T. Kumagai et al., ULTRASTRUCTURAL-LOCALIZATION AND BIOCHEMICAL-CHARACTERIZATION OF VITRONECTIN IN DEVELOPING RAT BONE, Histochemical Journal, 30(2), 1998, pp. 111-119
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.