Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy

Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 +/- 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 +/- 2.8 microm. In addition, we could calculate that each osteocyte spans an average of 4180 +/- 673 microm3 of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 +/- 0.02 microm per 1 microm3 bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 +/- 0.03 microm2 per 1 microm3 bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% +/- 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 +/- 145 microm, 1509 +/- 113 microm2, and 394 +/- 49 microm3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.