IMMUNOFLUORESCENCE AND CONFOCAL LASER-SCANNING MICROSCOPY STUDIES OF OSTEOBLAST GROWTH AND PHENOTYPIC-EXPRESSION IN 3-DIMENSIONAL DEGRADABLE SYNTHETIC MATRICES

In the development of three-dimensional cell-polymer synthetic matrice s for tissue regeneration, visualization of cells growing in these por ous structures can be difficult. The focus of this study was the devel opment and use of a novel method that would allow for visualization of osteoblasts inside opaque matrices. The morphologic responses and phe notypic characterization of osteoblasts as they attach, spread, and mi grate through a porous three-dimensional biodegradable polymer-ceramic matrix in vitro were studied using immunofluorescence and confocal la ser scanning microscopy (CLSM). CLSM offers several advantages over th e most commonly used imaging methods [traditional light microscopy and scanning electron microscopy (SEM)]. CLSM filters out-of-focus backgr ound and provides more structural details of cells. In addition, CLSM does not require extensive sample preparation as does SEM. When used i n conjunction with fluorescence-labeled antibodies to identify cells a nd 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 ma trix were found to have preserved osteoblast-like phenotype with growt h. The morphology of cells throughout the matrix was found to be simil ar to osteoblast cells grown on tissue culture polystyrene and consist ed of spread polygonal forms. Using the technique of CLSM with immunof luorescent antibodies, we have demonstrated for the first time that th ese three-dimensional degradable polymer matrices can support osteobla st growth and phenotypic expression throughout its structure. (C) 1995 John Wiley and Sons, Inc.