Fetal Rat Calvarial Cells Research Articles

Biocompatibility of Titanium Carbo-Nitride Coatings with Fetal Rat Calvarial Cells

Surface modification is a promising technique to improve mechanical properties of titanium and titanium alloys by changing the surface composition or microstructures. In this study, titanium carbo-nitride coating had been deposited on Ti-6Al-4V alloy by arc ion plating. The biocompatibility of TiN and TiCN coating on Ti-6Al-4V alloy with fetal rat calvarial cells was assessed by Enzyme Linked Immunoabsorbent Assay (ELISA) and Alkaline phosphatase (ALPase) activity. The effect of surface coating on IL-1β and IL-6 production was determined by ELISA. On IL-1β and IL-6 production, there were no statistical difference between control and thin coating surfaces. After 7 days of culture, the cells were provided to measure ALPase activity. ALPase activity of cells cultured on TiN and TiCN surface was similar to that seen on cp-Ti.

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

Rapamycin inhibits BMP‐7‐induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation.

Quercetin aglycone up‐regulates antioxidant response genes and proteins in osteoblast‐like fetal rat calvarial cells

Quercetin (QRC), a flavonol found in fruits and vegetables, has been shown to up‐regulate antioxidant response genes and proteins in a variety of cell types, but little is known about the effects of QRC on these genes and proteins in osteoblasts. The purpose of this study was to determine the extent that QRC alters expression of antioxidant genes and proteins in primary cultures of fetal rat calvarial cells, a well‐described model of osteoblast differentiation. Cells were treated up to 48 h starting at confluence with 0 or 20 μM QRC aglycone. Antioxidant gene and protein expression were analyzed using real‐time quantitative PCR and Western blotting, respectively. Quercetin significantly up‐regulated heme oxygenase 1 (HO‐1), catalase, and gamma‐glutamate‐cysteine ligase catalytic subunit (GCLC) (p < 0.05), but did not up‐regulate peroxiredoxins 3 and 5, and NADPH:quinone oxidoreductase (p >; 0.05). Protein expression was generally congruent with gene expression, with HO‐1 being the most markedly up‐regulated in response to 20 μM QRC. These data suggest that QRC up‐regulates some genes and proteins associated with the antioxidant response in osteoblasts. Since evidence suggests that oxidative stress may contribute to osteoporosis, QRC may have protective effects on bone health.Grant Funding Source: USDA Agricultural Research Services

Surface characteristics and osteoblastic cell response of alkali-and heat-treated titanium-8tantalum-3niobium alloy

PurposeThe aim of the present study was to evaluate the biological response of alkali- and heat-treated titanium-8tantalum-3niobium surfaces by cell proliferation and alkaline phosphatase (ALP) activity analysis.MethodsCommercial pure titanium (group cp-Ti) and alkali- and heat-treated titanium-8tantalum-3niobium (group AHT) disks were prepared. The surface properties were evaluated using scanning electron microscopy, energy dispersed spectroscopy and X-ray photoelectron spectroscopy (XPS). The surface roughness was evaluated by atomic force microscopy and a profilometer. The contact angle and surface energy were also analyzed. The biological response of fetal rat calvarial cells on group AHT was assessed by cell proliferation and ALP activity.ResultsGroup AHT showed a flake-like morphology microprofile and dense structure. XPS analysis of group AHT showed an increased amount of oxygen in the basic hydroxyl residue of titanium hydroxide groups compared with group cp-Ti. The surface roughness (Ra) measured by a profilometer showed no significant difference (P>0.05). Group AHT showed a lower contact angle and higher surface energy than group cp-Ti. Cell proliferation on group AHT surfaces was significantly higher than on group cp-Ti surfaces (P<0.05). In comparison to group cp-Ti, group AHT enhanced ALP activity (P<0.05).ConclusionsThese results suggest that group AHT stimulates osteoblast differentiation.

Response of Fetal Rat Calvarial Cells to Nanotubular Titanium Oxide Surface

This study examined the cell response to a TiO2 nanotubular surface (Ti-NT) for future biomedical applications. The level of cell attachment and spreading at 20 min and 60 min was evaluated by SEM. IL-6 and PGE2 secretion was evaluated by ELISA. In SEM analysis, the Ti-NT surface had more fully spread cells compared to the machined titanium surface (Ti-S). ELISA revealed that the level of IL-6 and PGE2 production was higher on the Ti-NT than on the Ti-S. These results suggest that a surface treatment with a nanotubular TiO2 surface enhances the early osteoblast responses, such as cell spreading and cytokine release, which are important for subsequent cell functions and bone healing in vivo.

The Effect of Nanotubular Titanium Surfaces on Osteoblast Differentiation

The biological response of fetal rat calvarial cells on a TiO2 nanotubular surface (Ti-NT) was evaluated by cell viability assay, alkaline phosphatase (ALP) activity and reverse transcription polymerase chain reaction (RT-PCR) analysis. The cell viability assay showed no significant difference between the Ti-NT and smooth titanium surfaces (Ti-S). Ti-NT had better cellular responses with regard to the ALP activity and bone-associated markers, such as bone sialoprotein and osteocalcin mRNA than Ti-S. These results suggest that Ti-NT stimulate the differentiation into osteoblasts of fetal rat calvarial cells, potentially contributing to rapid osseointegration.

Response of fetal rat calvarial cells on mineral trioxide aggregate after IL-1β stimulation

Purpose: The purpose of this study was to investigate the ability of Mineral trioxide aggregate(MTA) to support osteoclastic differentiation from fetal rat calvarial cell. Methods: In this study, response of IL-6, RANKL, and OPG in fetal rat calvarial cells stimulated with IL-1β on MTA was evaluated by ELISA and RT-PCR. Results: The results were as follows; there was no significant difference between glass and MTA at 5days. In ELISA analysis, Glass group and MTA group showed similar IL-6 expression, Glass+IL-1β group and MTA+IL-1β group showed similar IL-6 expression. In RT-PCR analysis, Glass group and MTA group showed similar IL-6, RANKL, OPG mRNA expression, MTA+IL-1β group and Glass+IL-1β group showed 3 fold increase of IL-6 and RNAKL mRNA expression when compared with MTA group. All groups showed similar OPG mRNA expression. Conclusions: MTA does not suppress cell proliferation and increase the proinflammatory cytokine that induce osteoclastogenesis. Thus, MTA is biocompatible material that could be used in various clinical conditions. (J Korean Acad Periodontol 2009;39:359-365)

Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF‐A independent mechanism

The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF-PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF-PEMF on angiogenesis. The hypothesis of this study is that ELF-PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)-A-based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF-PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF-PEMF increased endothelial proliferation 54-fold, whereas media from endothelial cells stimulated with ELF-PEMF did not affect osteoblast proliferation. We examined the role of the pro-angiogenic mediator VEGF-A in the mitogenic effect of ELF-PEMF-stimulated osteoblast media on endothelial cells. The production of VEGF-A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF-PEMF-induced osteoblast-derived endothelial mitogen observed in these studies was not VEGF-A, but some other soluble angiogenic mediator.

Platelet-Derived Growth Factor BB Treated Osteoprogenitors Inhibit Bone Regeneration

The study evaluates the ability of osteoprogenitors treated with platelet-derived growth factor BB (PDGF-BB) delivered on vinyl styrene microbeads (VSM) to regenerate rat calvarial critical-size defects (CSDs). Fetal rat calvarial cells were cultured and tested for their ability to attach to VSM using scanning electron microscopy. Twenty-five rats were equally divided into 5 groups; a negative control (GPI), vinyl styrene microbeads (GPII), PDGF-BB (GPIII), VSM plus osteoblastic progenitors (GPIV), and VSM plus PDGF-BB treated osteoblastic progenitors (GPV). CSDs were created and reconstructed according to the mentioned study design. After 16 weeks, animals were sacrificed and defect areas evaluated for bone regeneration. Cells attached to the microbeads; however, their morphology and topography were affected by the PDGF-BB. Transplanting the VSM/cells constructs to CSDs revealed significant reduction of bone regeneration upon pretreatment of the cells with PDGF-BB. However, short-term application of PDGF-BB to CSD stimulated bone regeneration. The ability of osteoprogenitor cells to regenerate bone was significantly reduced upon pretreatment with PDGF-BB in vitro. However, adding PDGF-BB at the time of surgery had stimulated bone regeneration.

Plasma surface modification of poly(D,L‐lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types

We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.

Thiazide Diuretics Directly Induce Osteoblast Differentiation and Mineralized Nodule Formation by Interacting with a Sodium Chloride Co-Transporter in Bone

Thiazide diuretics are used worldwide as a first-choice drug for patients with uncomplicated hypertension. In addition to their antihypertensive effect, thiazides increase bone mineral density and reduce the prevalence of fractures. Traditionally, these effects have been attributed to increased renal calcium reabsorption that occurs secondary to the inhibition of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal tubule. The aim of the current study was to determine whether thiazides exert a direct bone-forming effect independent of their renal action. We found that the osteoblasts of human and rat bone also express NCC, suggesting that these bone-forming cells may be an additional target for thiazides. In vitro, NCC protein was virtually absent in proliferating human and fetal rat osteoblasts, whereas its expression dramatically increased during differentiation. Thiazides did not affect osteoblast proliferation, but directly stimulated the production of the osteoblast differentiation markers runt-related transcription factor 2 (runx2) and osteopontin. Using overexpression/knockdown studies in fetal rat calvarial cells, we show that thiazides increase the formation of mineralized nodules, but loop diuretics do not. Overall, our study demonstrates that thiazides directly stimulate osteoblast differentiation and bone mineral formation independent of their effects in the kidney. Therefore, in addition to their use as antihypertensive drugs, our results suggest that thiazides may find a role in the prevention and treatment of osteoporosis.

Mechanism by which MLO-A5 Late Osteoblasts/Early Osteocytes Mineralize in Culture: Similarities with Mineralization of Lamellar Bone

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray analysis, and atomic force microscopy. At 3-4 days of culture, spheres of approximately 20-50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5-6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoid-osteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization.

Biocompatibility of Ti-8Ta-3Nb alloy with fetal rat calvarial cells

Titanium is the material of choice for oral implantology. The superiority of titanium comes from its mechanical, physicochemical, and biological properties, particularly immunotolerance and cytocompatibility1,2). However because of needs to improve its mechanical properties, titanium alloys such as Ti-6Al-4V, Ti-6Al-7Nb, have also been used for dental and orthopedic material3-5).1) Although titanium alloys are still widely used in medicine, some concern has been expressed over its use since it appears that small amounts of both vanadium and aluminum, released in the human body, induce possible cytotoxic effect and neurological disorders, respectively6-8). In terms of mechanical properties, Ti-6Al-4V alloy belongs to α+β type titanium alloy family. The rigidity of α+β type titanium alloy is much higher (about 120 GPa) than that of the cortical bone (Max 30 GPa)9). Such a strong rigidity (a large modulus of elasticity) causes insufficient loading of the bone adjacent to the implant. This is often referred to as the stress-shielding phenomenon and can lead to potential bone resorption and eventual failure of the implant10). Consequently the recent trend in research and development of titanium alloys for biomedical application is to develop low rigidity titanium alloys composed of non-toxic and non-allergenic elements with excellent mechanical properties and workability9,10). The most promising materials are the β-type titanium alloys because their elastic moduli are smaller than that of αor α+β type titanium family. Therefore new β-type titanium alloys which are composed of non-toxic elements like Tantalum (Ta), Niobium (Nb), Zirconium (Zr),

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Inhibition of MMP-13 mRNA expression by ginseng saponin in fetal rat calvarial cells

Inhibition of MMP-13 mRNA expression by ginseng saponin in fetal rat calvarial cells

Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones.

We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca2+]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca2+]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3beta, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor alpha1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [Ca2+]o, as was mineralized nodule formation. Alkaline phosphatase activity was maximal for [Ca2+]o between 1.2 and 1.8 mM. Inhibition of CaR by NPS 89636 blocked responses to the CaR agonists. In conclusion, we show that small deviations of [Ca2+]o from physiological values have a profound impact on bone cell fate, by means of the CaR and independently of systemic calciotropic peptides.

Effect of strain on bone nodule formation by rat osteogenic cells in vitro

The purpose of this study was to assess in vitro bone nodule formation by cells exposed to a range of microstrain, at a sub-optimal oscillation frequency for bone formation. Fetal rat calvarial cells experienced a Flexercell™ regimen within either FLEX I™ (deformable) or FLEX II™ (non-deformable) substrates. Cells in FLEX I™ plates were exposed to growth medium only; those in FLEX II™ plates were exposed to either growth medium only, or growth medium+10 −7 M IGF-1. Cell numbers were assessed from 1 to 6 days. Other cells were exposed to the Flexercell™ regimen (−2 kPa, 0.05 Hz) for 1–3 (Group 1), 3–6 (Group 2), 1–9 (Group 3) or 10–15 (Group 4) days and were maintained, at other times, under standard conditions. After 21 days, nodules were counted within each well and within the compression, <999, 1000–4900, 5000–9999, 10,000–14,999 and 15,000–25,000 microstrain regions of the FLEX I™ membrane. Cyclic deformation inhibited cell numbers from 1 to 6 days, compared to control or IGF-1 groups ( P<0.001). The number of nodules in Groups 2 and 4 were greater than Groups 1 or 3 ( P<0.001), but not different from control or IGF-1 groups. Compression or tensile microstrain significantly affected nodule formation in all groups, with Group 4 producing more nodules than other groups in most microstrain regions. Thus, the number of bone nodules produced by osteogenic cell cultures exposed to cyclic deformation was significantly affected by the timing of initiation and the characteristics and magnitude of the deformation regimen.

In vitro behavior of osteoblastic cells cultured in the presence of pseudowollastonite ceramic.

Pseudowollastonite ceramic (psW) is a bioactive ceramic that binds to bone when implanted in vivo and may be useful for the treatment of skeletal defects. However, there have been no studies that examined the interaction between psW and osteoblastic cells in vitro. This study investigated the suitability of psW as a substratum for cell attachment and the ability of the material to effect osteoblasts at a distance from the material surface. Fetal rat calvarial cells were plated onto the ceramic and examined by scanning electron microscopy. The findings reported show that cells attached and proliferated on the surface to the ceramic. Attachment by cells to the material can be enhanced by preincubation of psW in serum or media containing fibronectin. The adhesion of cells can be inhibited by addition of GRGDS peptides suggesting that adhesion to psW is mediated by integrin binding to adsorbed proteins. To study the effects of psW at a distance, cells were cultured in the presence but not in direct contact with the material. Subsequent changes in proliferation, alkaline phosphatase expression, and bone nodule formation were assessed. Cells grown in wells containing psW demonstrated an increase in both the rate and total numbers of bone nodules formed, although there were no differences in proliferation or alkaline phosphatase expression. Overall, these results suggest that psW is biocompatible and osteoconductive.

Effects of the MnCl2 on bone formation in fetal rat calvarial cell

Effects of the MnCl₂ on bone formation in fetal rat calvarial cell