Immunolocalization and quantification of noncollagenous bone matrix proteins in methylmethacrylate-embedded adult...

The noncollagenous proteins (NCPs) in the bone matrix comprise growth factors with distinct cellular effects and a series of proteins with less clear biological actions. In order to understand the role of these proteins in bone metabolism and in bone diseases, it is crucial to determine their localization and quantity in normal and pathological bone. We have developed an immunohistochemical method to detect osteopontin, osteocalcin, bone sialoprotein, osteonectin, decorin, biglycan, and the growth factors transforming growth factor-beta, insulin-like growth factor-I, and bone morphogenetic protein-2 both in bone matrix and in bone cells of adult human bone embedded in methylmethacrylate. Immunohistochemistry and standard bone histomorphometry in adjacent sections allows the localization of the proteins to metabolically active sites in bone. The protocol works with several fixatives and with bone specimens obtained and embedded to over 20 years ago. Most importantly, we developed a procedure to specifically stain the mineralized matrix green in combination with a red staining of the NCPs. Using digital image analysis it is possible to quantify the relative amounts of NCPs (microm2 NCP area/microm2 mineralized matrix area). Within one biopsy of normal bone cut at four different heights (at a distance of 100 microm), two adjacent sections were stained either for osteopontin or osteonectin. Thirty trabecular and 20 cortical microscopic fields were measured, and the NCP:mineralized matrix ratio was calculated. Stepwise analysis of the standard error of the mean of the NCP:mineralized matrix ratios showed that measuring about 50 microscopic fields is sufficient to obtain representative data with a small confidence interval. In conclusion, the present procedure enables to quantify NCPs and to relate their presence to metabolically active sites in bone. The quantification provides the opportunity to monitor differences in distribution (e.g., cortical vs. trabecular) and differences between normal and pathological conditions and to assess changes in matrix composition during treatment. This can be done by reanalyzing bone biopsies obtained in the past, e.g., during clinical trials. Therefore, the present technique will be a valuable tool for the study of noncollagenous bone matrix proteins in human bone.