Key Words: immunohistochemistry, osteocalcin, osteopontin, bone sialoprotein, alkaline phosphatase, procollagen type I, Technovit 9100 New, acrylate-embedding, undecalcified cancellous bone, histomorphology

Publication date: December 31st 2003

Abstract: Trabecular bone is routinely analysed by histomorphological-histometrical and immunohistochemical techniques as means of assessing the differentiation status of bone deposition and growth. Currently few embedding resins exist for which both morphological and immunohistochemical analyses can be performed on mineralised tissue. Paraffin, the standard embedding medium for bone enzyme and immunohistochemistry, can only be used on demineralised tissue, but then trabecular structure may be badly preserved. Methyl methacrylate (MMA), the resin of choice for undecalcified bone histology can only be used for bone immunohistochemistry if the usual, highly exothermic polymerisation procedure is avoided which destroys tissue antigenicity. Consequently, most current practices involve cutting samples in half to be processed in separate resins when more than one type of analysis is required. Technovit 9100 New® is a low temperature MMA embedding system that is purported to significantly improve tissue antigenicity preservation allowing polymerisation at -20°C. In this study, Technovit 9100 New®-embedded undecalcified trabecular bone samples (adult human, young bovine and ovine) yielded immunolabelling with several bone matrix markers and preserved morphological features in 7µm sections when stained with Masson-Goldner, von Kossa, or toluidine blue. Bone samples from all resins used were immunolabelled with antibodies against osteocalcin, alkaline phosphatase, osteopontin, osteonectin, bone sialoprotein and procollagen type I amino-terminal propeptide. Technovit-embedded bone yielded more reliable immunolabelling of the matrix proteins when compared with heat or cold-cured LR White or standard embedded MMA samples. Technovit 9100 New® provided better routine histology than LR White, and was comparable to MMA. Results demonstrated that Technovit 9100 New® can be used as a low-temperature acrylic resin embedding method for routine undecalcified bone histology, as well as for immunohistochemistry.

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