FLAT BED SCANNER IN THE QUANTITATIVE ASSAY OF 35SO4-INCORPORATION BY X-RAY-FILM AUTORADIOGRAPHY OF INTERVERTEBRAL-DISK SECTIONS

A rapid quantitation of proteoglycan synthesis distribution in intervertebral disc and endplates is described. Tissue blocks of disc (C7-Th1) in the midsagittal plane from ten female beagles were incubated in the presence of (SO4)-S-35 and prepared as histological slides. For comparison, sulphate incorporation rates in the C5-C6 discs were assayed by liquid scintillation. Autoradiographic film exposed against the labelled sections was developed and digitized for image analysis using a 256 grey level flat bed table scanner connected to a microcomputer. The film density versus dpm (disintegrations per minute) calibration was performed using a set of (SO4)-S-35-labelled glycosaminoglycan standards applied on the same film. Since section thickness, dpm calibration of the film density and the specific activity of sulphate in the medium were known, the incorporations per tissue volume could be calculated. The average incorporation rates of the anterior and posterior annulus fibrosus, nucleus pulposus and vertebral endplates were 5.2 + 0.9, 5.2 +/- 0.8, 4.5 +/- 0.6 and 4.1 +/- 0.8 pmol/mm3 per h (+/- SE, n = 10), respectively and closely corresponded to those obtained by liquid scintillation. This method offers a convenient and reproducible way to measure the rate of proteoglycan synthesis in large tissue sections but also in thin cartilaginous tissues such as the vertebral endplate.