ULTRASTRUCTURAL-LOCALIZATION AND BIOCHEMICAL-CHARACTERIZATION OF VITRONECTIN IN DEVELOPING RAT BONE

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in ea rly bone formation of developing rat mandible with rabbit antimurine v itronectin IgG. Developing jaws of foetuses were collected at embryoni c day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydra ted and embedded in paraffin, Spurr's resin or LR gold resin for morph ological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was obs erved in small elongated areas of intercellular matrix and osteoblasts . Concomitant with initiation of matrix mineralization at day 16, vitr onectin staining was similarly observed in small elongated areas conta ining intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the r ough-surfaced endoplasmic reticulum and the Golgi apparatus of osteobl asts as well as over demineralized bone matrix at day 16-18. In order to assess the presence of vitronectin in the mineral phase, mineral-bi nding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4 M guanidine HCl (G1 extract), aqueous EDTA wi thout guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed that the antibodies produced only a sing le band at an M-r of approximately 73 000 in both G1 and E extracts, i ndicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblas ts synthesize and release vitronectin, which is subsequently incorpora ted into the bone matrix and becomes a specific component of bone tiss ues. The observation of vitronectin in these critical stages of bone f ormation suggests that it may be involved in the regulation of bone fo rmation. (C) 1998 Chapman & Hall.