Oral and maxillofacial surgery
Characterization of primary osteocyte-like cells from rat mandibles

https://doi.org/10.1016/j.oooo.2016.08.020Get rights and content

Objective

The mandible is continuously undergoing remodeling as a result of mechanobiologic factors, such as chewing forces, tooth loss, orthodontic forces, and periodontitis. The effects of mechanical stress and biologic signals in bone homeostasis have been the focus of many investigations. However, much of this research utilized osteocytes derived from long bones, but little is known about the mandible-derived osteocytes. This study tests a protocol to isolate and grow osteocytes from rat mandible.

Study Design

Rat mandibles were harvested, sectioned into small pieces, and subjected to a sequence chemical treatment and enzymatic digestion. The treated tissues were cultured for a few weeks while cells emerged. Cells were sorted by using the osteocyte marker podoplanin, an early marker for osteocyte differentiation. The cells were then characterized according to morphology, biochemical markers (osteocalcin, podoplanin, and sclerostin), and alkaline phosphatase activity and compared with an isotype cell line MLO-Y4 cells.

Results

The mandibular osteocytic cells had stellate shape and were positive for osteocalcin, podoplanin, and sclerostin and lower alkaline phosphatase activity compared with MLO-Y4 osteocyte-like cells.

Conclusions

The protocol to isolate osteocyte-like cells will allow the investigators to investigate the mechanobiologic differences in biomechanical response between these mandibular and long bone osteocyte-like cells under various conditions.

Section snippets

Materials and Methods

This investigation conforms to the guide for the care and use of laboratory animals published by the US National Institutes of Health. The study was approved by Georgia Regents University Institutional Animal Care and Use Committee.

Results

Isolation of osteocyte-like cells from mandibular bone is a difficult process because these cells exist within the bone matrix. Multiple steps of bone cutting and sequential digestion process were performed to treat mandibular bone fragments. The fragments were cultured in petri dishes (see Figure 1A). After 2 weeks in culture, a high number of viable primary osteocyte-like cells migrated out of the explanted bone fragments (see Figure 1B). The cells showed stellate morphology characteristic of

Discussion

Cells isolated from rat mandibles exhibited the stellate morphology characteristic of osteocytes and were podoplanin (E11) positive. Podoplanin is one of the earliest osteocyte-selective markers, as the osteoblast is embedded and differentiates into an osteocyte. This protein first appears on the dentritic processes. It promotes dentritic process elongation and is important for osteocyte cell activity.39 Podoplanin expression is strong evidence of an osteocyte-like phenotype. Morphologically,

Conclusions

In summary, we propose that a protocol for isolation of mandibular cells exhibited cells with osteocyte-like morphology that tested positive for osteocytic biochemical markers osteocalcin, podoplanin (sorting and immunohistochemistry), and sclerostin. Furthermore, the cells demonstrated low alkaline phosphatase activity. This profile supports the identity of these primary mandibular cells as being osteocyte-like cells. Osteoblasts from the tibia and the mandible have differences in

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      Nuclei of at least 50 osteocytes were analyzed for each sample. For primary jawbone osteocyte-like cell isolation, soft tissues and teeth were removed and remaining bone fragments were processed using a modified protocol derived from Stern et al. and El Deeb Zakhary et al. combined methods [21,25]. Briefly, bone fragments from alveolar bone and ramus were independently digested with collagenase (Liberase; Roche Diagnostics GmbH, Mannheim, Germany) dissolved in α-MEM and incubated at 37 °C for 20 min (four incubations).

    This work was supported by a National Institutes of Health grant (R15 de022455-01 A1) to R.L.W.M. The MLO-Y4 cells were a gift from Lynda Bonewald.

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