CN103998070B - 纤维状脱钙骨基质的制备方法 - Google Patents

纤维状脱钙骨基质的制备方法 Download PDF

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CN103998070B
CN103998070B CN201180073750.6A CN201180073750A CN103998070B CN 103998070 B CN103998070 B CN 103998070B CN 201180073750 A CN201180073750 A CN 201180073750A CN 103998070 B CN103998070 B CN 103998070B
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bone
decalcification
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bone matrix
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宋锡范
郑求源
苏怔援
徐汉率
柳眩承
金贵南
金炳奭
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Cg Biotechnology Co Ltd
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Abstract

本发明涉及一种纤维状脱钙骨基质的制备方法，其包括下述步骤：（a）将分离到体外的骨在酸性溶液中进行1‑5小时的第1次脱钙；（b）将从步骤（a）得到的骨切成0.1‑3mm厚的片状骨；（c）将从步骤（b）得到的片状骨在酸性溶液中进行2‑6小时的第2次脱钙；及（d）将从步骤（c）得到的经过脱钙的骨进行粉碎。

Description

纤维状脱钙骨基质的制备方法
技术领域
本发明涉及纤维状脱钙骨基质的改善的制备方法，具体地，涉及包括部分脱钙、切割、完全脱钙及粉碎步骤的纤维状脱钙骨基质的改善的制备方法。
背景技术
将骨浸渍在酸中去除骨中的矿物质成分的部分称为脱钙骨基质（demineralized bone matrix，DBM）。脱钙骨基质大部分由高度交联的骨胶原构成，残存的非骨胶原性蛋白质包括转化生长因子-β（TGF–β）、血小板源性生长因子（PDGF）、骨桥蛋白、骨粘连蛋白、骨形态生成蛋白（bone morphogenetic protein，BMP）等。脱钙骨基质以用于修复骨缺损等为目的的骨移植用组合物的形态使用。
脱钙骨基质对分离到体外的骨进行脱钙步骤之后，将此粉碎为适当的大小，制备成颗粒形态。本发明发明人公开过具有0.05-250μm颗粒大小的脱钙骨基质；具有250-2000μm的颗粒大小的脱钙骨基质；及以特定比例混合水合物的骨再生用组合物，所述组合物具有优异的注入性及形态维持性（处理性）（韩国授权专利第10-1041784号）。
另外，皮质骨（cortical bone）平行于骨的长轴并由骨胶原纤维束（fiber bundles）构成，已知由此得到的纤维状脱钙骨基质在骨修复及其它整形外科的移植方面具有有用的性质。纤维状脱钙骨基质通过将分离到体外的皮质骨利用特殊粉碎机（例如，美国专利第5,607,269号）粉碎至得到纤维状颗粒，然后进行脱钙而制备。但是这种制备方法由于受到粉碎仪器的机械方面的限制，只能使用天然的皮质轴（intact cortical shafts）作为骨原料，且得到的纤维状脱钙骨收率低。为了改善上述问题，美国专利第7,323,193号及第7,939,108号中公开了将分离到体外的骨切断成适当大小，并在酸性溶液中进行6小时的脱钙后，再在酸性溶液中进行2天的脱钙，然后将其进行粉碎，从而制备纤维状脱钙骨基质的方法。但是上述美国专利第7,323,193号及第7,939,108号中公开的制备方法，需要进行长达2日，即48小时的长时间的脱钙步骤。
发明内容
发明目的
本发明发明人为了开发出具有有用的性质的纤维状脱钙骨基质的制备方法，尤其是能够改善长时间的脱钙步骤的制备方法，进行了多种研究。结果将部分脱钙而得到的骨进行切割制备成薄片状形态后，将此进行脱钙时，惊奇地发现能够将脱钙步骤显著缩短至约6小时左右，且脱钙骨基质具有纤维状形态的同时能够以高收率获得。
从而，本发明的目的在于通过依次进行部分脱钙、切割、完全脱钙及粉碎步骤的纤维状脱钙骨基质的制备方法。
技术方案
本发明涉及一种纤维状脱钙骨基质的制备方法，其包括下述步骤：（a）将分离到体外的骨在酸性溶液中进行1-5小时的第1次脱钙；（b）将从步骤（a）得到的骨切割（slicing）成0.1-3mm厚的片状骨；（c）将从步骤（b）得到的片状骨在酸性溶液中进行2-6小时的第2次脱钙；及（d）将从步骤（c）得到的经过脱钙的骨进行粉碎。
根据本发明的纤维状脱钙骨基质的制备方法，所述步骤（a）和步骤（c）的所述酸性溶液可以相互独立地为0.1-3N的HCl水溶液，优选所述步骤（a）和步骤（c）的所述酸性溶液可以为0.6N的HCl水溶液。此外，可以以获得具有1000-5000μm范围长度的纤维状脱钙骨基质来实施所述粉碎。
发明效果
根据本发明，依次进行部分脱钙、切割、完全脱钙及粉碎步骤时，发现能够将脱钙步骤显著缩短至约6小时左右。尤其通过引入切割步骤，不仅缩短了脱钙步骤的时间，而且还能够使最终得到的脱钙骨基质以具有纤维形态的脱钙骨基质高收率地获得。因此，本发明的制备方法能够适用于工业规模的大量生产。
附图说明
图1是表示将部分脱钙的骨进行切割后得到的骨（A）及将脱钙的骨进行粉碎后得到的骨（B）。
图2是表示纤维状脱钙骨基质（A）和颗粒状脱钙骨基质（B）的形态的光学显微镜测定照片。
具体实施方式
本发明提供一种纤维状脱钙骨基质的制备方法，其包括下述步骤：（a）将分离到体外的骨在酸性溶液中进行1-5小时的第1次脱钙；（b）将从步骤（a）得到的骨切割成0.1-3mm厚的片状骨；（c）将从步骤（b）得到的片状骨在酸性溶液中进行2-6小时的第2次脱钙；及（d）将从步骤（c）得到的经过脱钙的骨进行粉碎。
本发明的制备方法依次进行部分脱钙、切割、完全脱钙及粉碎步骤，尤其是通过部分脱钙及切割步骤，不仅能够将接下来的脱钙步骤显著缩短至约6小时左右，还能够高收率制备具有纤维形态的脱钙骨基质。
本发明的制备方法包括部分脱钙步骤，即将分离到体外的骨在酸性溶液中进行1-5小时的第1次脱钙的步骤（即步骤（a））。所述分离到体外的骨可以是包括人类的哺乳动物的骨。所述分离到体外的骨优选按照通常的方法将附着于骨的软组织、脂肪、骨髓等去除之后使用。所述去除通常可以使用60-90重量%的乙醇溶液进行，根据需要可以额外使用表面活性剂。所述酸性溶液通常可以使用HCl水溶液，可以使用0.1-3N的HCl水溶液，优选使用约0.6N的HCl水溶液。所述第1次脱钙时间可以是1-5小时，优选约3小时。
本发明的制备方法包括将部分脱钙的骨切割成片状形态的切片步骤，即，将从部分脱钙步骤中得到的骨切割成0.1-3mm厚的片状骨的步骤（即步骤（b））。所述切割只要是能够将骨切成薄片的机器均可使用，例如可以使用骨切割机（Bone Slicer）（YOU IL MC/CO.KR）等的骨切片器（bone slicer）。通过所述切割步骤得到的片状骨的厚度为0.1-3mm，优选0.2-1.0mm，更优选0.3-0.6mm，最优选约为0.5mm。厚度超过3.0mm时，骨形态为粉碎的颗粒形态而不是片状；厚度小于0.1mm时，虽然能够得到片状，但即使脱钙也无法得到纤维状而只能够得到颗粒状。
本发明包括将部分脱钙的片状骨完全脱钙的步骤，即将上述得到的片状骨在酸性溶液中进行2-6小时的第2次脱钙的步骤（即步骤（c））。所述酸性溶液通常可以使用HCl水溶液，可以使用0.1-3N的HCl水溶液，优选使用约0.6N的HCl水溶液。本发明公开了以下内容，第2次脱钙，即完全脱钙可以以相比以往的脱钙时间显著缩短的2-6小时，优选约3小时以内的时间进行。
本发明包括将完全脱钙的骨进行粉碎的步骤（即步骤（d））。所述粉碎步骤可以使用通常的粉碎机实施。其中，优选地以获得具有1000-5000μm，更优选地以获得2000-4000μm范围长度的纤维状脱钙骨基质来实施所述粉碎，所述粉碎可以根据粉碎机的种类来设定适合的粉碎条件，从而获得上述纤维状脱钙骨基质。
下面，通过实施例详细说明本发明。但是，以下实施例仅是例示本发明，本发明并不仅限于此。
实施例1
将来自人供体的骨（重量：约172g）利用手术器械去除附着在骨上的软组织，使用含有表面活性剂的组织洗涤剂去除软组织、脂肪及骨髓等杂质，然后切断成两半。将切断的骨在相对于每克骨重量为20ml的0.6N的HCl水溶液中浸渍3小时，从而进行部分脱钙。将部分脱钙的骨进行回收，在相对于每克骨重量为20ml的蒸馏水中浸渍，从而去除骨中的脱钙溶液。将得到的骨利用骨切片器（bone slicer，YOU IL MC/CO.KR）切片成0.5mm厚度的片状。得到的片状骨如图1A所示。将得到的片状骨在相对于每克骨重量为0.6N的HCl水溶液30ml中浸渍3小时，进行完全脱钙。将脱钙骨基质进行沉淀而分离后，放入粉碎机（IKA，M20通用粉碎机（Univeral mill），GR）中粉碎约10分钟，然后使用磷酸缓冲液（PBS）进行中和，用蒸馏水洗涤，然后进行冷冻干燥得到约31g的脱钙骨基质。利用光学显微镜观察得到的脱钙骨基质的照片如图2A所示。从图2A中可以确认得到的脱钙骨基质为纤维状脱钙骨基质。
比较例1
将来自人供体的骨（重量：约165g）利用手术器械去除附着在骨上的软组织，使用含有表面活性剂的组织洗涤剂去除软组织、脂肪及骨髓等杂质，然后切断成两半。将切断的骨在相对于每克骨重量为0.6N的HCl水溶液20ml中浸渍3小时，从而进行部分脱钙。将部分脱钙的骨进行回收，在相对于每克骨重量的蒸馏水20ml中浸渍，从而去除骨中的脱钙溶液。将得到的骨在相对于每克骨重量为0.6N的HCl水溶液30ml中浸渍3小时，再次进行脱钙步骤，但是脱钙几乎没有进行，无法得到脱钙骨基质。
比较例2
将来自人供体的骨（重量：约192g）利用手术器械去除附着在骨上的软组织，使用含有表面活性剂的组织洗涤剂去除软组织、脂肪及骨髓等杂质，然后切断成两半。将切断的骨在相对于每克骨重量为0.6N的HCl水溶液20ml中浸渍3小时，从而进行部分脱钙。将部分脱钙的骨进行分离，在相对于每克骨重量的蒸馏水20ml中浸渍，从而去除骨中的脱钙溶液。将得到的骨放入粉碎机（IKA，M20通用粉碎机，GR）中粉碎约60分钟得到粉碎的骨，其形态如图1B所示。将上述粉碎的骨在相对于每克骨重量为0.6N的HCl水溶液30ml中浸渍3小时，进行完全脱钙。脱钙骨基质进行沉淀分离后，使用磷酸缓冲液（PBS）进行中和，用蒸馏水洗涤，然后进行冷冻干燥得到约36.4g的脱钙骨基质。利用光学显微镜观察得到的脱钙骨基质的照片如图2B所示。从图2B中可以确认得到的脱钙骨基质为颗粒状脱钙骨基质。

Claims (3)

1.一种纤维状脱钙骨基质的制备方法，其特征在于，该方法包括下述步骤：(a)将分离到体外的骨在0.1-3N的HCl水溶液中进行1-5小时的第1次脱钙；(b)将从步骤(a)得到的骨切割成0.1-3mm厚的片状骨；(c)将从步骤(b)得到的片状骨在0.1-3N的HCl水溶液中进行2-6小时的第2次脱钙；及(d)将从步骤(c)得到的经过脱钙的骨进行粉碎的步骤。
2.根据权利要求1所述的纤维状脱钙骨基质的制备方法，其中，所述步骤(a)和步骤(c)中的所述HCl水溶液为0.6N的HCl水溶液。
3.根据权利要求1或2所述的纤维状脱钙骨基质的制备方法，其中，以获得具有1000-5000μm范围长度的纤维状脱钙骨基质来实施所述粉碎。
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