Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique.
BACKGROUND
The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position.


METHODS
The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2-3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures -30 degrees C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding.


RESULTS
Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intracellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them.


CONCLUSIONS
Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions.
Shapiro
F
F
Department of Orthopaedic Surgery, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115.
Cahill
C
C
Malatantis
G
G
Nayak
R C
RC
D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D001857:Bone Matrix / Q000737:chemistry / Q000648:ultrastructure; D017629:Gap Junctions / Q000648:ultrastructure; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D010006:Osteoblasts / Q000737:chemistry / Q000648:ultrastructure*; D010011:Osteocytes / Q000737:chemistry / Q000648:ultrastructure*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014746:Vimentin / Q000032:analysis / Q000648:ultrastructure*
D014746:Vimentin