Human Calvarial Osteoblasts Research Articles

Effect of Enterococcus faecalis OG1RF on human calvarial osteoblast apoptosis

BackgroundEnterococcus faecalis is a dominant pathogen in the root canals of teeth with persistent apical periodontitis (PAP), and osteoblast apoptosis contributes to imbalanced bone remodelling in PAP. Here, we investigated the effect of E. faecalis OG1RF on apoptosis in primary human calvarial osteoblasts. Specifically, the expression of apoptosis-related genes and the role of anti-apoptotic and pro-apoptotic members of the BCL-2 family were examined.MethodsPrimary human calvarial osteoblasts were incubated with E. faecalis OG1RF at multiplicities of infection corresponding to infection time points. Flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, caspase-3/-8/-9 activity assay, polymerase chain reaction (PCR) array, and quantitative real-time PCR were used to assess osteoblast apoptosis.ResultsE. faecalis infection increased the number of early- and late-phase apoptotic cells and TUNEL-positive cells, decreased the mitochondrial membrane potential (ΔΨm), and activated the caspase-3/-8/-9 pathway. Moreover, of all 84 apoptosis-related genes in the PCR array, the expression of 16 genes was upregulated and that of four genes was downregulated in the infected osteoblasts. Notably, the mRNA expression of anti-apoptotic BCL2 was downregulated, whereas that of the pro-apoptotic BCL2L11, HRK, BIK, BMF, NOXA, and BECN1 and anti-apoptotic BCL2A1 was upregulated.ConclusionsE. faecalis OG1RF infection triggered apoptosis in human calvarial osteoblasts, and BCL-2 family members acted as regulators of osteoblast apoptosis. Therefore, BCL-2 family members may act as potential therapeutic targets for persistent apical periodontitis.

The Biological Effect of Particulate Titanium Contaminants of Dental Implants on Human Osteoblasts and Gingival Fibroblasts.

To evaluate the effects of different titanium particle concentrations on viability of human calvarial osteoblasts and human gingival fibroblasts. Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and a live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-±) was estimated with Human IL-6 / Human TNF-± ELISA. Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 to 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared to control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles.

The Biological Effect of Particulate Titanium Contaminants of Dental Implants on Human Osteoblasts and Gingival Fibroblasts.

To evaluate the effects of different titanium particle concentrations on viability of human calvarial osteoblasts and human gingival fibroblasts. Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and a live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-±) was estimated with Human IL-6 / Human TNF-± ELISA. Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 to 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared to control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles.

Effects of equibiaxial mechanical stretch on extracellular matrix-related gene expression in human calvarial osteoblasts.

Mechanical stretch commonly promotes craniofacial suture remodeling during interceptive orthodontics. The mechanical responses of osteoblasts in craniofacial sutures play a role in suture remodeling. Moreover, the extracellular matrix (ECM) produced by osteoblasts is crucial for the transduction of mechanical signals that promote cell differentiation. Therefore, we aimed to investigate the effect of mechanical stretch on cell viability and ECM-related gene-expression changes in human osteoblasts. Human calvarial osteoblasts (HCObs) were subjected to 2% deformation. Caspase activity, MTT, and cell viability assays were used to estimate osteoblast apoptosis, proliferation, and viability, respectively. Real-time RT-PCR (RT2 -PCR) arrays were used to assess expression of cytoskeletal-, apoptosis-, osteogenesis-, and ECM-related genes. We found that mechanical stretch significantly increased osteoblast viability and cell proliferation, and decreased the activities of caspases 3 and 7. Moreover, the expression of 18 genes related to osteoblast differentiation, apoptosis, and ECM remodeling changed by more than two-fold in a time-dependent manner. Therefore, mechanical stretch promotes HCOb viability and alters expression of genes that are closely related to suture remodeling under mechanical stretch.

Amino acid composition of nanofibrillar self-assembling peptide hydrogels affects responses of periodontal tissue cells in vitro.

BackgroundThe regeneration of tissue defects at the interface between soft and hard tissue, eg, in the periodontium, poses a challenge due to the divergent tissue requirements. A class of biomaterials that may support the regeneration at the soft-to-hard tissue interface are self-assembling peptides (SAPs), as their physicochemical and mechanical properties can be rationally designed to meet tissue requirements.Materials and methodsIn this work, we investigated the effect of two single-component and two complementary β-sheet forming SAP systems on their hydrogel properties such as nanofibrillar architecture, surface charge, and protein adsorption as well as their influence on cell adhesion, morphology, growth, and differentiation.ResultsWe showed that these four 11-amino acid SAP (P11-SAP) hydrogels possessed physico-chemical characteristics dependent on their amino acid composition that allowed variabilities in nanofibrillar network architecture, surface charge, and protein adsorption (eg, the single-component systems demonstrated an ~30% higher porosity and an almost 2-fold higher protein adsorption compared with the complementary systems). Cytocompatibility studies revealed similar results for cells cultured on the four P11-SAP hydrogels compared with cells on standard cell culture surfaces. The single-component P11-SAP systems showed a 1.7-fold increase in cell adhesion and cellular growth compared with the complementary P11-SAP systems. Moreover, significantly enhanced osteogenic differentiation of human calvarial osteoblasts was detected for the single-component P11-SAP system hydrogels compared with standard cell cultures.ConclusionThus, single-component system P11-SAP hydrogels can be assessed as suitable scaffolds for periodontal regeneration therapy, as they provide adjustable, extracellular matrix-mimetic nanofibrillar architecture and favorable cellular interaction with periodontal cells.

Effects of Chromeceptin on Insulin-like Growth Factor II in Human Osteoblastic and Osteosarcoma Cells

The insulin-like growth factor system is a complex regulatory system of insulin-like growth factors I and II (IGF-I and IGF-II) as well as their cell surface receptors and a family of insulin growth factor binding proteins (IGF-BPs). Despite extensive research on the IGF system in skeletal tissue, there is still not a complete understanding particularly of the role of IGF-II in the regulation of normal and pathological osseous cells in human skeletal biology. The purpose of this study was to further delineate the possible involvement of IGF-II in the regulation of human normal calvarial osteoblasts and G292 human osteosarcoma cellular activity using exogenously added IGF-II as well as a drug, Chromeceptin that has been shown in some other cellular systems to down regulate endogenous IGF-II levels.The results presented here show a similar dose dependent effect of Chromeceptin on inhibition of endogenous IGF-II levels (measured with an immunoassay) and cellular activity (measured with the MTT assay) in both the normal and osteosarcoma cells while small significant increases in activity with exogenously added IGF-II were observed only in the osteosarcoma cells. Moreover, IGF-BP1 levels (measured with an immunoassay) were shown to significantly increase in the G292 cells in a similar manner previously reported in other cell systems with only minor non-significant effects of exogenously added IGF-BP1 in the osteosarcoma cells studied here. These studies suggest that endogenous IGF-II is a critical regulator of activity in both normal and osteosarcoma cells and Chromeceptin can be an effective pharmacological agent for further studies on the role of the growth factor in human skeletal systems.

Gingipains disrupt F-actin and cause osteoblast apoptosis via integrin β1.

The aim of this study was to explore the cellular mechanisms underlying gingipain-caused changes in cell morphology and apoptosis of osteoblasts. Human calvarial osteoblasts and mouse osteoblasts MC3T3-E1 were treated with gingipain extracts from Porphyromonas gingivalis stain W83. Apoptosis was detected with annexin V and propidium iodide flow cytometry analysis or terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling staining. F-actin was determined by immunostaining. Western blotting was used to detect protein expression. Knocking down and overexpressing approaches were used to determine the role of integrin β1. Osteoblasts exposed to gingipain extracts displayed increased apoptosis, accompanied by loss of F-actin integrity and cell shrinkage. The effects of gingipain extracts were abolished by the cysteine protease inhibitor N-tosyl-l-lysyl chloromethyl-ketone. Notably, gingipain extracts resulted in reduction of integrin β1, accompanied by diminished active RhoA whereas without effect on the total RhoA. Knockdown of integrin β1 resembled those seen in gingipain-treated osteoblasts. By contrast, the effects of gingipain extracts were abrogated by either overexpression of integrin β1 or presence of RhoA agonist CN03. Gingipain-induced F-actin disruption and apoptosis are mediated by the degradation of integrin β1 and inhibition of RhoA activity, which account for osteoblast apoptosis.

Signal-Regulated Protein Kinases/Protein Kinase B-p53-BH3-Interacting Domain Death Agonist Pathway Regulates Gingipain-Induced Apoptosis in Osteoblasts.

Gingipains are cysteine proteases produced by Porphyromonas gingivalis, the predominant pathogen in chronic periodontitis. The present study aims to examine the role of gingipains in promoting apoptosis in osteoblasts. Human calvarial osteoblasts and osteoblast MC3T3-E1 cells were treated with 8.348 U/L gingipains. Flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining were used to detect cell apoptosis. Protein expression was examined by Western blotting, and gene expression was detected by real-time polymerase chain reaction. Small interfering (si)RNA transfection was used to knock down BH3-interacting domain death agonist (Bid) expression. Treatment with 8.348 U/L gingipains from 4 to 72 hours increased apoptosis, accompanied by elevated cleaved caspase-3 levels. Notably, gingipain-induced apoptosis was associated with increase of Bid and its truncated form, tBid, as well as p53. Transfection with Bid siRNA resulted in suppression of gingipain-induced apoptosis. The p53 inhibitor, Pifithrin-α, blocked the gingipain-induced Bid. The ability of gingipains to stimulate p53 and Bid expression was mimicked by PD-0325901 and MK-2206, the specific extracellular signal-regulated protein kinases (ERK) and protein kinase B (PKB) inhibitors, respectively. Furthermore, treatment with gingipains reduced phospho-ERK and phospho-PKB levels, an effect correlated to gingipain-induced increase in p53 and tBid expression. The present findings suggest that Bid plays an essential role in gingipain-induced osteoblast apoptosis, which is dependent on inhibition of ERK and PKB phosphorylation, followed by the activation of p53.

Up‐regulation of expression of selected genes in human bone cells with specific capacitively coupled electric fields

The objective of the described experiments was to determine the electrical parameters that lead to optimal expression of a number of bone-related genes in cultured human bone cells exposed to a capacitively coupled electric field. Human calvarial osteoblasts were grown in modified plastic Cooper dishes in which the cells could be exposed to various capacitively coupled electric fields. The optimal duration of stimulation and optimal duration of response to the electrical field, and the optimal amplitude, frequency and duty cycle were all determined for each of the genes analyzed. Results indicated that a capacitively coupled electric field of 60 kHz, 20 mV/cm, 50% duty cycle for 2 h duration per day significantly up-regulated mRNA expression of a number of transforming growth factor (TGF)-β family genes (bone morphogenetic proteins (BMP)-2 and -4, TGF-β1, - β2 and -β3) as well as fibroblast growth factor (FGF)-2, osteocalcin (BGP) and alkaline phosphatase (ALP). Protein levels of BMP-2 and -4, and TGF-β1 and - β2 were also elevated. The clinical relevance of these findings in the context of a noninvasive treatment modality for delayed union and nonunion fracture healing is discussed.

Differential Growth Factor Adsorption to Calvarial Osteoblast-Secreted Extracellular Matrices Instructs Osteoblastic Behavior

Craniosynostosis (CS), the premature ossification of cranial sutures, is attributed to increased osteogenic potential of resident osteoblasts, yet the contribution of the surrounding extracellular matrix (ECM) on osteogenic differentiation is unclear. The osteoblast-secreted ECM provides binding sites for cellular adhesion and regulates the transport and signaling of osteoinductive factors secreted by the underlying dura mater. The binding affinity of each osteoinductive factor for the ECM may amplify or mute its relative effect, thus contributing to the rate of suture fusion. The purpose of this paper was to examine the role of ECM composition derived from calvarial osteoblasts on protein binding and its resultant effect on cell phenotype. We hypothesized that potent osteoinductive proteins present during sutural fusion (e.g., bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1)) would exhibit distinct differences in binding when exposed to ECMs generated by human calvarial osteoblasts from unaffected control individuals (CI) or CS patients. Decellularized ECMs produced by osteoblasts from CI or CS patients were incubated in the presence of BMP-2 or TGF-β1, and the affinity of each protein was analyzed. The contribution of ECM composition to protein binding was interrogated by enzymatically modulating proteoglycan content within the ECM. BMP-2 had a similar binding affinity for each ECM, while TGF-β1 had a greater affinity for ECMs produced by osteoblasts from CI compared to CS patients. Enzymatic treatment of ECMs reduced protein binding. CS osteoblasts cultured on enzymatically-treated ECMs secreted by osteoblasts from CI patients in the presence of BMP-2 exhibited impaired osteogenic differentiation compared to cells on untreated ECMs. These data demonstrate the importance of protein binding to cell-secreted ECMs and confirm that protein-ECM interactions have an important role in directing osteoblastic differentiation of calvarial osteoblasts.

A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre–Chotzen syndrome

Genetic mutations of Twist, a bHLH transcription factor, induce premature fusion of cranial sutures (craniosynostosis) in the Saethre-Chotzen syndrome (SCS). The mechanisms by which Twist haploinsufficiency may alter osteoblast differentiation are poorly understood. In this study, we investigated the role of fibroblast growth factor receptor-2 (Fgfr2) in the abnormal osteoblast differentiation in SCS. Cranial osteoblasts from an SCS patient with a Y103X mutation inducing deletion of the Twist bHLH domain showed decreased Fgfr2 mRNA levels associated with decreased expression of Runx2, bone sialoprotein (BSP) and osteocalcin (OC), markers of differentiated osteoblasts, compared with wild-type osteoblasts. Transfection with Twist or Runx2 expression vectors, but not with Runx2 mutant which impairs DNA binding, restored Fgfr2, Runx2, BSP and OC expression in Twist mutant osteoblasts. EMSA analysis of mutant osteoblast nuclear extracts showed reduced Runx2 binding to a target OSE2 site in the Fgfr2 promoter. ChIP analyses showed that both Twist and Runx2 in mutant osteoblast nuclear extracts bind to a specific region in the Fgfr2 promoter. Significantly, forced expression of Fgfr2 restored Runx2 and osteoblast marker genes, whereas a dominant-negative Fgfr2 further decreased Runx2 and downstream genes in Twist mutant osteoblasts, indicating that alteration of Fgfr2 results in downregulation of osteoblast genes in Twist mutant osteoblasts. We conclude that Twist haploinsufficiency downregulates Fgfr2 mRNA expression, which in turn reduces Runx2 and downstream osteoblast-specific genes in human calvarial osteoblasts. This provides genetic and biochemical evidence for a role of Fgfr2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the SCS.

Cbl-mediated ubiquitination of α5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation

Fibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, we investigated integrin-mediated attachment and cell survival in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduced osteoblast attachment on fibronectin. This was associated with reduced expression of the alpha5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored alpha5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis showed that alpha5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restored alpha5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in alpha5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha5 integrin expression was associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of alpha5 integrin rescued cell attachment and corrected both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. We show that Cbl recruitment induced by FGFR2 activation triggers alpha5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by alpha5 integrin degradation.

Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.

Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of TNFalpha overexpression using anti-TNFalpha or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8 and caspases-3, -6 and -7 in M-Tw osteoblasts. These studies provide novel evidence that Twist haploinsufficiency in SCS promotes osteoblast apoptosis by a TNFalpha-caspase-2-caspase-8-caspases-3, -6, -7 cascade, and uncover a molecular mechanism in which Twist plays an anti-apoptotic role in human calvarial osteoblasts.