Isolation of calcospherulites from the mineralization front of bone.
Isolation of calcospherulites from the mineralization front of bone.
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DOI：
10.1159/000152914
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Gorski JP
中科院分区：
文献类型：
--
作者：
Midura RJ;Vasanji A;Su X;Midura SB;Gorski JP
含钙球体（钙球粒）存在于骨的矿化前沿，被认为在骨形成中发挥作用。现有的分离钙球晶的方法涉及严酷的处理，以去除大部分有机物。这项研究试图使用破坏性较小的方法来分离它们，以更好地保存其有机成分。幼年大鼠被注射低剂量的钙黄绿素，以标记体内骨矿化前沿新形成的矿物质。将骨膜从胫骨骨干上完全解剖，并通过胶原酶去除未矿化的类骨质基质，以暴露钙球粒。沿着矿化前沿观察到平均直径约为 0.5 μm 的钙黄绿素标记的钙球晶，它们的原位 Ca/P 比为 1.3。通过短暂的分散酶消化，钙球晶从矿化前沿释放出来，并通过荧光流式分选进行分离。 X 射线衍射显示它们含有磷灰石晶体（c 轴长度为 17.5 ± 0.2 nm），并且它们的 Ca/P 比在分离过程中保持不变。用冰冷乙醇处理的钙球晶表现出 1.6 的 Ca/P 比，表明存在一些可提取的磷脂。从分离的钙球晶中提取的蛋白质通过 SDS-PAGE 分解成 20 多个不同的条带。蛋白质印迹分析显示这些制剂中存在基质蛋白。这些结果表明钙球石可以从骨的矿化前沿分离出来，其形式可用于研究其蛋白质组和脂质组成。
Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 μm average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 ± 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.
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影响因子：
4.1
作者：
Midura, Ronald J.;Vasanji, Arnit;Gorski, Jeff P.
通讯作者：
Gorski, Jeff P.
DOI：
10.1023/a:1003707909842
发表时间：
1999-07-01
期刊：
HISTOCHEMICAL JOURNAL
影响因子：
--
作者：
Aaron, JE;Oliver, B;Carter, DH
通讯作者：
Carter, DH
DOI：
10.1007/bf02013233
发表时间：
1978-01-01
期刊：
CALCIFIED TISSUE RESEARCH
影响因子：
--
作者：
BOYDE, A;SELA, J
通讯作者：
SELA, J
影响因子：
4.8
作者：
Midura, RJ;Wang, AM;Gorski, JP
通讯作者：
Gorski, JP
影响因子：
4.8
作者：
Gorski, JP;Wang, AM;Midura, RJ
通讯作者：
Midura, RJ