Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.
The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energydispersive X-ray analysis, and atomic force microscopy. At 3-4 days of culture, spheres of approximately 20-50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5-6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoidosteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization. KeywordsMLO-A5 cells; Mineralization; Osteoblast; Osteoid; Osteocyte Bone cells such as osteoblasts, osteoid-osteocytes, and osteocytes may play different roles in the initiation and regulation of bone mineralization. As early as 1976 and 1981, Bordier and coworkers [1] and Nijweide and coworkers [2] proposed that osteoid-osteocytes play an important role in the initiation and control of matrix calcification. Osteoid-osteocytes were described by Palumbo [3] in 1986 to be cells actively making matrix and calcifying this matrix. Like osteoblasts, their activity was polarized toward the mineralization front to which their cellular processes were oriented. Cellular processes oriented toward blood vessels only began to appear when mineralization began to spread around the cell. She described the cell body reducing in size in parallel with the formation of cytoplasmic processes. This reduction was about 30% at the osteoid-osteocyte stage and 70% with complete maturation of the osteocyte.Correspondence to: C. Barragan-Adjemian; E-mail: Barraganc@umkc.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptOwen [4] stated that during the time for an osteoblast to become an osteocyte, the cell has manufactured three times its own volume in matrix.Mikuni-Takagaki and colleagues [5] proposed that casein kinase II, produced in high amounts by osteoid-osteocytes and not osteoblasts, is responsible for phosphorylation of the matrix proteins necessary for mineralization. Phosphoproteins appear t...
Mineralization of bone matrix and osteocyte differentiation occur simultaneously and appear interrelated both spatially and temporally. Although these are dynamic events, their study has been limited to using static imaging approaches, either alone or in combination with chemical and biochemical analysis and/or genetic manipulation. Here we describe the application of live cell imaging techniques to study mineralization dynamics in primary osteoblast cultures compared to a late osteoblast/early osteocyte-like cell line, MLO-A5. Mineral deposition was monitored using alizarin red as a vital stain for calcium. To monitor differentiation into an osteocyte-like phenotype, the calvarial cells were isolated from transgenic mice expressing green fluorescent protein (GFP) driven by an 8-kb dentin matrix protein-1 (Dmp1) promoter that gives osteocyte-selective expression. Time lapse imaging showed that there was a lag phase of 15–20 h after β-glycerophosphate addition, followed by mineral deposition that was rapid in primary osteoblast cultures but more gradual in MLO-A5 cultures. In primary osteoblast cultures, mineral was deposited exclusively in association with clusters of cells expressing Dmp1-GFP, suggesting that they were already differentiating into osteocyte-like cells. In MLO-A5 cells, the first indication of mineralization was the appearance of punctate areas of alizarin red fluorescence of 4–7 μm in diameter, followed by mineral deposition throughout the culture in association with collagen fibrils. A high amount of cell motility was observed within mineralizing nodules and in mineralizing MLO-A5 cultures. These studies provide a novel approach for analyzing mineralization kinetics that will enable us to dissect in a time-specific manner the essential players in the mineralization process.
Intravenous bisphosphonate (BP) therapy has become the standard of care for the treatment of cancers that metastasize to bone. BPs are associated with osteonecrosis of alveolar bones, a condition known as osteonecrosis of the jaw (ONJ). The incidence or pathogenesis of ONJ is largely unknown. The lesions are characterized by areas of exposed necrotic bone that do not heal after 8 weeks in the absence of radiation to the head and neck. ONJ lesions have been recalcitrant to conventional therapies. Lesions in cancer patients treated with BPs develop in association with periodontal disease, tooth extraction and/or in association with increased mechanical force due to partial/complete dentures. We hypothesized that intravenous BPs in cancer patients impair normal bone remodeling, thereby increasing the incidence of osteonecrotic lesions and that these lesions can be detected using cone beam computerized tomography (CBCT). From CBCTs taken at the University of Missouri at Kansas City School of Dentistry, 26 subjects had a cancer diagnosis and were on BP therapy. From these 26 subjects, 18 presented visible, exposed necrotic bone. We observed both sclerotic and radiolucent lesions. Lesions could be detected and measured in reconstructed images where most were found to expand to large areas of the bone. We were able to identify necrotic bodies or ‘involucrums’ within the ONJ lesions, suggesting that this could be the mechanism for the formation of a clinically visible sequestrum. We propose that CBCT can potentially identify and follow the progression of both pre- and postclinical lesions in ONJ patients, allowing better diagnosis and assessment of disease status.
Methyl methacrylate used in bone cements has drawbacks of toxicity, high exotherm, and considerable shrinkage. A new resin, based on silorane/oxirane chemistry, has been shown to have little toxicity, low exotherm, and low shrinkage. We hypothesized that silorane-based resins may also be useful as components of bone cements as well as other bone applications and began testing on bone cell function in vitro and in vivo. MLO-A5, late osteoblast cells, were exposed to polymerized silorane (SilMix) resin (and a standard polymerized bisGMA/TEGDMA methacrylate (BT) resin and compared to culture wells without resins as control. A significant cytotoxic effect was observed with the BT resin resulting in no cell growth, whereas in contrast, SilMix resin had no toxic effects on MLO-A5 cell proliferation, differentiation, nor mineralization. The cells cultured with SilMix produced increasing amounts of alkaline phosphatase (1.8-fold) compared to control cultures. Compared to control cultures, an actual enhancement of mineralization was observed in the silorane resin-containing cultures at days 10 and 11 as determined by von Kossa (1.8–2.0 fold increase) and Alizarin red staining (1.8-fold increase). A normal bone calcium/phosphate atomic ratio was observed by elemental analysis along with normal collagen formation. When used in vivo to stabilize osteotomies, no inflammatory response was observed, and the bone continued to heal. In conclusion, the silorane resin, SilMix, was shown to not only be non cytototoxic, but actually supported bone cell function. Therefore, this resin has significant potential for the development of a nontoxic bone cement or bone stabilizer.
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