Davies J E, Lowenberg B, Shiga A
Centre for Biomaterials, University of Toronto, Ontario, Canada.
J Biomed Mater Res. 1990 Oct;24(10):1289-306. doi: 10.1002/jbm.820241003.
Commercially pure 5-mm-diameter titanium (cpTi) discs received droplet inoculations of cells derived from rat bone marrow and were maintained in supplemented culture medium for 2-3 weeks. The cells and extracellular matrix (ECM) were processed for observation by light (LM), scanning (SEM), and transmission electron (TEM) microscopy. The latter was achieved by freeze-fracturing the solid metal from the resin-embedded tissue using a method which preserved the interface. Surface staining of whole discs revealed cells separated from the metal substratum by areas of ECM which stained positively using von Kossa's method to identify mineralization. At SEM, the ECM comprised dense interwoven collagen fiber networks which were partially obscured by globular masses (GMs). Individual GMs were associated with collagen fibers, especially at fiber intersections. EDAX line scan analysis confirmed the presence of Ca and P in these areas which were assumed to be spheritic foci of calcification since the Ca and P peaks diminished in areas which demonstrated only collagen fibers or the underlying cpTi. TEM examination confirmed the presence of globular mineralization and also revealed the presence of an interfacial zone between the metal substratum and the mineralized ECM elaborated by osteoblasts during the culture period. The interfacial zone comprised two layers, a bonding zone containing few collagen fragments and a ruthenium red positive layer containing more densely packed collagen fibers. We believe that this is the first report of both the formation of bonelike tissue on solid titanium substrata in vitro and demonstration of an interface which bears close morphological similarities to that known to develop in vivo.
将直径5毫米的商业纯钛（cpTi）圆盘接种源自大鼠骨髓的细胞液滴，并在补充培养基中培养2 - 3周。对细胞和细胞外基质（ECM）进行处理，以便通过光学显微镜（LM）、扫描电子显微镜（SEM）和透射电子显微镜（TEM）观察。后者是通过使用一种保留界面的方法对树脂包埋组织中的固体金属进行冷冻断裂来实现的。对整个圆盘进行表面染色显示，细胞通过ECM区域与金属基质分离，这些ECM区域使用冯·科萨法染色呈阳性以鉴定矿化。在扫描电子显微镜下，ECM由密集交织的胶原纤维网络组成，这些网络部分被球状团块（GMs）遮挡。单个GMs与胶原纤维相关联，尤其是在纤维交叉处。能量色散X射线光谱（EDAX）线扫描分析证实这些区域存在钙和磷，由于在仅显示胶原纤维或下层cpTi的区域钙和磷峰减少，这些区域被认为是钙化的球状体病灶。透射电子显微镜检查证实了球状矿化的存在，还揭示了在培养期间金属基质与成骨细胞形成的矿化ECM之间存在一个界面区。界面区由两层组成，一层是含有少量胶原碎片的结合区，另一层是含有更密集排列的胶原纤维的钌红阳性层。我们认为，这是关于体外在固体钛基质上形成类骨组织以及展示与体内已知形成的界面具有紧密形态相似性的界面的首次报道。
