A NOVEL TECHNIQUE FOR QUANTITATIVE DETECTION OF MESSENGER-RNA EXPRESSION IN HUMAN BONE-DERIVED CELLS CULTURED ON BIOMATERIALS

A nonisotopic and quantitative in situ hybridization technique was ada pted to investigate the effect of biomaterials on the cellular express ion of mRNA from human bone derived cells (HBD cells). HBD cells were cultured for 24 or 48 h on tissue culture plastic, alumina, and ion mo dified alumina. Osteocalcin, osteopontin, alkaline phosphatase, type I collagen al, and type I collagen alpha 2 mRNAs were quantified. Prote in expression for collagen types I, III, and V, and for anti-human mac rophages CD68 (DAKO-CD68, KP1) and CD68 (PG-M1), and anti-human myeloi d/histiocyte antigen (DAKO-MAC 387) were determined immunohistochemica lly using monoclonal antibodies. At 24 and 48 h, levels of mRNA for al kaline phosphatase and osteonectin were greater than mRNA levels for o steopontin, osteocalcin, collagen type I alpha 1, and collagen type I alpha 2 for cells grown on the three substrata. However, at 48 h mRNA levels for alkaline phosphatase and osteonectin were significantly hig her on the modified ceramic substrata relative to the native alumina. HBD cells appear to express CD68-KP1 when cultured for 24 h. The techn iques provide a sensitive and reproducible assay to evaluate gene and protein expression of cells grown on different substrata. (C) 1996 Joh n Wiley & Sons, Inc.