Quantification of the bone-related mRNAs at the bone/prosthetic interface

Aseptic loosening is one of the major reasons for failure of joint prosthes es. The periprosthetic tissue has previously been described microscopically ; however, little work has been devoted towards quantitating genes expresse d by cells at the materials/tissue interface. This study aims to characteri ze the phenotypic expression of osteoblasts and test the feasibility of qua ntifying the level of gene expression in periprosthetic tissue sections by combining in situ hybridization and image analysis techniques. There are ma ny factors to consider when quantifying mRNA, in that comparing labeling be tween different cDNA probes, these should have comparable length and base c omparison. The probes should be labeled with the same specific activity, th at is the amount of probe to label added is the same, both between differen t probes and between batches of the same probe. Chromagen color reactions a re variable in that the color development is not always linear and more lik ely follows a sigmoidal curve. Samples should only be compared when it is k nown that the reaction has been in the linear range. The image analysis of such staining introduces further factors which should be considered and con trolled. Color analysis is a very complex problem with respect to reproduci bly analyzing histological sections. The brightness component of the image should be independent of the colors within the image, in conventional RGB ( red, green and blue) signalling mode this is not possible, while when using HSI (hue saturation and intensity) mode this becomes possible, and factors like staining intensity and brightness of the image become much more accou ntable and controllable. With these factors identified, we consider that th e quantitative image analysis approach does allow comparison of patterns of bone-related mRNAs and demonstrates differences in expression in these ost eogenic factors depending on distance from the prosthesis, tissue type, pat ient and device. (C) 1998 Kluwer Academic Publishers.