Attawia M A, Devin J E, Laurencin C T
Helen I. Moorehead-Laurencin Biomaterials Research Laboratory, Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, USA.
J Biomed Mater Res. 1995 Jul;29(7):843-8. doi: 10.1002/jbm.820290709.
In the development of three-dimensional cell-polymer synthetic matrices for tissue regeneration, visualization of cells growing in these porous structures can be difficult. The focus of this study was the development and use of a novel method that would allow for visualization of osteoblasts inside opaque matrices. The morphologic responses and phenotypic characterization of osteoblasts as they attach, spread, and migrate through a porous three-dimensional biodegradable polymer-ceramic matrix in vitro were studied using immunofluorescence and confocal laser scanning microscopy (CLSM). CLSM offers several advantages over the most commonly used imaging methods [traditional light microscopy and scanning electron microscopy (SEM)]. CLSM filters out-of-focus background and provides more structural details of cells. In addition, CLSM does not require extensive sample preparation as does SEM. When used in conjunction with fluorescence-labeled antibodies to identify cells and their products, it can characterize morphology of growing cells and successfully determine phenotypic function. Using monoclonal antibody to osteocalcin, a bone cell-specific protein, cells throughout the matrix were found to have preserved osteoblast-like phenotype with growth. The morphology of cells throughout the matrix was found to be similar to osteoblast cells grown on tissue culture polystyrene and consisted of spread polygonal forms. Using the technique of CLSM with immunofluorescent antibodies, we have demonstrated for the first time that these three-dimensional degradable polymer matrices can support osteoblast growth and phenotypic expression throughout its structure.
在用于组织再生的三维细胞 - 聚合物合成基质的研发过程中，观察在这些多孔结构中生长的细胞可能具有挑战性。本研究的重点是开发和使用一种新方法，该方法能够实现对不透明基质内成骨细胞的可视化观察。我们利用免疫荧光和共聚焦激光扫描显微镜（CLSM），研究了体外培养时成骨细胞附着、铺展并迁移通过多孔三维可生物降解聚合物 - 陶瓷基质的形态学反应和表型特征。与最常用的成像方法（传统光学显微镜和扫描电子显微镜（SEM））相比，CLSM具有多项优势。CLSM可滤除离焦背景并提供细胞的更多结构细节。此外，CLSM不像SEM那样需要大量的样品制备。当与荧光标记抗体联合使用以识别细胞及其产物时，它能够表征生长中细胞的形态并成功确定表型功能。利用针对骨钙素（一种骨细胞特异性蛋白）的单克隆抗体，发现整个基质中的细胞在生长过程中保持了成骨细胞样表型。发现整个基质中细胞的形态与在组织培养聚苯乙烯上生长的成骨细胞相似，由铺展的多边形形态组成。通过使用带有免疫荧光抗体的CLSM技术，我们首次证明了这些三维可降解聚合物基质能够在其整个结构中支持成骨细胞生长和表型表达。
