Deegan Anthony J, Aydin Halil M, Hu Bin, Konduru Sandeep, Kuiper Jan Herman, Yang Ying
Institute for Science and Technology in Medicine, School of Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK.
Biomed Eng Online. 2014 Sep 16;13:136. doi: 10.1186/1475-925X-13-136.
Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required.
We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT).
Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture.
This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.
骨组织中的矿化涉及细胞间和细胞与细胞外基质（ECM）的逐步相互作用。调节成骨细胞培养微环境可调整成骨细胞的增殖和矿化速率，以及最终钙化组织的质量和/或数量。因此，迫切需要一种体外模型来研究这些影响因素。
我们开发了一种简便的体外模型，利用成骨细胞系和聚集体培养（通过修饰培养孔表面）来模拟膜内骨矿化。通过成骨基因表达（PCR）和矿化标志物（组织学染色、扫描电子显微镜-能谱分析（SEM-EDX）和显微计算机断层扫描（micro-CT）），研究了培养环境（包括培养持续时间（用于快速矿化研究最长72小时）和聚集体大小（单层培养作为对照））对矿化速率以及矿物质数量/质量的影响。
通过这种简便技术可高产量获得两种大小的聚集体（平均而言，大聚集体为745μm，小聚集体为79μm）。聚集体培养中的细胞在24小时内产生了可见且可量化的矿化基质，而单层培养中的细胞在72小时内未能产生。参与骨形成的重要ECM分子，如I型胶原蛋白、碱性磷酸酶、骨桥蛋白和骨钙素的基因表达随时间变化，在单层培养和聚集体培养之间存在差异，且取决于聚集体大小。单层标本在最初24小时处于增殖阶段，直至72小时仍处于基质合成阶段；而小聚集体在最初24小时和48小时培养时处于成熟阶段，然后在72小时时进入矿化阶段。大聚集体在所有这三个时间点均处于矿化阶段，并且在培养仅7后，产生的骨结节比小聚集体中的大36%，钙含量更高。
本研究证实聚集体培养足以诱导快速矿化，且聚集体大小决定矿化速率。矿物质含量取决于聚集体大小和培养持续时间。因此，我们的培养系统可能为研究成骨细胞谱系不同发育阶段的调节因子提供一个良好的模型。
