Calvarial Osteoblast Cultures Research Articles

Osteoprotective effect by interleukin-4 (IL-4) on lipoprotein-induced periodontitis

Lipoproteins are immunostimulatory bacterial components suggested to participate in inflammation-induced bone loss in periodontal disease through stimulation of osteoclast differentiation. Toll-like receptor 2 activation by Pam2CSK4 (PAM2), known to mimic bacterial lipoproteins, was previously shown to enhance periodontal bone resorption in mice. The anti-inflammatory cytokine interleukin-4 (IL-4) is a known inhibitor of RANKL-induced bone resorption in vitro. Here, we have investigated whether IL-4 could decrease PAM2-induced periodontal bone loss and osteoclastogenesis in vivo. In a model of periodontitis induced by gingival injections of PAM2 in mice, concomitant injections of IL-4 reduced bone loss. Histologically, IL-4 reduced the recruitment of inflammatory cells and the formation of TRAP+ osteoclasts stimulated by PAM2. Mouse bone marrow macrophages (BMMs) and neonatal calvarial osteoblasts were used to assess the effect of IL-4 on PAM2-induced osteoclastogenesis in vitro. In RANKL-primed BMMs stimulated by PAM2 Nfatc1, Ctsk, and Acp5 gene expression was up-regulated and resulted in robust formation of TRAP+ multinucleated osteoclasts, effects which were impaired by IL-4. These effects were mediated by impairment in PAM2-induced c-fos expression. In primary calvarial osteoblast cultures, IL-4 decreased PAM2-induced Tnfsf11 (encoding RANKL) mRNA and enhanced Tnfrsf11b (encoding OPG) expression. Our data demonstrate that the osteoprotective effect by IL-4 on lipoprotein-induced periodontal disease occurs through the inhibition of osteoclastogenesis by three mechanisms, one by acting directly on osteoclast progenitors, another by acting indirectly through decreasing the expression of osteoclast-regulating cytokines in osteoblasts and a third by decreasing inflammation.

Role of Discoidin Domain Receptor 2 in Craniofacial Bone Regeneration

Bone loss caused by trauma, neoplasia, congenital defects, or periodontal disease is a major cause of disability and human suffering. Skeletal progenitor cell–extracellular matrix interactions are critical for bone regeneration. Discoidin domain receptor 2 (DDR2), an understudied collagen receptor, plays an important role in skeletal development. Ddr2 loss-of-function mutations in humans and mice cause severe craniofacial and skeletal defects, including altered cranial shape, dwarfing, reduced trabecular and cortical bone, alveolar bone/periodontal defects, and altered dentition. However, the role of this collagen receptor in craniofacial regeneration has not been examined. To address this, calvarial subcritical-size defects were generated in wild-type (WT) and Ddr2-deficient mice. The complete bridging seen in WT controls at 4 wk postsurgery was not observed in Ddr2-deficient mice even after 12 wk. Quantitation of defect bone area by micro–computed tomography also revealed a 50% reduction in new bone volume in Ddr2-deficient mice. Ddr2 expression during calvarial bone regeneration was measured using Ddr2-LacZ knock-in mice. Expression was restricted to periosteal surfaces of uninjured calvarial bone and, after injury, was detected in select regions of the defect site by 3 d postsurgery and expanded during the healing process. The impaired bone healing associated with Ddr2 deficiency may be related to reduced osteoprogenitor or osteoblast cell proliferation and differentiation since knockdown/knockout of Ddr2 in a mesenchymal cell line and primary calvarial osteoblast cultures reduced osteoblast differentiation while Ddr2 overexpression was stimulatory. In conclusion, Ddr2 is required for cranial bone regeneration and may be a novel target for therapy.

Isolation and Culture of Neonatal Mouse Calvarial Osteoblasts.

This chapter describes the isolation and culture of neonatal mouse calvarial osteoblasts. This primary cell population is obtained by sequential enzymatic digestion of the calvarial bone matrix and is capable of differentiating in vitro into mature osteoblasts that deposit a collagen extracellular matrix and form mineralized bone nodules. Maturation of the cultures can be monitored by gene expression analyses and staining for the presence of alkaline phosphatase or matrix mineralization. This culture system, therefore, provides a powerful model in which to test how various experimental conditions, such as the manipulation of gene expression, may affect osteoblast maturation and/or function.

The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71-257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase-dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase-positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

Role of estrone on the regulation of osteoblastogenesis

Although estradiol bone contribution has been deeply studied, little is known about the action of estrone. We investigated the direct action of estrone on osteoblasts growth and differentiation, with focus on the biochemical mechanism displayed by the estrogen. Murine calvarial osteoblast cultures in vitro exposed to 10 nM estrone were employed. Estrone enhanced gene expression of the osteogenic differentiation marker, Runx2 mRNA (150% above control). The hormone significantly increased cell proliferation (38% above control), nitric oxide production (108% above control), alkaline phosphatase activity (50% above control), in addition to stimulation of extracellular matrix mineralization. Using specific antagonists, we found that the mechanism of action of estrone involves estrogen receptor, nitric oxide synthase and MAPK signalling pathways participation. The hormone acts by its own and probably not via conversion to estradiol, since 17 B HSD inhibition did not affect the hormonal action. This work shows a novel action of estrone on bone cells promoting osteoblastogenesis.

Abstract 5527: Characterizing the role of WWOX dysregulation in canine osteosarcoma

Abstract Introduction: The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that is frequently deleted or reduced in human osteosarcoma (OS) tumors and loss of WWOX in OS cells promotes a highly tumorigenic phenotype. The purpose of this study was to investigate the functional consequences of WWOX deficiency in canine OS, a well-established spontaneous large animal model of human OS. Methods: Real-time PCR and Western blotting was performed to evaluate WWOX expression was in primary canine OS tumors and canine OS cell lines. Full-length canine Wwox cDNA was amplified from normal canine testes and cloned into the pCDH-cop-GFP lentiviral vector (Systems Biosciences). Canine OS cell lines expressing low basal levels of WWOX were transduced with either empty or WWOX lentiviral vectors and stably transduced cells were sorted based on GFP positivity. Overexpression of canine WWOX lentiviral constructs was confirmed by Western blotting and OS cell lines were evaluated for differences in proliferative capacity, expression of bone differentiation markers, and the ability to migrate through matrigel. Transgenic mice (Tg) carrying a conditional allele of Wwox (Wwoxfl/fl-Tg) were crossed with mice expressing Cre recombinase under the osteoblast-specific Collagen1α1 promoter (Col1a1-Cre-Tg), restricting deletion of WWOX to osteoblasts. Calvarial osteoblast cultures were generated from Wwoxfl/fl and Col1a1-Cre; Wwoxfl/fl mice and the effects of WWOX deletion on osteoblast functions was evaluated using assays described above. Results and Conclusions: Western blotting demonstrated that WWOX protein is absent or reduced in primary canine OS tumors and canine OS cell lines. To assess the functional consequences of WWOX deletion on normal osteoblast behavior, the canine OSA8 and Abrams OS cell lines which express low levels of WWOX were transduced with WWOX lentiviral constructs resulting in high levels of WWOX expression as demonstrated by real time PCR and Western blotting. Restoration of WWOX in canine OS cells resulted in decreased cellular proliferation and invasion. To more critically evaluate the consequences of Wwox deficiency in normal osteoblast biology without the confounding influence of altered Wwox expression in other tissues, Col1a1-Cre; Wwoxfl/fl-Tg mice were generated in which Wwox deletion is restricted to osteoblasts. Consistent with our findings in canine OS cell lines, deletion of WWOX in osteoblasts derived from Col1a1-Cre; Wwoxfl/fl-Tg mice resulted in enhanced cellular proliferation. Studies are underway to identify WWOX binding partners and investigate regulatory pathways altered by WWOX that promote cell proliferation and invasion in normal and malignant osteoblasts. In summary, these data provide evidence supporting a role for WWOX in canine OS cell proliferation and invasion, and suggest that dysregulation of WWOX may be fundamental to the disease process in both human and canine OS. Citation Format: Justin Breitbach, Feng Xu, F. Kay Huebner, Cheryl A. London, Joelle M. Fenger. Characterizing the role of WWOX dysregulation in canine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5527.

Synthesis and study of benzofuran-pyran analogs as BMP-2 targeted osteogenic agents

Twenty-four novel benzofuran-pyran derivatives were synthesized and evaluated for their anti-osteoporotic activity in primary cultures of rat calvarial osteoblasts in vitro. Among all the compounds screened for the alkaline phosphatase activity, three compounds 4e, 4j and 4k showed potent activity at picomolar concentrations in osteoblast differentiating stimulation. Additionally, these compounds were found effective in mineralization, assessed by alizarin red-S staining assay. Compounds were again validated through a series of other in vitro experiments. Moreover, molecular dynamics simulations demonstrated that both benzofuran and pyran moieties are requisite to fit into the active site of BMP-2 receptor, a key target of the osteogenic agents. The obtained results strongly convey that compound 4e is a potential bone anabolic agent among synthesized series, which can be further explored as a drug lead for treating osteoporosis.

NAMPT expression in osteoblasts controls osteoclast recruitment in alveolar bone remodeling.

In bone remodeling, osteoclasts are recruited via increased production of RANKL (receptor activator of nuclear factor-κB ligand) and migrate to the bone surface, aided by matrix metalloproteinases (MMPs). NAMPT (nicotinamide phosphoribosyl transferase), which catalyzes the rate-limiting step in the NAD+ salvage pathway, increases during in vitro osteogenic differentiation and inhibits RANKL-induced osteoclast differentiation. Alveolar bone loss, due to disturbance of the remodeling process, is a major feature of periodontitis. Thus, we investigated the role of NAMPT in a synchronized alveolar bone remodeling rat model. NAMPT expression increased in osteogenic cells during the remodeling activation phase, in parallel with RANKL and MMP-2 expression. Inhibition of NAMPT activity, by systemic delivery of its selective inhibitor FK866, decreased the recruitment of osteoclasts, but not their activity. In vitro, NAMPT mRNA, and protein expression also increased during osteoblast differentiation in primary calvarial osteoblast cultures. Recombinant NAMPT and NMN, its direct metabolite, dose-dependently increased bone marker expression, including that of sialoprotein (BSP) and osteocalcin (OC), whereas their expression was inhibited by FK866 treatment. Recombinant NAMPT did not regulate MMP-2, -9, MMP-13, or RANKL/OPG mRNA expression in osteoblasts. Our data suggest that de novo NAMPT synthesis in osteoblasts controls cell differentiation through osteoclast recruitment during the activation of bone remodeling.

Phytochemical investigation of Kigelia pinnata leaves and identification of osteogenic agents

Phytochemical investigation of n-butanol fraction of Kigelia pinnata leaves lead to the isolation of six known compounds. Among them two compounds polybotrin (4), scutellarin 7-O-β-D-glucopyranoside (6) have been isolated for the first time from this plant. The structure of compounds was elucidated by extensive spectroscopic methods, including one-dimensional, two-dimensional nuclear magnetic resonance experiments, and mass spectrometry analysis. All the isolated compounds (1–6) were assessed for osteogenic activity in primary calvarial osteoblast cultures. Compound 6 increased alkaline phosphatase activity and mineralization at 10 nM and 1 μM concentrations, respectively.

Osteoprotective effects of osthole in a mouse model of 5/6 nephrectomy through inhibiting osteoclast formation.

The present study aimed to investigate the effects of osthole on osteoclast formation and bone loss in a mouse model of 5/6 nephrectomy. The mice in control and osthole groups were treated 1month following 5/6 nephrectomy with either a placebo or osthole, respectively. At 2months post‑nephrectomy, the L4 vertebrae were harvested. The bone mineral density (BMD) of cancellous bone was measured using micro‑CT and tartrate‑resistant acid phosphatase (TRAP) staining was performed to evaluate osteoclast formation. Immunohistochemistry staining and reverse transcription‑quantitative polymerase chain reaction were performed to detect the expression of nuclear factor of activated T‑cells, cytoplasmic‑1 (NFATc‑1), c‑Fos, cathepsinK, Trap, matrix metalloproteinase9 (Mmp9), osteoprotegerin (Opg) and receptor activator for nuclear factor‑κB ligand (Rankl). Bone marrow cells were cultured with osthole, and osteoclast formation was shown by TRAP staining. Primary calvaria osteoblasts were cultured with osthole, and expression levels of Opg and Rankl were detected. Compared with the sham group, the BMD of mice in model group was significantly reduced. The numbers of osteoclasts and the expression levels of NFATc‑1, c‑Fos, cathepsinK and Mmp9 were significantly increased. Compared with the control group, the mice in the osthole group exhibited increased BMD of the L4 vertebrae, a reduction in osteoclast numbers and decreased expression levels of NFATc‑1, c‑Fos, cathepsinK and Mmp9. Invitro experiments also showed that osteoclast formation was decreased following treatment with osthole. Osteoprotegerin (Opg)/receptor activator for nuclear factor‑κB ligand (Rankl) was upregulated by osthole treatment in the L4 vertebrae and in primary cultures of calvarial osteoblasts. Osthole inhibited osteoclast formation and partially reversed the bone loss induced by 5/6 nephrectomy in mice through the upregulation of OPG/RANKL.

Peroxisomes in Different Skeletal Cell Types during Intramembranous and Endochondral Ossification and Their Regulation during Osteoblast Differentiation by Distinct Peroxisome Proliferator-Activated Receptors

Ossification defects leading to craniofacial dysmorphism or rhizomelia are typical phenotypes in patients and corresponding knockout mouse models with distinct peroxisomal disorders. Despite these obvious skeletal pathologies, to date no careful analysis exists on the distribution and function of peroxisomes in skeletal tissues and their alterations during ossification. Therefore, we analyzed the peroxisomal compartment in different cell types of mouse cartilage and bone as well as in primary cultures of calvarial osteoblasts. The peroxisome number and metabolism strongly increased in chondrocytes during endochondral ossification from the reserve to the hypertrophic zone, whereas in bone, metabolically active osteoblasts contained a higher numerical abundance of this organelle than osteocytes. The high abundance of peroxisomes in these skeletal cell types is reflected by high levels of Pex11β gene expression. During culture, calvarial pre-osteoblasts differentiated into secretory osteoblasts accompanied by peroxisome proliferation and increased levels of peroxisomal genes and proteins. Since many peroxisomal genes contain a PPAR-responsive element, we analyzed the gene expression of PPARɑ/ß/ɣ in calvarial osteoblasts and MC3T3-E1 cells, revealing higher levels for PPARß than for PPARɑ and PPARɣ. Treatment with different PPAR agonists and antagonists not only changed the peroxisomal compartment and associated gene expression, but also induced complex alterations of the gene expression patterns of the other PPAR family members. Studies in M3CT3-E1 cells showed that the PPARß agonist GW0742 activated the PPRE-mediated luciferase expression and up-regulated peroxisomal gene transcription (Pex11, Pex13, Pex14, Acox1 and Cat), whereas the PPARß antagonist GSK0660 led to repression of the PPRE and a decrease of the corresponding mRNA levels. In the same way, treatment of calvarial osteoblasts with GW0742 increased in peroxisome number and related gene expression and accelerated osteoblast differentiation. Taken together, our results suggest that PPARß regulates the numerical abundance and metabolic function of peroxisomes via Pex11ß in parallel to osteoblast differentiation.

BMP-2 Enhances Lgr4 Gene Expression in Osteoblastic Cells.

Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism, and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP-induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. J. Cell. Physiol. 231: 887-895, 2016. © 2015 Wiley Periodicals, Inc.

Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing.

SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts.

BackgroundOsteoporosis is one of the prevalent diseases in ageing populations. Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder. Punica granatum (PG) represent a potent fruit-bearing medicinal herb which exerted valuable anti-osteoporotic activities. The present study was carried out to validate the in vitro osteogenic effects of the PG seed extract in primary calvarial osteoblast cultures harvested from neonatal rats.MethodsThe ethanolic extract of PG was subjected to evaluate cell proliferation, regeneration, mineralization and formation of collagen matrix using MTT, alkaline phosphatase, Alizarin Red-S staining and Sirius Red dye, respectively. Cell cycle progression and osteogenic gene Runx2 expression were carried out by flow cytometry and real time PCR, respectively.ResultsExposure of different concentrations (10–100 μg/ml) of the extract on osteoblastic cells showed characteristic morphological changes and increment in cell number. A significant growth in cell proliferation, ALP activity, collagen contents and matrix mineralization of osteoblasts in a dose dependent manner (p < 0.05), suggested that PG has a stimulatory effect on osteoblastic bone formation or potential activity against osteoporosis. In addition, PG extract also enhanced DNA content in S phase of cell cycle and Runx2 gene expression level in osteoblasts.ConclusionThe data clearly indicated that PG promoting bone cell proliferation and differentiation in primary osteoblasts might be due to elevating the osteogenic gene Runx2 expression. The present study provides an evidence for PG could be a promising herbal medicinal candidate that able to develop drugs for osteoporosis.

Isolation and culture of neonatal mouse calvarial osteoblasts.

This chapter describes the isolation and culture of neonatal mouse calvarial osteoblasts. This primary cell population is obtained by sequential enzymatic digestion of the calvarial bone matrix and is capable of differentiating in vitro into mature osteoblasts that deposit a collagen extracellular matrix and form mineralized bone nodules. Maturation of the cultures can be monitored by gene expression analyses and staining for the presence of alkaline phosphatase or matrix mineralization. This culture system, therefore, provides a powerful model to test how various experimental conditions, such as the manipulation of gene expression, may affect osteoblast maturation and/or function.

Characterization of distinct classes of differential gene expression in osteoblast cultures from non-syndromic craniosynostosis bone.

Craniosynostosis, the premature fusion of one or more skull sutures, occurs in approximately 1 in 2500 infants, with the majority of cases non-syndromic and of unknown etiology. Two common reasons proposed for premature suture fusion are abnormal compression forces on the skull and rare genetic abnormalities. Our goal was to evaluate whether different sub-classes of disease can be identified based on total gene expression profiles. RNA-Seq data were obtained from 31 human osteoblast cultures derived from bone biopsy samples collected between 2009 and 2011, representing 23 craniosynostosis fusions and 8 normal cranial bones or long bones. No differentiation between regions of the skull was detected, but variance component analysis of gene expression patterns nevertheless supports transcriptome-based classification of craniosynostosis. Cluster analysis showed 4 distinct groups of samples; 1 predominantly normal and 3 craniosynostosis subtypes. Similar constellations of sub-types were also observed upon re-analysis of a similar dataset of 199 calvarial osteoblast cultures. Annotation of gene function of differentially expressed transcripts strongly implicates physiological differences with respect to cell cycle and cell death, stromal cell differentiation, extracellular matrix (ECM) components, and ribosomal activity. Based on these results, we propose non-syndromic craniosynostosis cases can be classified by differences in their gene expression patterns and that these may provide targets for future clinical intervention.

Synthesis and evaluation of new coumarin–pyridine hybrids with promising anti-osteoporotic activities

Anti-osteoporotic effects of the newly synthesized coumarin–pyridine hybrids were evaluated in primary cultures of rat calvarial osteoblasts in vitro. Compounds 6a, i, j and k were potent in stimulating osteoblast differentiation and mineralization as assessed by the alkaline phosphatase production and alizarin red-S staining assay, respectively. These compounds were also found to be nontoxic in osteoblast cells as compared to the control group in an MTT assay. Furthermore, the effect of these compounds on the transcript levels of osteogenic genes revealed that the compound 6j robustly enhanced mineralization of the osteogenic genes in calvarial osteoblasts. In this context, compound 6j was selected as a potential lead for further structural optimization in the development of new anti-osteoporotic agents.

Bone-forming cultures of rat and mouse calvarial osteoblasts: key differences in protocols

Bone-forming cultures of rat and mouse calvarial osteoblasts: key differences in protocols

Expression and Function of Enamel-related Gene Products in Calvarial Development

Enamel-related gene products (ERPs) are detected in non-enamel tissues such as bone. We hypothesized that, if functional, ERP expression corresponds with distinct events during osteoblast differentiation and affects bone development and mineralization. In mouse calvariae and MC3T3 cells, expression profiles of enamel-related gene products (ERPs) correlated with key events in post-natal calvarial development and MC3T3 cell mineralization. Developing skulls from both Amel- and Ambn-deficient animals were approximately 15% shorter when compared with those of wild-type controls, and their sutures remained patent for a longer period of time. Analysis of Amel- and Ambn-deficient calvariae and calvarial osteoblast cultures revealed a dramatic reduction in mineralized nodules, a significant reduction in Runx2, Sp7, Ibsp, and Msx2 expression, and a reduction in Alx4 in Amel-deficient calvariae vs. an increase in Alx4 in Ambn-deficient calvariae. Analysis of these data indicates that ERP expression follows defined developmental profiles and affects osteoblast differentiation, mineralization, and calvarial bone development. We propose that, in parallel to their role in the developing enamel matrix, ERPs have retained an evolutionary conserved function related to the biomineralization of bones.

Osteoclast progenitor cells present in significant amounts in mouse calvarial osteoblast isolations and osteoclastogenesis increased by BMP-2

Enzymatically released cells from neonatal mouse calvarial bones are frequently used as primary mouse osteoblasts for in vitro studies. We, here, report that although these cells lack mRNA expression of the osteoclastic genes Calcr, Acp5 and Mmp-9 at early time points after their isolation, these transcripts are gradually upregulated when the calvarial osteoblast cultures are differentiated to more mature osteoblasts in long term cultures. Similarly, Calcr, Acp5, Mmp-9, as well as Rank and Nfatc1 mRNA expressions are robustly increased when the osteoblast cultures were induced to differentiate by treatment with bone morphogenetic protein (BMP-2). The increased Calcr mRNA resulted in functionally active calcitonin receptors. Enhanced osteoblastic differentiation was associated with increased Rankl mRNA expression and decreased Opg and Cfs1 mRNA expression. Treatment of the osteoblastic cells with BMP-2 or RANKL, either on plastic dishes or bone slices, resulted in the formation of multinucleated tartrate-resistant acid phosphatase positive cells, which were able to excavate resorption pits and release CTX from the bones. In contrast, increased osteoblastic differentiation induced by BMP-2 in the mouse calvarial osteoblastic cell line MC3T3-E1 was not associated with increased mRNA expression of Calcr, Acp5, Rank, c-Fms or Oscar. Interestingly, Ctsk mRNA was increased during osteoblastic differentiation in both mouse calvarial osteoblast cultures and in MC3T3-E1 cultures. Also osteoblasts isolated from mouse long bones by outgrowth from explant cultures were contaminated with osteoclast progenitors able to differentiate into bone resorbing osteoclasts. These data indicate that mouse calvarial osteoblast cultures contain osteoclast progenitor cells, which will be differentiated along the osteoclastic lineage during osteoblastic differentiation. Moreover, the data show that BMP-2 not only stimulates osteoblastic differentiation but can also induce osteoclastogenesis through increased RANKL.