Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 microM xylenol orange and 30 microM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.

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BMP‐7 is a member of the BMP family of signaling molecules that are thought to play key roles in mediating inductive events during embryogenesis. In the present study the possible roles of BMP‐7 in mediating inductive events during the initiation phase of odontogenesis and mandibular morphogenesis were investigated. To do so, we have examined the effects of agarose beads soaked in recombinant BMP‐7 on E11 mouse molar‐forming mesenchyme and stage 23 chick mandibular mesenchyme, and analyzed the patterns of expression of Bmp‐7 in developing mouse and chick first branchial arches. Beads releasing BMP‐7 induced a translucent zone, cellular proliferation, and expression of Msx‐1, Msx‐2, and Bmp‐4 in molar‐forming mesenchyme after 24 hr. The effects of BMP‐7 on molar‐forming mesenchyme are similar to the effects of BMP‐4 and are consistent with their overlapping patterns of expression in the thickened epithelium of the early developing tooth buds, which is suggestive of cooperative and/or redundant roles of BMPs in mediating the inductive interactions during the early stages of odontogenesis. Our studies in the developing chick mandible showed that Bmp‐7 is expressed in the mandibular epithelium. In the absence of mandibular epithelium, BMP‐7 beads maintained cell proliferation and Msx expression in the medial mandibular mesenchyme and were able to induce cell proliferation, cell death, and Msx expression in the lateral chick mandibular mesenchyme. The effects of BMP‐7 on the expression of Msx genes in lateral chick mandibular mesenchyme, although different from the effects of lateral mandibular epithelium, are similar to the effects of epithelium from the medial region where multiple Bmps are expressed. We also showed that laterally placed BMP‐7 beads induced ectopic expression of Msx genes and changes in the development of posterior skeletal elements in the maxillary and mandibular arches. However, despite its proliferative effects on mandibular mesenchyme, BMP‐7 did not support the directional outgrowth of the mandible. These observations suggest that epithelial–mesenchymal interactions in the medial region of the mandibular arch regulating directional outgrowth of the mandibular mesenchyme are mediated by cooperative interactions between BMPs and other growth factors. Our observations also indicated that EGF, another growth factor implicated in mediating epithelial–mesenchymal interactions in the initiation phase of odontogenesis and morphogenesis of the developing mandible, induces an extensive translucent zone and cellular proliferation in the E11 mouse molar‐forming mesenchyme and stage 23 chick mandibular mesenchyme. However, in contrast to BMPs, EGF did not induce Msx‐1, Msx‐2, and Bmp‐4, but modulated the effects of BMPs on the expression of Msx‐1 and Msx‐2 in these mesenchymes. Our combined data suggest that BMP‐7 is a component of the signaling network mediating epithelial–mesenchymal interactions during the initiation phase of odontogenesis and morphogenesis of the mandibular arch. Dev Dyn 1999;21...

Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5-to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo, as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites.Bone loss under the influence of bacterial virulence factors is thought to occur through engagement of receptors for pathogen-associated molecular pattern (PAMP) molecules, resulting in the stimulation of osteoclasts and/or the inhibition of osteoblasts. A frequently cited example is the engagement of Toll-like receptor 4 (TLR4) by bacterial lipopolysaccharide (LPS) that is reported to mediate bone loss in destructive periodontal diseases through activation of osteoclasts and inhibition of osteoblasts (9,21,27,40,44,45). A subgingival organism strongly associated with destructive periodontal disease, Porphyromonas gingivalis, is a Gram-negative anaerobe that produces LPS, but this LPS is unique in that it has been shown to engage both TLR4 and TLR2 (4, 10, 20, 24, 37), although these reports are sometimes conflicting. Though inflammatory bone loss in experimental periodontal diseases can be produced by engagement of TLR4 by LPS (28), recent evidence indicates that periodontal bone loss in experimental animals mediated by live P. gingivalis requires engagement of TLR2 (16). Furthermore, previous report...

Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population.Introduction: Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures. Materials and Methods: Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression. Results: Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH. Conclusion: GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.

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Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential to promote TLR2-dependent bone loss as is reported in experimental periodontitis following oral infection with P. gingivalis. These results also support the conclusion that serine dipeptide lipids are involved in alveolar bone loss in chronic periodontitis.

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