The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.

The coordinated actions of three different types of bone cells are required for bone remodeling in response to mechanical loading. On the surface of bone are osteoblasts, which form new bone, and osteoclasts, which remove bone. Located deeper within the bone matrix and housed in cave-like lacunae, are osteocytes, which function as the mechanosensory system within bone. Osteocytes have a dendritic morphology with many cell processes extending through canaliculi to form a highly connected communication network between themselves and the bone surface cells. When mechanical loads are applied to bone, they are sensed by osteocytes, which translate signals provided by mechanostimulation into biochemical signals. These signals are believed to regulate the actions of osteoblasts and osteoclasts, thereby providing a mechanism to regulate bone deposition and absorption according to the local mechanical requirements of the bone (1-3).

Osteocytes are the most abundant of the three bone cell types; however, the least is known about them. While their location deep within the bone matrix makes them ideally situated to sense bone strain, it also makes their observation and study in vivo difficult. Additionally, primary osteocytes, particularly those within the long bones of skeletally mature animals, have proven difficult to obtain and study ex vivo. Furthermore, once primary osteocytes are obtained, their study is often limited by their inability to proliferate as they are considered terminally differentiated cells.

To facilitate the study of osteocytes in vitro, Kato et al. created the immortalized osteocyte-like MLO-Y4 line from osteocytes isolated from the long bones of 6-week-old transgenic mice, in which the SV40 large T-antigen oncogene is expressed under the control of an osteocalcin promoter (4, 5). The MLO-Y4 cell line is well-characterized and represents the phenotype of early osteocytes (4, 6). This osteocyte-like cell line has been used to study communication between osteocytes, as well as communication between osteocytes and bone surface cells (7-13). MLO-Y4 cells have also been used in the investigation of the response of osteocytes to various types of physical stimulation, including fluid flow and mechanical perturbations (7-20). Although the MLO-Y4 cell line is a very powerful tool for the study of osteocytes in vitro, there are known differences between primary osteocytes and the immortalized MLO-Y4 cell line. For example, MLO-Y4 cells express low to undetectable levels of Dentin matrix protein 1 (Dmp1) and Sclerostin (Sost), while osteocytes are known to express these genes in vivo (6, 21-29). The MLO-A5 cell line has characteristics of a postosteoblast/preosteocyte. These cells are very large, over 100 nm, express all of the markers of the late osteoblast such as extremely high alkaline phosphatase (ALP), bone sialoprotein, PTH type 1 receptor, and osteocalcin, and rapidly mineralize in sheets, not nodules (30). In culture, these cells begin to express markers of osteocytes as they generate cell processes such as E11 (31), but express low levels of Sost.

Recently, a cell line has been generated that differentiates from the late osteoblast to the late osteocyte called IDG-SW3, made by crossing the Immortomouse with the 8-kb Dmp1-GFP transgenic mouse line. IDG-SW3 cells synthesize and mineralize a “honeycomb-like” matrix rich in type-I collagen similar to MLO-A5 cells. Similar to osteocytes in vivo, these cells express osteocyte marker genes from the early osteocyte marker E11 to the mature osteocyte marker sclerostin, down-regulate Sost expression with PTH treatment and increase Fgf23 mRNA expression in response 1,25-dihydroxyvitamin D₃ (32). This cell line faithfully recapitulates the differentiation process from osteoblast to late osteocyte as observed in vivo.

While these cell lines are convenient and useful, an ideal strategy for the study of osteocyte function ex vivo, and validation of results obtained with cell lines is to develop better methodology for the isolation and study of primary osteocytes.

Primary osteocytes have most commonly been isolated from 16-or 18-day-old chick calvaria (15, 33-44) or from newborn through 4-day-old rat calvaria, 12-day-old mouse calvaria (45), and 3-to 4-week-old mouse calvaria and long bones (46). More recently, osteocytes have also been isolated from the long bones of 3-day-old Sprague-Dawley rats (33). Calvaria from young chicks and neonatal rats and mice, as well as the long bones from neonatal rats and young mice (3–4 weeks) are all very thin and easily processed using sequential digestions with EDTA and collagenase. Studies utilizing these primary osteocytes can provide insight to the behavior of osteocytes during development but do not aid in the study of osteocytes from skeletally mature animals or enable the comparison between osteocytes isolated from skeletally mature but relatively young mice (4-to 6-month) and aged mice (>22-month-old).