In situ measurement of collagen synthesis by human bone cells with a Sirius Red-based colorimetric microassay:: effects of transforming growth factor β2 and ascorbic acid 2-phosphate

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid beta were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples.