Osteoblast Transcription Factor Research Articles

Deletion of Amp-activated Protein Kinase (AMPK)-Alpha 1 catalytic subunit in mice impairs the in vivo response of bones to hormonal challenges

Deletion of Amp-activated Protein Kinase (AMPK)-Alpha 1 catalytic subunit in mice impairs the in vivo response of bones to hormonal challenges

Oxytocin receptor nuclear translocation mediates upregulation of osteoblast specific genes

Oxytocin receptor nuclear translocation mediates upregulation of osteoblast specific genes

Crosstalk between tyrosine kinase receptors and BMP2 response during osteoblastic differentiation in mesenchymal stem cells

It is well documented that bone morphogenic proteins (BMPs) promote mesenchymal stem cell (MSCs) osteogenic differentiation, whereas platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) activate their proliferation. Understanding of the interactions between proliferation and differentiation of MSCs by different growth factors is an important issue for potentially improving the process of bone repair. In this study, we investigated the effects of PDGF or FGF signalling on BMP2 response during the osteogenic differentiation in human MSCs. Inhibition of PDGF and/or FGF receptor signalling with selective receptor inhibitors (1 μM) enhanced BMP2-induced alkaline phosphatase activity (from 1.6 to 2.4 fold) and expression of the osteoblastic transcription factor Osterix. These data were associated with an increase in Smad1/5/8 phosphorylation and BMP responsive element (BRE) transcriptional activity induced by BMP2. Since the phosphorylation of the Smad linker region at MAPK and GSK3 sites [1] has been shown to regulate the ubiquitination/degradation of Smads, signalling and interactions of PDGF and FGF receptor pathways with Smad activation by BMP2 were investigated in MSCs. PDGF and FGF activated MAPKs, in particular ERK1/2 but reduced GSK3α activity. BRE luciferase transcriptional activity was increased in the presence of ERK (U0126), JNK (SB600125) and GSK3 (SB216753) inhibitors, but not with the p38 inhibitor. Increased BMP2 response induced by PDGF and FGF receptor inhibitors was further increased in the presence of the GSK3 inhibitor (from 50 to 85% higher than without the GSK3 inhibitor). These data strongly suggest that activation of PDGF and FGF receptors negatively regulates BMP signalling through phosphorylation of Smad by activation of ERK and JNK in MSCs. In conclusion, inhibition of receptor tyrosine kinase signalling enhances BMP2-induced Smad activity and MSCs differentiation by inhibiting ERK and JNK signalling. This effect can be potentiated in the presence of a GSK3 inhibitor. Selective inhibition of these pathways might be of potential interest to improve the differentiation of MSCs during the process of bone repair. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

Divergent upstream osteogenic events contribute to the differential modulation of MG63 cell osteoblast differentiation by MMP-1 (collagenase-1) and MMP-13 (collagenase-3)

Previously we showed that MMP-1 (collagenase-1) and MMP-13 (collagenase-3) differentially regulate the expression of osteoblastic markers in a heterogenous population of primary human periodontal ligament cells. The mechanisms for these differential responses are not known, but may result from divergence in regulation of early osteogenic transcription factors. The purpose of this study was to elucidate where in the hierarchy of osteoblast-specific transcription factors and markers the differences in MMP-1- and -13-mediated regulation of osteoblastic differentiation arise. We found that the overexpression of MMP-1 resulted in significant decreases in BMP-2, Dlx5, AP, OP and BSP and increases in TGF-β1 and MSX2. In contrast, MMP-13 overexpression resulted in significant decreases in Runx2, OP and BSP, and increases in TGF-β1, MSX2 and OC. The knockdown of MMP-1 caused significant increases in all osteoblastic markers. MMP-13 knockdown produced significant increases only in TGF-β1, MSX2 and Osx, but decreases in Runx2 and OC. Suppression of both MMPs together resulted in significant increases of all osteoblastic markers except Runx2. MMP-1 had a more robust and generalized effect in regulating osteoblast transcription factors and markers than MMP-13. Finally, of the markers and transcription factors assayed, Runx2 is the most early stage transcription factor induced by suppression of MMP-1, while Osx and MSX2 are the most early stage transcription factors regulated by MMP-13. These data show that MMP-1's and -13's differential regulation of osteoblastic markers in MG63 cells likely results from their modulation of divergent signaling pathways involved in osteoblastic differentiation.

The effect of strontium incorporation in hydroxyapatite on osteoblasts in vitro

The purpose of this study was to test the effects of a series of strontium-substituted HA (Sr-HA) ceramics (0, 1, 5, and 10 mol% Sr substitution) on osteoblasts, thereby demonstrating whether strontium incorporation with HA would favor osteoblast metabolism. Rat primary osteoblasts were cultured with culture media containing ions released from the Sr-HA ceramics as they dissolved. MTT test, alkaline phosphatase activity, osteoblast transcription factor gene (cbfa1) expression and Alizarin Red staining were conducted at different time-points. There is no significant difference in cell proliferation between groups. However, compared with HA group, Sr-HA groups presented significant enhancement with regard to ALP activity, cbfa1 mRNA expression, and mineralization nodules. Among Sr-HA groups, 5 and 10% groups showed much better performances in ALP activity, cbfa1 mRNA expression, and mineralization nodules than 1% group, however, no significant difference was found between 5 and 10% groups. This study has demonstrated that Sr incorporation in HA ceramic enhanced osteoblastic cell differentiation and mineralization. However, further detailed studies are needed to understand the mechanistic effects of this Sr incorporation on osteoblastic cells and the optimal percentage of calcium should be substituted with strontium in HA.

Role of Melastatin Transient Receptor Potential 7 Channels in the Osteoblastic Differentiation of Murine MC3T3 Cells

Adequate bone formation is assured by the coordinated proliferation, migration, differentiation, and secretory functions of osteoblasts. Epidemiological studies have linked insufficient dietary magnesium (Mg) intake to osteoporosis. Here, we investigated the role of melastatin-like transient receptor potential 7 (TRPM7), a calcium (Ca) and Mg channel, in osteoblastic differentiation of the murine MC3T3 cell line. Osteoblastic differentiation was monitored by alkaline phosphatase activity, osteocalcin gene expression, and extracellular matrix mineralization. Gene expression of TRPM7 increased with osteoblastic differentiation, suggesting the importance of intracellular Ca/Mg homeostasis to cell differentiation. Alteration of intracellular Ca/Mg homeostasis by culture conditions with low extracellular Ca or Mg significantly reduced the osteoblastic differentiation markers alkaline phosphatase activity and osteocalcin gene expression. In accordance, matrix mineralization was reduced under low extracellular Ca or Mg levels. Nevertheless, expression of collagen type I, the predominant matrix protein, was increased in low-Mg culture conditions, indicating that dysfunction of matrix protein production cannot account for the reduced mineralization. Silencing TRPM7 expression during the differentiation period also reduced osteoblastic differentiation and the extent of matrix mineralization. Gene expression of osteoblastic transcription factor Runx2 was reduced by conditions of culture under low extracellular Ca or Mg levels, as well as by TRPM7 silencing. Our results indicate that intracellular Ca and Mg homeostasis ensured by TRPM7 expression is important for the osteoblastic differentiation of MC3T3 cells. Thus, Mg deficiency, a common condition among the population, may be associated with altered osteoblastic differentiation leading to inadequate bone formation and the development of osteoporosis.

Steady and Oscillatory Fluid Flows Produce a Similar Osteogenic Phenotype

Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, which may include regulation of transcription factors that support osteoblast differentiation and function. Flow effects on osteoblast transcription factors have generally been evaluated after short exposures. In this work, we assayed flow effects on osteogenic genes at early and late time points in a preosteoblast (CIMC-4) cell line and evaluated both steady and oscillatory flows. Four hours of steady unidirectional flow decreased the level of RANKL mRNA 53 ± 7% below that of nonflowed cells, but increases in Runx2 and osterix mRNA (44 ± 22% and 129 ± 12%, respectively) were significant only after 12-19 h of continuous flow. Late flow effects on RANKL and osterix were also induced by an intermittent flow-rest protocol (four cycles of 1 h on/1 h off + overnight rest). Four hours of oscillatory flow decreased RANKL mRNA at this early time point (63 ± 2%) but did not alter either osterix or Runx2. When oscillatory flow was delivered using the intermittent flow-rest protocol, Runx2 and osterix mRNA increased significantly (85 ± 19% and 161 ± 22%, respectively). Both β-catenin and ERK1/2, known to be involved in RANKL regulation, were rapidly activated by steady flow. Inhibition of flow-activated ERK1/2 prevented the increase in osterix mRNA but not Runx2; Runx2 phosphorylation was increased by flow, an effect which likely contributes to osterix induction. This work shows that both steady and oscillatory fluid flows can support enhancement of an osteogenic phenotype.

Improved pre‐osteoblast response and mechanical compatibility of ultrafine‐grained Ti–13Nb–13Zr alloy

Metallic implantation materials having high yield strength, low elastic modulus, and non-cytotoxic alloying elements would be advantageous for the long-term stability of implants. This study assessed the surface and mechanical properties, and also in vitro osteoconductivity of ultrafine-grained (UFG) Ti-13Nb-13Zr alloy produced by dynamic globularization without any severe deformation for future biomedical applications as an endosseous implant material. The surface characteristics and mechanical properties were investigated by orientation image microscopy, contact angle measurements, optical profilometry, and uniaxial tension tests. Mouse calvaria-derived pre-osteoblastic cell (MC3T3-E1) attachment, spreading, viability, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on UFG Ti-13Nb-13Zr alloy were compared with coarse-grained (CG) Ti-13Nb-13Zr and CG Ti-6Al-4V alloys. Dynamic globularized Ti-13Nb-13Zr alloy has an ultrafine grain size (0.3 μm) and an excellent combination of yield strength and elastic modulus compared with CG alloys, which displayed significantly lower water contact angles compared with CG alloys (P<0.05). The UFG and CG Ti-13Nb-13Zr alloys displayed significantly increased cellular attachment compared with CG Ti-6Al-4V alloy (P<0.05). The UFG Ti-13Nb-13Zr supported better cell spreading and more numerous focal adhesions. ALP activity (P<0.05) and mRNA expressions of the osteoblast transcription factor genes (osterix, Runx2) and marker gene for osteoblast differentiation (osteocalcin) were markedly increased in cells grown on the UFG substrate compared with CG substrates at early incubation timepoints. Enhanced pre-osteoblast response to UFG Ti-13Nb-13Zr substrate is attributable to the non-cytotoxic alloying elements and the submicron scale grain size contributes to the superior surface hydrophilicity and abundant grain boundaries favorable for cell behavior. These findings indicate that dynamic globularized UFG Ti-13Nb-13Zr alloy is promising for load-bearing endosseous implant material because of excellent mechanical and biological compatibilites.

The effect of strontium incoporated with hydroxyapatite on osteoblastic differentiation of bone masenchymal stromal cells

Objective To determine if strontium-containing hydroxyapatite (Sr-HA) will favor osteoblastic differentiation of bone masenchymal stromal cells (BMSCs). Methods Rat BMSCs were cultured with culture media containing ions released from the strontium-substituted HA ceramics as they dissolved. We had 3 experimental groups and one blank control group. In group A, leaching liquor containing 10 mol% of Sr-HA was used;in group B, leaching liquor containing 5 mol% of Sr-HA was used;in group C,leaching liquor containing 1 mol% of Sr-HA was used;in group D, leaching liquor containing no Sr-HA was used. MTT test, alkaline phosphatase (ALP) activity and osteoblast transcription factor gene (Cbfα1) expression examinations were conducted at different time-points. Results MTT test showed that there were no significant differences in absorbance between the 4 groups after cell culture for 1, 3, 5, 7, 10 days( P ＞ 0. 05),but there were significant differences in absorbance between different time-points within the same group ( P ＜0.05). The ALP activity in groups A and B was significantly greater than in groups C and D ( P ＜ 0. 05) at 6, 9,12, 15 days. At 9 and 12 days, there were significant differences in Cbfα1 mRNA expression between the 4 groups ( P ＜ 0. 05). Compared with the blank control group, Sr-HA groups presented a significant enhancement with regard to ALP activity and Cbfα1 mRNA expression in a concentration-dependent manner. Conclusion Incorportion of strontium in HA ceramics may not promote proliferation of BMSCs, but may stimulate osteoblastic differentiation of BMSCs. Key words: Strontium; Hydroxyapatites; Bone marrow cells; Cell differentiation

Effect of omeprazole on the expression of transcription factors in osteoclasts and osteoblasts

The use of proton pump inhibitors (PPIs) seems to be related to increased fracture risk but the mechanism is unclear. In an effort to clarify the mechanism, we evaluated the effect of omeprazole, a representative of the PPIs, on the expression of transcription factors in osteoclasts and osteoblasts. Murine RAW264.7 and MC3T3-E1 cells were used for osteoclast and osteoblast analysis, to which various concentrations of omeprazole were added. RAW264.7 cells with ≥3 nuclei were considered tartrate-resistant acid phosphatase (TRAP)-positive, i.e. activated osteoclasts. Expressions of the calcitonin receptor (CTR), c-fos, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and matrix metalloproteinase (MMP)-9 mRNA in osteoclasts were evaluated. Gene expression of osteocalcin and of the osteoprotegerin/receptor activator of NF-κB ligand (OPG/RANKL) ratio in osteoblasts was examined and Western blotting of NFATc1 was performed. Treating the osteoclasts with increasing doses of omeprazole did not affect TRAP positivity, but significantly decreased the expressions of CTR, c-fos, NFATc1, and MMP-9 regardless of the omeprazole concentration. The expression of osteocalcin and of the OPG/RANKL ratio in osteoblasts was augmented with increasing omeprazole concentrations. The result of the Western blot analysis with NFATc1 was similar to that of the expression of NFATc1 mRNA. Omeprazole decreased the activation of osteoclasts but increased that of osteoblasts in vitro, in part causing an osteopetrosis-like effect. Together with the effect of omeprazole on calcium homeostasis, increased fracture risk may be due to the osteopetrorickets-like effect of omeprazole.

Tissue‐specific calibration of extracellular matrix material properties by transforming growth factor‐β and Runx2 in bone is required for hearing

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-β (TGFβ)-responsive pathway that controls osteoblast differentiation. Deregulated TGFβ or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFβ signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

High-phosphate-induced calcification is related to SM22α promoter methylation in vascular smooth muscle cells

Hyperphosphatemia is closely related to vascular calcification in patients with chronic kidney disease. Vascular smooth muscle cells (VSMCs) exposed to high phosphate concentrations in vitro undergo phenotypic transition to osteoblast-like cells. Mechanisms underlying this transdifferentiation are not clear. In this study we used two in vitro models, human aortic smooth muscle cells and rat aortic rings, to investigate the phenotypic transition of VSMCs induced by high phosphate. We found that high phosphate concentration (3.3 mmol/L) in the medium was associated with increased DNA methyltransferase activity and methylation of the promoter region of SM22α. This was accompanied by loss of the smooth muscle cell-specific protein SM22α, gain of the osteoblast transcription factor Cbfa1, and increased alkaline phosphatase activity with the subsequent in vitro calcification. The addition of a demethylating agent (procaine) to the high-phosphate medium reduced DNA methyltransferase activity and prevented methylation of the SM22α promoter, which was accompanied by an increase in SM22α expression and less calcification. Additionally, downregulation of SM22α, either by siRNA or by a methyl group donor (S-adenosyl methionine), resulted in overexpression of Cbfa1. In conclusion, we demonstrate that methylation of SM22α promoter is an important event in vascular smooth muscle cell calcification and that high phosphate induces this epigenetic modification. These findings uncover a new insight into mechanisms by which high phosphate concentration promotes vascular calcification.

In vitro biocompatibility of magnesium-incorporated submicro-porous titanium oxide surface produced by hydrothermal treatment

This study investigated the surface characteristics and in vitro biocompatibility of titanium (Ti) oxide surface incorporating magnesium ions (Mg), produced by hydrothermal treatment using an alkaline Mg-containing solution, for future biomedical applications. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and optical profilometry. Mouse calvaria-derived osteoblastic cell (MC3T3-E1) attachment, spreading, proliferation, alkaline phosphatase (ALP) activity, and osteoblastic gene expression on Mg-containing surfaces were compared with untreated Ti surfaces. Hydrothermal treatment resulted in Mg-incorporated Ti oxide layer with submicro-porous surface structures approximately 2 μm in thickness. ICP-AES analysis revealed Mg ions release from treated surfaces into the solution. The Mg-incorporated surface displayed significantly increased cellular attachment and ALP activity compared with untreated surface ( p < 0.05), and supported better cell spreading. Real-time polymerase chain reaction analysis showed notably higher mRNA expression of the osteoblast transcription factor genes (Dlx5, Runx2) and the osteoblast phenotype genes (ALP, bone sialoprotein and osteocalcin) in cells grown on the Mg-incorporated surfaces than untreated surfaces. These results demonstrate that the Mg-incorporated submicro-porous Ti oxide surface produced by hydrothermal treatment may improve implant osseointegration by enhancing the attachment, spreading and differentiation of osteoblastic cells.

Riboflavin and photoproducts in MC3T3-E1 differentiation

Photoderivatives of riboflavin can modulate the proliferation and survival of cancer cells. In this work, we examined the influence of riboflavin and photoderivatives on osteoblast differentiation induced by ascorbic acid and β-glycerophosphate. These compounds decreased the osteoblast proliferation, increased the alkaline phosphatase activity, promoted a reduction in matrix metalloproteinase-2 activity and the decreased in the OPG/RANKL ratio. The effects of flavins on osteoblasts were unrelated to the antioxidant activity of these compounds. The biological activity of osteogenic medium containing riboflavin and its photoderivatives involved the activation of different signaling pathways (AKT, FAK, CaMKII), caspases-3, -8 and -9, and up-regulation of the expression and/or stabilization of osteoblastic transcription factors (Runx2 and β-catenin). These findings suggest a potential use of flavins as adjuvants to improve bone metabolism.

Suppressive effects of nicotine on the cytodifferentiation of murine periodontal ligament cells

Tobacco smoking has been suggested to be one of the important risk factors of developing periodontal disease. Although epidemiological studies have shown the detrimental effects of smoking on periodontal disease, the effects of smoke compounds on gingival tissue are not well understood. The aim of this study was to evaluate the effects of nicotine, which is the major component of the thousands of chemicals that constitute cigarette smoke, for cytodifferentiation of murine periodontal ligament (MPDL) cell. Expression of nAChR subunits on MPDL cells was examined using RT-PCR. The effects of nicotine on gene expression of extracellular matrices and osteoblastic transcription factors were evaluated by quantitative RT-PCR. Mineralized nodule formation of nicotine-treated MPDL cells was characterized by alizarin red staining. Murine periodontal ligament cells expressed several subunits of nAChR, which have functional calcium signals in response to nicotine. Gene expression of extracellular matrices and osteoblastic transcription factors were reduced in nicotine-treated MPDL cells. In addition, mineralized nodule formation was inhibited in MPDL cells in the presence of nicotine. Our findings indicate that nicotine may negatively regulate the cytodifferentiation and mineralization of MPDL cells.

Osteoblast response to magnesium ion‐incorporated nanoporous titanium oxide surfaces

This study investigated the surface characteristics and in vitro osteoconductivity of a titanium (Ti) surface incorporated with the magnesium ions (Mg) produced by hydrothermal treatment for future application as an endosseous implant surface. Mg-incorporated Ti oxide surfaces were produced by hydrothermal treatment using Mg-containing solution on two different microstructured surfaces--abraded minimally rough (Ma) or grit-blasted moderately rough (RBM) samples. The surface characteristics were evaluated using scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, and inductively coupled plasma atomic emission spectroscopy (ICP-AES). MC3T3-E1 pre-osteoblast cell attachment, proliferation, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on Ma, RBM, Mg-incorporated Ma (Mg), and Mg-incorporated grit-blasted (RBM/Mg) Ti surfaces were evaluated. Hydrothermal treatment produced an Mg-incorporated Ti oxide layer with nanoporous surface structures. Mg-incorporated surfaces showed surface morphologies and surface roughness values almost identical to those of untreated smooth or micro-rough surfaces at the micron scale. ICP-AES analysis showed Mg ions released from treated surfaces into the solution. Mg incorporation significantly increased cellular attachment (P=0 at 0.5 h, P=0.01 at 1 h) on smooth surfaces, but no differences were found on micro-rough surfaces. Mg incorporation further increased ALP activity in cells grown on both smooth and micro-rough surfaces at 7 and 14 days of culture (P=0). Real-time polymerase chain reaction analysis showed higher mRNA expressions of the osteoblast transcription factor gene (Dlx5), various integrins, and the osteoblast phenotype genes (ALP, bone sialoprotein and osteocalcin) in cells grown on micro-rough (RBM) and Mg-incorporated (Mg and RBM/Mg) surfaces than those on Ma surfaces. Mg incorporation further increased the mRNA expressions of key osteoblast genes and integrins (α1, α2, α5, and β1) in cells grown on both the smooth and the micro-rough surfaces. These results indicate that an Mg-incorporated nanoporous Ti oxide surface produced by hydrothermal treatment may improve implant bone healing by enhancing the attachment and differentiation of osteoblastic cells.

Enhanced osteoblast response to hydrophilic strontium and/or phosphate ions‐incorporated titanium oxide surfaces

This study investigated the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces incorporated with strontium ions (Sr) and/or phosphate ions (P) produced by hydrothermal treatment for future applications as endosseous implant surfaces. Sr and/or P-incorporated Ti oxide surfaces were produced by hydrothermal treatment. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, contact angle and surface energy measurements, inductively coupled plasma atomic emission spectroscopy, and profilometry. MC3T3-E1 pre-osteoblast cell attachment, morphology of spread cells, viability, and quantitative analysis of osteoblastic gene expression on grit-blasted microrough (RBM), P-incorporated (P), and P- and Sr-incorporated (SrP) Ti surfaces were evaluated. Microstructured P and SrP surfaces showed significantly higher wettability and surface energy compared with RBM surfaces (P<0.01). After immersion in Hank's balanced salt solution, considerable apatite deposition was observed on the P and SrP surfaces. Sr incorporation significantly increased cellular attachment and viability compared with other surfaces (P<0.05). Real-time polymerase chain reaction analysis showed markedly higher mRNA expressions of the osteoblast transcription factor gene (Runx2) and the osteoblast phenotype genes (alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin) in cells grown on the P and SrP surfaces compared with those on the RBM surface. These results demonstrate that Sr- and P-incorporated Ti oxide surfaces may improve implant bone healing by enhancing attachment, viability, and differentiation of osteoblastic cells.

Bio-Oss®acts on Stem cells derived from Peripheral Blood

This study aims to study how Bio-Oss® can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells markers using real time Reverse Transcription-Polymerase Chain Reaction. PB-hMSCs stem preparations were obtained for gradient centrifugation from peripheral blood of healthy anonymous volunteers, using the Acuspin System-Histopaque 1077. The samples were then cultured for 7 days for RNA processing, and the expression was quantified using real time PCR. Bio-Oss® caused an induction of osteoblast transcriptional factor like RUNX2 and of bone related genes; SPP1 and FOSL1. In contrast, the expression of ENG was significantly decreased in stem cells treated with Bio-Oss® with respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. The results obtained can be relevant to enhance the understanding of the molecular mechanism of bone regeneration and can act as a model for comparing other materials with similar clinical effects.

Medpor&amp;#174; Acts on Stem Cells Derived from Peripheral Blood

Porous polyethylene (PP or Medpor®) is an alloplastic material used worldwide for craniofacial reconstruction. Although several clinical studies are available, how this material alters osteoblast activity to promote bone formation is poorly understood. To study how PP can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction. PP causes induction of osteoblast transcriptional factor RUNX2 and of the bone related genes osteocalcin (BGLAP) and alkaline phosphatase (ALPL). In contrast the expression of ENG was decreased in stem cells treated with PP respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. The obtained results can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Severe cleidocranial dysplasia and hypophosphatasia in a child with microdeletion of the C‐terminal region of RUNX2

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia due to mutations causing haploinsufficiency of RUNX2, an osteoblast transcription factor specific for bone and cartilage. The classic form of CCD is characterized by delayed closure of the fontanels, hypoplastic or aplastic clavicles and dental anomalies. Clinical reports suggest that a subset of patients with CCD have skeletal changes which mimic hypophosphatasia (HPP). Mutations in RUNX2 are detected in approximately 65% of cases of CCD, and microdeletions occur in 13%. We present clinical and radiological features in a 6-year-old child with severe CCD manifested by absence of the clavicles marked calvarial hypomineralization, osteoporosis and progressive kyphoscoliosis. HPP features included Bowdler spurs, severe osteopenia, and low alkaline phosphatase. Following negative mutation analysis of RUNX2, comparative genomic hybridization (CGH) microarray was performed. The result revealed a microdeletion in RUNX2, disrupting the C-terminal part of the gene.