A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy

Abstract

Osteocytes are the most numerous cells in bone, embedded within the mineralized bone matrix. Their slender cytoplasmic processes form a complex intercellular network. In addition, these processes are thought to be important structures in the response to mechanical stress. This study provides an extensive analysis of the three-dimensional structure of the osteocyte and its processes in 16-day-old embryonic chick calvariae, based on nondestructive subsurface histotomography using both confocal laser scanning (CLS) microscopy and differential interference contrast (DIC) microscopy. OB7.3, a chicken osteocyte-specific monoclonal antibody, and Texas Red-X-conjugated phalloidin were used to confirm the osteocyte phenotype and to identify whole cells in the calvariae, respectively. Serial CLS images revealed morphological changes in bone cells up to 20 μm in depth. Osteocytes had widely spread their processes into the osteoblast layer, and we found for the first time that some of these processes had elongated to the vascular-facing surface of the osteoblast layer. Furthermore, stereotype images reconstructed from CLS images could show the three-dimensional distribution of these processes. Using the stereopair image, we could evaluate the frequency of processes between osteocytes and osteoblasts. Complementation of DIC microscopy revealed canaliculi and lacunae with high contrast. The distributional pattern of canaliculi generally coincided with that of the osteocyte processes. We consider that the combination method of CLS microscopy and DIC microscopy using a laser scanning microscope is a very useful new technical approach for investigating osteocytes in bone.

Introduction

The great majority of bone cells are osteocytes derived from osteoblasts. There are approximately 10 times as many osteocytes as osteoblasts in normal human bone.21 Therefore, several functions have been proposed for osteocytes.1 These include osteocytes as a calcium-sensor,8, 9, 26 regulator for osteoid matrix maturation and mineralization,14 and mechanosensor.2, 4, 11, 24, 25 It is thought that osteocytes acquire such functions with after having become embedded in the bone matrix. During the process of encasement, the prospective osteocytes change their shape and generate long processes that comprise a complex intercellular communication network.5 Especially, osteocytes processes are thought to be an important means by which cells translate the mechanical stress into biological response.15 In addition, these processes have now become recognized to play an important role in modulating osteoblast activity, recruiting osteoblasts that become transformed into osteocyte.17, 19 Thus, the study of the anatomical status of osteocyte processes is significant.

To date, most previous studies on osteocyte processes have been based on conventional transmission electron microscopy of demineralized bone tissue, where the tissue needs to be cut into 60–100-nm-thin slices.6, 7, 18, 19 As a result, comprehension of three-dimensional (3D) relationship between structures is difficult. To study the osteocyte environment in undecalcified specimen, confocal laser scanning (CLS) microscopy, which allows the nondestructive histotomography of bone, was employed.3 Although this breakthrough improved the accessibility for investigating the osteocyte environment, the 3D network of the osteocyte processes and osteoblasts is still unknown. Furthermore, the solo use of CLS microscopy was not sufficient to determine the relationship between osteocyte processes and the surrounding tissue because of the lack of simultaneous information about the lacunar wall and canalicular walls. On the other hand, differential interference contrast (DIC) microscopy is a potent tool for obtaining 3D images without any preparation of the specimen.10, 23

The present study is based on the combination of CLS microscopy and DIC microscopy using a laser scanning microscope for observation of osteocytes in mineralized embryonic chicken calvariae. CLS microscopy images identified whole cells stained with Texas-Red-X phalloidin as well as osteocytes immunofluorescently labeled by monoclonal antibody OB7.3, which specially identifies chicken osteocytes.16 Complementation of CLS microscopy with DIC microscopy in the same confocal layer revealed the 3D organization of the cells, the lacunar wall, and canalicular walls. In the present study, we found for the first time that osteocytes elongate their processes to the vascular-facing surface of osteoblast layer through the intercellular spaces of osteoblasts and evaluated the frequency of processes between osteocytes and osteoblasts.