Abstract
Presently, no intra-operative method for a direct assessment of bone vitality exists. Therefore, we set out to test the applicability of tetrazolium-based staining on bone samples. The explanted femoral heads of 37 patients were used to obtain either cancellous bone fragments or bone slices. Samples were stained with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue, MTT) at different times (one to twelve hours) after explantation. Staining was quantified either spectrophotometrically after extraction of the dyes or by densitometric image analysis. TTC-staining of cancellous bone fragments and bone slices, respectively, indicated the detectability of vital cells in both types of samples in a window of up to six hours after explantation. Staining intensity at later time-points was indistinguishable from the staining of untreated samples or sodium azide treated samples, which represent dead cells. In contrast, MTT-staining of bone slices revealed intense unspecific staining, which obscured the evaluation of the vitality of the samples. The lack of a detectable increase of colour intensity in TTC-stained bone samples, which were treated more than six hours after explantation, corresponds to reduced fracture healing. The described simple procedure could provide a basis for an intraoperative decision by the orthopaedic surgeon.
Highlights
Macroscopic-histological examination via 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) is an established method for the determination of structural damages in organs
Samples were stained with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at different times after explantation
The lack of a detectable increase of colour intensity in TTC-stained bone samples, which were treated more than six hours after explantation, corresponds to reduced fracture healing
Summary
Macroscopic-histological examination via 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) is an established method for the determination of structural damages in organs. Hitherto, this staining method has been used on tissue samples of the brain and the heart, to promptly assess the vitality of the respective tissue after organ removal. The conversion of the dye’s precursor to a visible dye is hindered in damaged tissue because of a lack of mitochondrial membrane potential (concomitant with shortage of adenosine triphosphate (ATP)) in ischemic areas This mechanism allows a discrimination of damaged or dead tissue (remains unstained) and healthy tissue (reddening) through TTC staining, and enables a quantitative comparison of the formerly named (dead and healthy, respectively) tissues. The results are rated as a reliable parameter for an early visualization of area-specific under-perfusion [1,2,3]
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