - 一种仿生骨膜、骨膜-骨替代物及制备方法 - Google Patents
一种仿生骨膜、骨膜-骨替代物及制备方法 Download PDFInfo
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Abstract
本发明要求保护一种用于大段骨缺损修复的仿生骨膜、骨膜‑骨替代物及制备方法。所述的仿生骨膜是在聚二甲基硅氧烷表面培养成骨前体细胞至形成细胞片后,再脱除细胞获得细胞外基质(ECM)片。将仿生骨膜缠绕在可降解的骨支架材料表面即形成骨膜‑骨替代物。本发明所制备的成骨前体细胞来源的ECM片不仅具有生物活性,而且在体内外均表现出骨诱导作用。此外,该ECM片对骨髓间充质干细胞具有趋化作用。通过结合细胞来源的ECM片与可降解骨支架材料,可改善目前临床上用于修复大面积节段性骨缺损的诱导膜技术,制备方法简单,可作为优异的骨膜‑骨替代材料。
Description
技术领域
本发明涉及一种生物医用材料技术领域,具体涉及一种用于修复大段骨缺损的仿生骨膜、骨膜-骨替代物及制备方法。
背景技术
严重创伤、肿瘤或感染引起的大段骨缺损修复在骨科仍是一大挑战,修复过程中往往出现愈合延迟甚至不愈合。众所周知,当缺陷超过临界尺寸时,不能通过自身的愈合过程完全修复。
骨膜完整性对骨折愈合和骨再生至关重要。骨膜是一层覆盖于骨外表面的薄组织膜,将70-80%的血供输送到骨皮质,提供成骨细胞、前体细胞和骨膜干细胞,在骨形成和再生中发挥着重要作用。骨膜可以通过多种生物过程促进愈合,如细胞增殖和分化,或通过旁分泌信号,这些信号可以招募和激活宿主骨祖细胞。因此,如果人造骨膜可以发挥与天然骨膜相似的功能,将有助于修复大段骨缺损。
细胞外基质(ECM)具有作为人工骨膜的巨大潜力。ECM是由蛋白质和蛋白多糖组成的网络,含有大量生长因子和其他信号分子。此外,它为细胞生长提供黏附位点,并为细胞与邻近基质相互作用提供结构支持。此外,ECM可以提供调控细胞行为和功能的微环境。
发明内容
本发明的目的在于提供一种可用于修复大段骨缺损的仿生骨膜、骨膜-骨替代物及制备方法。
为了解决上述技术问题,本发明提供了一种仿生骨膜-骨替代物,可以有效修复大段骨缺损。其中,制备得到的ECM片将发挥类似骨膜的作用,促进周围骨膜细胞的黏附、迁移和增殖,并募集干细胞以快速再生缺损处骨膜和骨骼,而骨支架材料可提供临时支撑、避免纤维组织侵入和支持仿生骨膜以及周围细胞的生长和成骨。
为达到上述目的,本发明的第一方面提供一种用于修复大段骨缺损的仿生骨膜,其通过在聚二甲基硅氧烷(PDMS)表面培养成骨前体细胞,细胞生长成熟为细胞片后将细胞去除而得到ECM片。
本发明优选技术方案中,仿生骨膜的ECM片为成骨前体细胞、骨髓间充质干细胞、脂肪来源干细胞等,厚度为10-50μm。
本发明第二方面提供骨膜-骨替代物,其由成骨前体细胞来源的ECM片和骨支架材料构成。优选地,所述仿生骨膜缠绕在骨支架材料表面。
本发明优选技术方案中,所述骨支架材料选自但不限于甲基丙烯酸酐化明胶凝胶或海藻酸钠水凝胶等生物材料,它们都具有良好的生物相容性和可降解性能。
本发明骨膜-骨替代物,通过具有生物活性和成骨诱导活性的ECM片发挥类似骨膜的作用促进骨再生,与起力学支撑作用及可降解的骨支架材料相结合后,可作为优异的骨修复材料用于骨组织工程。
骨膜-骨替代物,可将制备好的ECM片包裹在骨支架材料表面以构建骨膜-骨替代物。其中,仿生骨膜占骨膜-骨替代物的质量百分比为0.01-10%。
骨支架材料可以选用甲基丙烯酸酐化明胶凝胶或者是海藻酸钠水凝胶,当然还可以选用本领域技术人员公知的其他使用的可降解的骨支架材料。配制甲基丙烯酸酐化明胶(GelMA)凝胶的方法如下:先配制GelMA溶液,加入光引发剂(苯基-2,4,6-三甲基苯甲酰膦酸锂),将该溶液注入石英模具中,用波长为365nm的蓝光光源手电筒照射,获得了GelMA水凝胶。配制海藻酸钠水凝胶的方法如下:分别配制海藻酸钠溶液和氯化钙溶液,将两者混合并注入石英模具中,即可获得海藻酸钠水凝胶。GelMA的浓度优选为0.1w/v%-10w/v%;海藻酸钠的浓度优选为0.1w/v%-2w/v%。骨支架材料形状优选为圆柱形、块状,但也可为不规则形态。
本发明的第三方面提供了仿生骨膜的制备方法,包括以下步骤:
(1)准备聚二甲基硅氧烷,将聚二甲基硅氧烷置于等离子体清洗机内,使其在氧气氛围下清洗,灭菌后,加入纤连蛋白或明胶进行孵化;
(2)将成骨前体细胞接种于步骤(1)得到的聚二甲基硅氧烷上进行细胞培养,得到细胞片;
(3)将培养好的细胞片用PBS清洗后,先用Triton-X100和NH4OH的混合溶液对细胞片进行脱细胞处理,然后使用DNA酶和RNA酶消化残余的DNA和RNA,最后将ECM片与PDMS分离即得。
本发明优选技术方案中,步骤(1)中,纤连蛋白的浓度为10~20μg/mL,明胶的浓度为0.5%-2%wt/v,孵化时间为6~8h。
本发明优选技术方案中,步骤(2)中,待细胞生长至聚二甲基硅氧烷表面80%以上时,优选如90%以上,向培养基中加入抗坏血酸,以促进细胞外基质的分泌,继续培养14天直到细胞片成熟。所述成骨前体细胞为MC3T3-E1细胞、骨髓间充质干细胞或脂肪间充质干细胞,接种密度为0.5×104/cm2~2×104/cm2,抗坏血酸的浓度为10~50μg/mL。
本发明优选技术方案中,步骤(3)中,Triton-X的浓度为0.1%~0.25%,NH4OH溶液的浓度为10~50mM,脱细胞处理时间为5-20min;DNA酶,如DNase I的浓度为10~50U/mL,RNA酶,如RNase A的浓度为0.5~2.5μL/mL。
本发明优选技术方案中,步骤(3)中,将培养好的细胞片用PBS清洗后,用Triton-X100和NH4OH溶液对细胞进行脱细胞处理,然后使用DNaseI和RNase A在37℃处理2h以去除残余细胞DNA和RNA,将ECM片与PDMS分离,然后置于4℃保存备用。
本发明第四方面提供上述仿生骨膜-骨替代物用途,可用于作制备修复骨缺损医用材料,尤其是大段骨缺损修复的医用材料的制备。
本发明通过在聚二甲基硅氧烷(PDMS)表面培养成骨前体细胞,细胞成熟后利用脱细胞技术将细胞去除获得ECM片并将其缠绕在光交联后的GelMA水凝胶表面以构建骨膜-骨仿生骨替代物。细胞来源的ECM由于具有较高的生物诱导活性并且与天然骨膜ECM具有相似的结构和组成,将促进周围骨膜细胞的粘附、迁移和增殖,并募集干细胞以快速再生缺损的骨膜和骨骼。另外,骨支架材料将起到临时支撑作用,避免纤维组织侵入,并支持仿生骨膜以及周围细胞的生长和成骨。
本发明中,聚二甲基硅氧烷(PDMS)可以是普通市售产品,还可以是制备得到;如将道康宁184试剂盒中的前聚体和固化剂按照质量比为5:1~30:1混合,固化制得聚二甲基硅氧烷,优选地,前聚体硅弹性体基材与固化剂的质量比为10:1。
本发明的优点在于:
(1)本发明通过脱细胞技术制备了成骨前体细胞来源的ECM片(图1),然后将其缠绕包裹在水凝胶表面制得仿生骨膜-骨,该仿生骨膜-骨可有效修复大段骨缺损;
(2)本发明中的ECM片,不仅可促进细胞黏附和增殖,在体外具有细胞趋化作用,而且在体内外都表现出良好的成骨诱导效果,其能直接替代现有膜诱导技术诱导生成的组织膜,发挥类似骨膜的作用,从而简化现有的膜诱导技术的操作流程并缩短治疗时间,具有非常好的应用前景;
(3)本发明的仿生骨膜-骨中,以可降解的、生物相容性良好的骨支架材料替代膜诱导技术中植入的不可降解的聚甲基丙烯酸酯骨水泥,一方面可以起到临时支持和避免纤维组织长入的问题,另一方面能省去膜诱导技术需要二期手术取出骨水泥的步骤。
附图说明
图1A为光学显微镜下ECM片的图像;图1B为脱细胞后ECM片的DAPI染色;图1C为脱细胞后ECM片的SEM图像。
图2骨膜-骨替代物的外观(2A)和SEM图像(2B)。
图3为将仿生骨膜-骨植入到兔子桡骨节段性骨缺损中,第12周时各组的H&E染色图片,图3A为缺损后未处理组;图3B为水凝胶植入组;图3C为骨膜-骨替代物植入组。其中IM:植入材料;NB:新骨;BM:骨髓。
具体实施方式
以下结合具体实施例对上述方案做进一步说明。应理解,这些实施例是用于说明本发明而不限于限制本发明的范围。实施例中采用的实施条件可以根据具体厂家的条件做进一步调整,未注明的实施条件通常为常规实验中的条件。
介绍和概述
本发明通过举例而非给出限制的方式来进行说明。应注意的是,在本公开文件中所述的“一”或“一种”实施方式未必是指同一种具体实施方式,而是指至少有一种。
下文将描述本发明的各个方面。然而,对于本领域中的技术人员显而易见的是,可根据本发明的仅一些或所有方面来实施本发明。为说明起见,本文给出具体的编号、材料和配置,以使人们能够透彻地理解本发明。然而,对于本领域中的技术人员将显而易见的是,本发明无需具体的细节即可实施。在其他例子中,为不使本发明费解而省略或简化了众所周知的特征。
将各种操作作为多个分立的步骤而依次进行描述,且以最有助于理解本发明的方式来说明;然而,不应将按次序的描述理解为暗示这些操作必然依赖于顺序。
将根据典型种类的反应物来说明各种实施方式。对于本领域中的技术人员将显而易见的是,本发明可使用任意数量的不同种类的反应物来实施,而不只是那些为说明目的而在这里给出的反应物。此外,也将显而易见的是,本发明并不局限于任何特定的混合示例。
本发明中硅弹性体基材为道康宁市售产品,MC3T3-E1细胞为市售产品,骨髓间充质干细胞和脂肪间充质干细胞是从大鼠组织中提取。
实施例1:
(1)将道康宁184试剂盒中的前聚体和固化剂按照质量比为10:1混合,放置于60℃固化2h,制得PDMS。将PDMS置于等离子体清洗机内,使其在氧气氛围下清洗。PDMS在细胞培养前,对PDMS底物进行紫外线杀菌,然后用20μg/mL的纤连蛋白孵化6-8h;
(2)将MC3T3-E1细胞接种于孵化后的PDMS上,接种密度为0.5×104/cm2,待细胞生长至表面80%时,向培养基中加入20μg/mL的抗坏血酸,继续培养;
(3)14天后,将培养好的细胞片用PBS清洗3遍,首先使用0.25%的Triton-X100和25mM的NH4OH溶液对细胞脱细胞处理10min,然后使用25U/mL DNase I和1.5μL/mL RNase A在37℃处理2h以去除残余细胞DNA和RNA,将制备好的ECM片置于4℃保存备用,ECM片的形貌见说明书附图1;
(4)向GelMA固体海绵中加入PBS,然后置于37℃水浴锅中溶解至透明状液体,接着加入光引发剂(苯基-2,4,6-三甲基苯甲酰膦酸锂),溶解后得到浓度为5%的GelMA溶液。将GelMA溶液注入石英模具中,用波长为365nm的蓝光光源手电筒照射1min,获得了长为10mm,直径为10mm的GelMA柱;
(5)将制备好的ECM片包裹在GelMA柱表面以构建仿生骨膜-骨结构。
实施例2:
(1)将道康宁184试剂盒中的前聚体和固化剂按照质量比为20:1进行混合,然后固化制得PDMS,并通过等离子体处理。在细胞培养前,对制备的PDMS进行紫外线杀菌,然后用明胶溶液孵化8h;
(2)将骨髓间充质干细胞接种于孵化后的PDMS上,接种密度为2×104/cm2,待细胞生长至表面90%时,向培养基中加入50μg/mL的抗坏血酸促进细胞外基质的分泌,继续培养直到细胞片成熟;
(3)将培养好的细胞片用PBS清洗3遍,首先使用0.25%的Triton-X100和50mM的NH4OH溶液对细胞脱细胞处理5min,然后使用50U/mL DNaseI和2.5μL/mL RNase A在37℃处理2h以去除残余细胞DNA和RNA,ECM片具有良好的生物相容性;
(4)向GelMA固体海绵中加入PBS,然后置于37℃水浴锅中溶解至透明状液体,接着加入光引发剂(苯基-2,4,6-三甲基苯甲酰膦酸锂),溶解后得到浓度为10%的GelMA溶液。将GelMA溶液注入石英模具中,用波长为405nm的蓝光光源手电筒照射1min,获得了长为15mm,直径为5mm的GelMA柱;
(5)将制备好的ECM片包裹在GelMA柱表面以构建仿生骨膜-骨结构,仿生骨膜-骨的外观和SEM图像见图2。相比于对照组和纯GelMA组,仿生骨膜-骨组具有良好的修复效果(图3)。
实施例3:
(1)将道康宁184试剂盒中的前聚体和固化剂按照质量比为5:1混合,并固化,制得PDMS,并通过等离子体处理。在细胞培养前,对PDMS底物进行紫外线杀菌,然后用10μg/mL的纤连蛋白孵化6-8h;
(2)将脂肪间充质干细胞接种于孵化后的PDMS上,接种密度为1×104/cm2,待细胞生长至表面90%时,向培养基中加入40μg/mL的抗坏血酸促进细胞外基质的分泌,继续培养直到细胞片成熟;
(3)将培养好的细胞片用PBS清洗3遍,首先使用0.1%的Triton-X100和40mM的NH4OH溶液对细胞脱细胞处理20min,然后使用40U/mLDNase I和2.0μL/mL RNase A在37℃处理2h以去除残余细胞DNA和RNA,将制备好的ECM片置于4℃保存备用;
(4)配制2%的海藻酸钠溶液和10mM的氯化钙溶液,将其混合后加入模具中,制备直径为5mm,高度为10mm的水凝胶柱;
(5)将制备好的ECM片包裹在水凝胶柱表面以构建仿生骨膜-骨结构。
以上所述具体实施例仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进或替换,这些改进或替换也应当视为本发明的保护范围。
Claims (11)
1.一种仿生骨膜,其特征在于,其通过在聚二甲基硅氧烷表面培养成骨前体细胞,细胞生长成熟为细胞片后将细胞去除而得到ECM片。
2.一种骨膜-骨替代物,其特征在于,其由权利要求1的仿生骨膜和骨支架材料构成。
3.根据权利要求2所述的骨膜-骨替代物,其特征在于,所述仿生骨膜缠绕在骨支架表面。
4.根据权利要求2或3所述的骨膜-骨替代物,其特征在于,所述骨支架材料是可降解材料。
5.根据权利要求2或3所述的骨膜-骨替代物,其特征在于,所述骨支架材料形状选自圆柱形、块状或不规则形态。
6.根据权利要求2或3所述的骨膜-骨替代物,其特征在于,仿生骨膜占骨膜-骨替代物的质量百分比为0.01-10%。
7.根据权利要求2或3所述的骨膜-骨替代物,其特征在于,所述骨支架材料选自甲基丙烯酸酐化明胶凝胶、海藻酸钠水凝胶。
8.制备如权利要求1所述的仿生骨膜的方法,包括以下步骤:
(1)准备聚二甲基硅氧烷,将聚二甲基硅氧烷通过等离子处理,灭菌后,加入纤连蛋白或明胶进行孵化;
(2)将成骨前体细胞接种于步骤(1)得到的聚二甲基硅氧烷上进行细胞培养,得到细胞片;
(3)将培养好的细胞片用PBS清洗后,先用Triton-X100和NH4OH的混合溶液对细胞片进行脱细胞处理,然后使用DNA酶和RNA酶消化残余的DNA和RNA,最后将ECM片与聚二甲基硅氧烷分离即得。
9.根据权利要求8所述的方法,其特征在于,步骤(2)中,待细胞生长至聚二甲基硅氧烷表面80%以上时,向培养基中加入抗坏血酸,继续培养至细胞片成熟。
10.根据权利要求8所述的方法,其特征在于,步骤(2)中,成骨前体细胞选自MC3T3-E1、骨髓间充质干细胞或脂肪间充质干细胞。
11.如权利要求1的仿生骨膜或权利要求2的骨膜-骨替代物用途,用于制备修复骨缺损的医用材料。
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