Calcospherulites isolated from the mineralization front of bone induce the mineralization of type I collagen
Calcospherulites isolated from the mineralization front of bone induce the mineralization of type I collagen
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DOI：
10.1016/j.bone.2007.08.036
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发表时间：
2007-12-01
期刊：
影响因子：
4.1
通讯作者：
Gorski, Jeff P.
中科院分区：
文献类型：
--
作者：
Midura, Ronald J.;Vasanji, Arnit;Gorski, Jeff P.
以前的工作表明，“钙球粒”积极参与骨发育和愈合的矿化。本研究试图通过开发一种分离钙球晶的方法并分析其对纤维状胶原蛋白矿化的能力来直接测试这一假设。未成年大鼠胫骨骨干的骨膜表面富含类似于0.5 μ m直径的球晶，表现出Ca/P比为1.3。体内注射后24小时，其在胫骨矿化前沿吸收钙黄绿素，证实了其作为钙球粒的身份。切开骨膜，用胶原酶去除未矿化的类骨质，以暴露钙球粒。然后通过分散酶消化从矿化前沿释放钙黄绿素标记的钙球粒，并通过荧光流式分选分离。X射线衍射分析显示它们含有磷灰石晶体（c轴长度为17.5 +/-0.2nm），尽管它们的Ca/P比为1.3低于羟基磷灰石。它们的大部分非矿物磷含量被冰冷的乙醇除去，使它们的Ca/P比升高到1.6，表明磷脂的存在。蛋白质印迹分析表明，在这些制剂中的骨基质蛋白和I型胶原蛋白的存在。在胶原蛋白水凝胶中孵育分离的钙球粒表明，它们可以在体外对I型胶原纤维进行矿化反应。超微结构分析显示胶原纤维上的晶体沿纤维长度沿着均匀分布。此外，在远离所观察到的钙球粒的距离处观察到晶体。我们的研究结果直接支持钙球粒在诱导骨矿化前沿的I型胶原纤维矿化中的积极作用。(C)2007年爱思唯尔公司All rights reserved.
Previous work has suggested that "calcospherulites" actively participate in the mineralization of developing and healing bone. This study sought to directly test this hypothesis by developing a method to isolate calcospherulites and analyzing their capacity to seed mineralization of fibrillar collagen. The periosteal surface of juvenile rat tibial diaphysis was enriched in spherulites of similar to 0.5-mu m diameter exhibiting a Ca/P ratio of 1.3. Their identity as calcospherulites was confirmed by their uptake of calcein at the tibial mineralization front 24 h following in vivo injection. Periosteum was dissected and unmineralized osteoid removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites were then released from the mineralization front by dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction analysis revealed they contained apatite crystals (c-axis length of 17.5 +/- 0.2 nm), though their Ca/P ratio of 1.3 is lower than that of hydroxyapatite. Much of their nonmineral phosphorous content was removed by ice-cold ethanol, elevating their Ca/P ratio to 1.6, suggesting the presence of phospholipids. Western blot analyses showed the presence of bone matrix proteins and type I collagen in these preparations. Incubating isolated calcospherulites in collagen hydrogels demonstrated that they could seed a mineralization reaction on type I collagen fibers in vitro. Ultrastructural analyses revealed crystals on the collagen fibers that were distributed rather uniformly along the fiber lengths. Furthermore, crystals were observed at distances well away from the observed calcospherulites. Our results directly support an active role for calcospherulites in inducing the mineralization of type I collagen fibers at the mineralization front of bone. (C) 2007 Elsevier Inc. All rights reserved.