Improvements in Dehydration and Cement Line Staining for Methacrylate Embedded Human Bone Biopsies

Abstract

Undemineralized methacrylate embedded bone biopsies and other bone specimens can be processed much more rapidly by application of acidified 2,2-dimethoxypropane (DMP) dehydration, which requires two hours, than by traditional graded ethanol dehydration, which requires at least four days. This shortened processing time is valuable when biopsy results are urgently needed to detect osteomalacia or to determine bone features prior to possible parathyroidectomy. We have processed over 200 bone specimens with DMP and have compared DMP dehydration to graded ethanol dehydration in 11 biopsies in which two plugs were available from the same patient. DMP dehydration does not compromise the following: tetracycline retention, Goldner's stain, acid phosphatase localization or histochemical identification of aluminum. Cement lines, which provide a record of past remodelling, are useful in clinical interpretation of bone biopsies. We have adapted two stains, toluidine blue and methylene blue/basic fuchsin, for improved cement line identification. Five-micrometer sections individually demineralized in acetate buffer prior to cement line staining show best results with toluidine blue at pH 5.5 and with methylene blue/basic fuchsin at pH 2.5-3.5.

Interactions between microstructural and geometrical adaptation in human cortical bone

“…Following staining, the undecalcified cross sections were sectioned and polished to 100 mm thickness. The use of two different stains for proximal and distal middiaphyseal sections was for identifying microdamage by basic fuchsin 18 and cement lines by toluidine blue 19 which were unrelated to this investigation.…”

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail

“…Many modifications of protocols have been made through the years to optimize the use of MMA for light microscopy of various tissues, especially bone (Gruber et al 1985, Mueller et al 2000, ChungChing 1987, Wolf et al 1992and Erben 1997. We have modified more recent protocols further to develop a procedure suitable for use with newborn mouse calvaria.…”

Application of Stains-All for Demarcation of Cement Lines in Methacrylate Embedded Bone

“…Bone specimens were fixed in 10% neutral buffered formalin and embedded in glycol methacrylate or dehydrated and embedded in methyl methacrylate as previously described (Gruber et al 1985(Gruber et al , 1988. Briefly, the glycol methacrylate method used the JB-4 embedding system (Polysciences, Inc., Warrington, PA) and specimens were infiltrated in a graded series of distilled HzO-glycol methacrylate and embedded on plastic block holders.…”

“…The application of Stains-all we report here for demarcation of cement lines in bone is simple and, a s an advantage over our previously reported methods (Gruber et al 1985), does not require prior section demineralization.…”