School of Geography and Earth Sciences, Canada; School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada.
Department of Materials Science and Engineering, McMaster University, Hamilton, Ontario, Canada.
Bone. 2020 Jun;135:115304. doi: 10.1016/j.bone.2020.115304. Epub 2020 Mar 5.
Bright-field transmission electron microscope (TEM) images of ion milled or focused ion beam (FIB) sections of cortical bone sectioned parallel to the long axis of collagen fibrils display an electron-dense phase in the gap zones of the fibrils, as well as elongated plates (termed mineral lamellae) comprised of apatite crystals, which surround and lie between the fibrils. Energy dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) studies by others have shown that the material in the gap zones is calcium phosphate. Dark-field (DF) images are capable of revealing the projected position of crystals of apatite in a section of bone. We obtained bright field (BF) images of ion milled sections of bovine femoral cortical bone cut parallel to fibril axes (longitudinal view), and compared them with DF images obtained using the (002) apatite reflection to test a widely held theory that most of the mineral in bone resides in the gap zones. Most apatite crystals which were illuminated in DF images and which projected onto gap zones were extensions of crystals that also project onto adjacent overlap zones. However, in BF images, overlap zones do not appear to contain significant amounts of mineral, implying that the crystals imaged in DF are actually in the interfibrillar matrix but projected onto images of fibrils. However a small number of "free" illuminated crystals did not extend into the overlap zones; these could be physically located inside the gap zones. We note that projections of gap zones cover 60% of the area of any longitudinal field of view; thus these "free" crystals have a high random probability of appearing to lie on a gap zone, wherever they physically lie in the section. The evidence of this study does not support the notion that most of the mineral of bone consists of crystals in the gap zone. This study leaves uncertain what is the Ca-P containing material present in gap zones; a possible candidate material is amorphous calcium phosphate.
明场透射电子显微镜（TEM）图像显示，沿胶原原纤维长轴平行切割的皮质骨离子铣削或聚焦离子束（FIB）切片在原纤维的间隙区显示电子致密相，以及由磷灰石晶体组成的拉长板（称为矿物薄片），这些晶体围绕并位于原纤维之间。其他人的能量色散 X 射线光谱（EDS）和电子能量损失光谱（EELS）研究表明，间隙区的物质是磷酸钙。暗场（DF）图像能够揭示骨切片中磷灰石晶体的投影位置。我们获得了沿原纤维轴（纵向）切割的牛股骨皮质骨离子铣削切片的明场（BF）图像，并将其与使用（002）磷灰石反射获得的 DF 图像进行了比较，以测试一个广泛持有的理论，即骨骼中的大部分矿物质都存在于间隙区。在 DF 图像中被照亮并投射到间隙区的大多数磷灰石晶体是也投射到相邻重叠区的晶体的延伸。然而，在 BF 图像中，重叠区似乎不含有大量矿物质，这意味着在 DF 图像中成像的晶体实际上位于原纤维之间的基质中，但投射到原纤维的图像上。然而，少数“自由”照亮的晶体没有延伸到重叠区；它们可能位于间隙区内部。我们注意到，间隙区的投影覆盖了任何纵向视场的 60%的区域；因此，无论它们在切片中的物理位置如何，这些“自由”晶体都有很高的随机概率出现在间隙区上。本研究的证据不支持大多数骨骼矿物质由间隙区中的晶体组成的观点。本研究使间隙区中存在的含 Ca-P 物质不确定；一种可能的候选物质是无定形磷酸钙。
