Proliferation Of Osteoblastic MC3T3-E1 Cells Research Articles

In situ precipitation of amorphous calcium phosphate nanoparticles within 3D porous collagen sponges for bone tissue engineering.

Amorphous calcium phosphate (ACP) plays an important role in biomineralization within the three-dimensional (3D) collagen network in human hard tissues, and exhibits osteoconductivity. Porous collagen sponges coated with ACP nanoparticles could be considered as potential scaffolds for use in bone tissue engineering. In this study, such composite materials were fabricated via homogeneous ACP precipitation using a supersaturated calcium phosphate (CaP) solution. Homogeneous ACP precipitation was induced in situ within the sponges by a temperature-controlled coating process composed of two steps. In the first step, the CaP solution was cooled to 4°C to suppress precipitation until the solution penetrated fully into the sponge's internal pores. In the second step, the CaP solution was warmed up to 25°C with continuous shaking to induce ACP precipitation within the sponges. The resulting sponges were therefore coated with ACP nanoparticles on their inner and outer surfaces. A simulated body fluid (SBF) test indicated osteoconductivity of the collagen sponges coated with ACP nanoparticles. Further, ACP-coated collagen sponges immobilizing basic fibroblast growth factor (bFGF) were fabricated using the CaP solution supplemented with bFGF. The fabricated sponges allowed the sustained release of bFGF in a culture medium and enhanced proliferation of osteoblastic MC3T3-E1 cells. Such ACP-coated collagen sponges have the potential to be used as scaffolds in bone tissue engineering if pursued for further in vitro and in vivo studies.

Lycium barbarum polysaccharides promoted proliferation and differentiation in osteoblasts.

Osteoblasts have the capacity to differentiate into several different cell types, including adipocyte, chondrocyte, and muscle lineages. Therefore, osteoblast can be potentially applied in the treatment of bone diseases. The factors controlling osteoblast differentiation is complex. Recently, it has been reported that some natural products regulate the differentiation in osteoblasts and promote bone formation. Based on these findings, this study demonstrated that Lycium barbarum polysaccharides (LBP) could promote proliferation of osteoblast MC3T3-E1 cells through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Besides, expression of key proteins correlated with cellular proliferation such as proliferating cell nuclear antigen (PCNA) and Ki67 was enhanced in the presence of LBP. We also detected the increased expression of bone-specific matrix proteins such as morphogenetic protein 2 (BMP2), bone Gla protein (BGP), osteopontin (OPN), and α-1 type-I collagen (COL1A1) when treated with LBP. This process was mediated by some signals, such as smad1, smad8, Runt-related transcription factor 2 (RUNX2), and Osterix. Furthermore, RUNX2 silencing inhibited osteoblast differentiation by decreasing expression of bone-specific matrix proteins. Collectively, we proposed a previously unidentified function of LBP in osteoblast differentiation, suggesting its potential clinical role in bone disease treatment.

Isolation and Characterization of Lactoferrin Peptides with Stimulatory Effect on Osteoblast Proliferation

Lactoferrin is reported to be a potential food protein with osteogenic activity. However, the activity of lactoferrin peptides is questionable. In the present study, we isolated and characterized peptides from lactoferrin with stimulatory effect on osteoblast proliferation. Peptides from the lactoferrin pepsin hydrolysate were purified using cation-exchange and gel-filtration chromatography. Effects of different hydrolysates and peptides on the proliferation of osteoblast MC3T3-E1 cells were compared by MTT assay. Results showed that fraction P5-a from Superdex Peptide 10/300 GL gel chromatography showed better activity. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry confirmed that two peptides components of P5-a corresponded to fractions of 20-78 and 191-277 amino acids in Bos taurus lactoferrin molecule (GI: 221706349). These results will provide some theoretical and practical data for the preparation and application of osteogenic peptides in functional food industry.

Novel hydroxyethyl chitosan/cellulose scaffolds with bubble-like porous structure for bone tissue engineering

Hydrogels fabricated from natural polysaccharides may serve as ideal scaffolds for tissue engineering because of their similarity to the extracellular matrices. In this study, novel hydrogel scaffolds with bubble-like porous structure were prepared from hydroxyethyl chitosan (HECS) and cellulose (CEL) by a combination of chemical crosslinking, particle-leaching using silicon dioxide particles as porogen and freeze-drying method. The morphology, compression stress-strain curves, wettability, and swelling and rheological behaviors of the HECS/CEL scaffolds were characterized by SEM, mechanical test, contact angle measurement and rheometer. HECS/CEL scaffolds had good comprehensive performances and could reach equilibrium swelling state in water within 20s. The results from in vitro biocompatibility evaluated using SEM, live/dead cell viability and MTT assays demonstrated that the HECS/CEL scaffolds could well support the attachment, spreading and proliferation of osteoblastic MC3T3-E1 cells and showed good biocompatibility. Therefore, the novel HECS/CEL scaffolds may be promising for bone tissue engineering applications.

Highly dispersed nanoscale hydroxyapatite on cellulose nanofibers for bone regeneration

A successful method for fabrication of electrospun hydroxyapatite (HAp) coated cellulose nanofiber scaffold (HAp/CMC) for tissue engineering is reported. The cellulose acetate nanofibers (CANFs) were deacetylated to produce cellulose nanofibers (CNFs) which were subsequently converted to sodium carboxy methyl cellulose (Na-CMC). The Na-CMC nanofibers mat was then coated with HAp for bone regeneration applications. The FTIR results confirmed conversion of CANFs to CNFs and CNFs to Na-CMC. HAp nanocrystal growth was observed in SEM images, with crystal becoming minute and numerous upon increasing HCO3- in the simulated body fluid (SBF) solution. The proliferation of osteoblastic MC3T3-E1cells on HAp-coated CMC nanofiber mat was observed to increase with seeding time. The results confirm suitability of the HAp/CMC nanofiber mat for bone regeneration.

대황 추출물이 조골세포와 파골세포의 성장과 활성에 미치는 영향

The effects of Eisenia bicyclis extracts on osteoblast differentiation and osteoclast formation were investigated. The proliferation of MC3T3-E1 osteoblastic cells was tested in an MTT assay. Treatment with E. bicyclis ethanol extract increased cell proliferation by approximately 128% at a concentration of 10 μg/ml. The ALP activities in the MC3T3-E1 cells was 179% higher when the E. bicyclis ethanol extract was processed at a concentration of 50 μg/ml. The proliferation of RAW 264.7 osteoclastic cells decreased significantly in response to treatment with the E. bicyclis extracts. Moreover, the proliferation of the RAW 264.7 osteoclastic cells treated with E. bicyclis hot water extract decreased by nearly 80%. In addition, the E. bicyclis extract reduced the number of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from osteoclastic RAW 264.7 cells. These results indicate that E. bicyclis extracts have an anabolic effect on bone through the promotion of osteoclast differentiation and suggest that the extracts could be used in the treatment of common metabolic bone diseases.

Osteogenic activity and antibacterial effect of zinc ion implanted titanium

Titanium (Ti) and its alloys are widely used as orthopedic and dental implants. In this work, zinc (Zn) was implanted into oxalic acid etched titanium using plasma immersion ion implantation technology. Scanning electron microscopy and X-ray photoelectron spectroscopy were used to investigate the surface morphology and composition of Zn-implanted titanium. The results indicate that the depth profile of zinc in Zn-implanted titanium resembles a Gaussian distribution, and zinc exists in the form of ZnO at the surface whereas in the form of metallic Zn in the interior. The Zn-implanted titanium can significantly stimulate proliferation of osteoblastic MC3T3-E1 cells as well as initial adhesion, spreading activity, ALP activity, collagen secretion and extracellular matrix mineralization of the rat mesenchymal stem cells. The Zn-implanted titanium presents partly antibacterial effect on both Escherichia coli and Staphylococcus aureus. The ability of the Zn-implanted titanium to stimulate cell adhesion, proliferation and differentiation as well as the antibacterial effect on E. coli can be improved by increasing implantation time even to 2h in this work, indicating that the content of zinc implanted in titanium can easily be controlled within the safe concentration using plasma immersion ion implantation technology. The Zn-implanted titanium with excellent osteogenic activity and partly antibacterial effect can serve as useful candidates for orthopedic and dental implants.

In Vitro Analysis of Nanoparticulate Hydroxyapatite/Chitosan Composites as Potential Drug Delivery Platforms for the Sustained Release of Antibiotics in the Treatment of Osteomyelitis

Nanoparticulate composites of hydroxyapatite (HAp) and chitosan were synthesized by ultrasound-assisted sequential precipitation and characterized for their microstructure at the atomic scale, surface charge, drug release properties, and combined antibacterial and osteogenic response. Crystallinity of HAp nanoparticles was reduced because of the interference of the surface layers of chitosan with the dissolution/reprecipitation-mediated recrystallization mechanism that conditions the transition from the as-precipitated amorphous calcium phosphate phase to the most thermodynamically stable one—HAp. Embedment of 5–10 nm sized, narrowly dispersed HAp nanoparticles within the polymeric matrix mitigated the burst release of the small molecule model drug, fluorescein, bound to HAp by physisorption, and promoted sustained-release kinetics throughout the 3 weeks of release time. The addition of chitosan to the particulate drug carrier formulation, however, reduced the antibacterial efficacy against Saureus. Excellent cell spreading and proliferation of osteoblastic MC3T3-E1 cells evidenced on microscopic conglomerates of HAp nanoparticles in vitro also markedly diminished on HAp/chitosan composites. Mitochondrial dehydrogenase activity exhibited normal values only for HAp/chitosan particle concentrations of up to 2 mg/cm2 and significantly dropped, by about 50%, at higher particle concentrations (4 and 8 mg/cm2). The gene expression of osteocalcin, a mineralization inductor, and the transcription factor Runx2 was downregulated in cells incubated in the presence of 3 mg/cm2 HAp/chitosan composite particles, whereas the expression of osteopontin, a potent mineralization inhibitor, was upregulated, further demonstrating the partially unfavorable osteoblastic cell response to the given particles. The peak in the expression of osteogenic markers paralleling the osteoblastic differentiation was also delayed most for the cell population incubated with HAp/chitosan particles. Overall, the positive effect of chitosan coating on the drug elution profile of HAp nanoparticles as carriers for the controlled delivery of antibiotics in the treatment of osteomyelitis was compensated for by the lower bacteriostatic efficiency and the comparatively unviable cell response to the composite material, especially at higher dosages.

Verification of Estrogen Like Activities of Herbal Medicines Using an In Vitro Detection System

Estrogen like activities were evaluated using ethanol and hot water extracts of herbal medicines by using an in vitro detection system. Bokryung (Poria cocos), Sanyak (root of Dioscorea batatas) and Mokdanpi (root skin of Paeonia suffruticosa) represented statistically significant estrogen-like activities (p<0.001), while Omija (fruit of Schizandra chinensis), Taeksa (root of Alisma canaliculatum A. BR.), Jihwang (root of Rhemannia glutinosa), and Sansuyu (fruit of Cornus officinalis) did not. Estrogen-like activities of Bokryung hot water extract (500 μg/ml) and ethanol extract (50 μg/ml) were almost same as that of a 10<SUP>-7</SUP> M 17β-estradiol. Furthermore, estrogen-like activities of ethanol extracts (500 μg/ml) of Bokryung and Mokdanpi were stronger than that of 10<SUP>-7</SUP> M 17β-estradiol. These results suggest that Bokryung, Sanyak and Mokdanpi show estrogen-like activities. Especially, Sanyak and Mokdanpi represented promotive effect on the proliferation of MC3T3-E1 osteoblastic cells. Bokryung, Sanyak and Mokdanpi also exhibited superior inhibitory effect on the viability of RAW 264.7 cells. In conclusion, these three herbal medicines might be interpreted as candidates for the further study or development of functional foods or medicine to prevent or avoid postmenopausal symptoms of women.

Synthesis of bioactive and machinable miserite glass-ceramics for dental implant applications

ObjectivesTo synthesize and characterize machinable, bioactive glass-ceramics (GCs) suitable for dental implant applications. MethodsA glass in the SiO2–Al2O3–CaO–CaF2–K2O–B2O3–La2O3 system was synthesized by wet chemical methods, followed by calcination, melting and quenching. Crystallization kinetics were determined by differential thermal analysis (DTA). GC discs were produced by cold pressing of the glass powder and sintered using schedules determined by DTA. The crystalline phases and microstructure of GC samples were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Dynamic Young's modulus (E), true hardness (Ho), fracture toughness (KIC) and brittleness index (BI) were evaluated. Bioactivity was studied by examining the formation of hydroxyapatite (HA) on the GC surfaces after soaking in simulated body fluid (SBF). Attachment and proliferation of MC3T3-E1 osteoblastic cells were assessed in vitro. ResultsMiserite [KCa5(Si2O7)(Si6O15)(OH)F] was the main crystalline phase of the GC with additional secondary phases. Microstructural studies revealed interlocking lath-like crystalline morphology. E, Ho, and KIC values for the GCs were 96±3GPa, 5.27±0.26GPa and 4.77±0.27MPam0.5, respectively. The BI was found to be 1.11±0.05μm−0.5, indicating outstanding machinability. An HA surface layer was formed on the GC surfaces when soaked in SBF, indicating potential bioactivity. MC3T3-E1 cells exhibited attachment, spreading and proliferation on GC surfaces, demonstrating excellent biocompatibility. SignificanceWe present a novel approach for the synthesis of miserite GC with the physical and biological properties required for non-metallic dental implant applications.

BMP-2-loaded silica nanotube fibrous meshes for bone generation

Silica nanotube fibrous meshes were fabricated as multiple functional matrices for both delivering bone morphological protein-2 (BMP-2) and supporting osteoblast attachment and proliferation. The meshes were fabricated via a collagen-templated sol–gel route and consisted of tubular silica with open ends. BMP-2 was loaded to the meshes by soaking in BMP-2 solution. The meshes effectively enabled the attachment and proliferation of osteoblast MC3T3-E1 cells and delivered bioactive BMP-2 to stimulate cell differentiation. These results demonstrate the potential use of the meshes in bone generation applications.

Olive polyphenol hydroxytyrosol prevents bone loss

Polyphenols reportedly exert physiological effects against diseases such as cancer, arteriosclerosis, hyperlipidemia and osteoporosis. The present study was designed to evaluate the effects of oleuropein, hydroxytyrosol and tyrosol, the major polyphenols in olives, on bone formation using cultured osteoblasts and osteoclasts, and on bone loss in ovariectomized mice. No polyphenols markedly affected the proliferation of osteoblastic MC3T3-E1 cells at concentrations up to 10μM. Oleuropein and hydroxytyrosol at 10 to 100μM had no effect on the production of type I collagen and the activity of alkaline phosphatase in MC3T3-E1 cells, but stimulated the deposition of calcium in a dose-dependent manner. In contrast, oleuropein at 10 to 100μM and hydroxytyrosol at 50 to 100μM inhibited the formation of multinucleated osteoclasts in a dose-dependent manner. Furthermore, both compounds suppressed the bone loss of trabecular bone in femurs of ovariectomized mice (6-week-old BALB/c female mice), while hydroxytyrosol attenuated H2O2 levels in MC3T3-E1 cells. Our findings indicate that the olive polyphenols oleuropein and hydroxytyrosol may have critical effects on the formation and maintenance of bone, and can be used as effective remedies in the treatment of osteoporosis symptoms.

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

Isolation of two triterpenes from Lythrum salicaria L. and the effect of betulinic acid on the proliferation of osteoblastic MC3T3-E1 cells

Purple loosestrife –Lythrum salicaria L. (Lythraceae) is well known as a medicinal plant, which grows commonly in damp places of moderate climate in Asia, Europe and North America. Its pharmacological activity is mostly due to its phenolic constituents, mainly tannins [1]. It is used for its beneficial health effects against diarrhea, chronic intestinal catarrh, hemorrhoids, eczema, varicose veins and bleeding of the gums [2]. The powdered herbal parts of Lythrum salicaria L. (Lythraceae) were extracted with 80% methanol. The concentrated methanol extracts were suspended in water and partitioned with ethyl acetate and then n-butanol, sequentially. The ethyl acetate extract was applied to silica gel column chromatography and afforded one sterol and two triterpene compounds. Their chemical structures were determined by mass and NMR spectroscopy as β-sitosterol (1), betulinic acid (2), and 3β-hydroxy-20(29)-lupen-28-oic acid methyl ester (3). These two triterpene compounds were isolated for the first time from Lythrum salicaria L. The effect of betulinic acid (2) on the proliferation of osteoblastic MC3T3-E1 cells was examined by checking the cell viability. Betulinic acid (2) showed a tendency of increasing the growth of osteoblastic MC3T3-E1 cells.

Carbonated apatites obtained by the hydrolysis of monetite: Influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells

The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO 4, DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075 mol l −1. Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24 h and 7 days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9 wt.% and levels out to an acceptable value (±50% compared to the control) for carbonate contents between 6.9 and 16.1 wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.

Blockers of K+/Cl- transporter/channels diminish proliferation of osteoblastic cells

In the preset report, we studied if blockers of the K+/Cl- cotransporter, and Cl- and K+ channels modify the proliferation of MC3T3-E1 osteoblastic cells. A blocker of the K+/Cl- cotransporter, DIOA (100 and 500 microM), diminished the cell proliferation. NPPB (100 and 300 microM, a blocker of Cl- channel) and quinine (100 and 500 microM, a blocker of K+ channel) also suppressed the proliferation of MC3T3-E1 cells. These blockers of the K+/Cl- cotransporter/channels (DIOA, NPPB and quinine) would increase the intracellular Cl- concentration ([Cl-]i). On the other hand, our previous study (Biochem. Biophys. Res. Commun. 361, 1038-1043, 2007) reported that a decrease in [Cl-]i diminished the proliferation of MC3T3-E1 osteoblastic cells. Taken together, the observations indicate that both increases and decreases in [Cl-]i diminish the proliferation of MC3T3-E1 osteoblastic cells.

Chloride-dependent acceleration of cell cycle via modulation of Rb and cdc2 in osteoblastic cells

In the present study, we investigated if Cl − regulates the proliferation of the MC3T3-E1 osteoblastic cells. The proliferation of MC3T3-E1 osteoblastic cells was diminished by lowering the extracellular Cl − concentration ([Cl −] o) in the culture medium. The lowered in [Cl −] o increased the periods of the G 0/G 1 and the G 2/M phases in cell cycle. We further studied the effects of [Cl −] o on the key enzymes, Rb and cdc2, playing key roles in checking points of the G 0/G 1 and the G 2/M phases in cell cycle. The lowered in [Cl −] o diminished the active forms of enzymes, Rb and cdc2. We further found that the action of lowered [Cl −] o on the cell proliferation, the cell cycle, Rb and cdc2 was abolished by the presence of 2 mM glutamine, but not by that of pyruvate as another Krebs cycle substrate. Taken together, these observations indicate here for the first time that Cl − modulates Rb and cdc2, enhancing the proliferation of the MC3T3-E1 osteoblastic cells.

The Influences of β-Glucan Associated with BMP-7 on MC3T3-E1 Proliferation and Osteogenic Differentiation

b-glucan, an immunomodulator, can selectively enhance the immunobiological activities of neutrophils and macrophages without stimulating proinflammatory cytokine production. Biologic response modifiers, like beta-glucan, will modulate immunity, modify neoplastic disease and increase resistance to microbial challenge. Therefore, beta-glucan polymers can be applied in bone induction and regeneration model and have a possibility of association with bone morphogenetic protein (BMP) because of tissue-regenerative and antimicrobial effects of those polymers. In this report, we studied an E. coli expression system for BMP-7 production and the biological activities of b-glucan associated with BMP-7. The proliferation of MC3T3-E1 osteoblastic cells was enhanced by treatment with Aureobasidium b-glucan, while neither mushroom b-glucan nor barley b-glucan increased the cell proliferation. Mushroom b-glucan alone or associated with BMP-7 increased alkaline phosphatase (ALP) activity of MC3T3-E1 cells, one of the osteoblast phenotype markers, but the other b-glucans did not affect ALP activity of the cells. In mineralization assay, a highly significant increase in nodular staining was observed in cultures treated with both mushroom and Aureobasidium b-glucans in the presence of BMP-7 compared with nontreated controls, while barley b-glucan showed a significant decrease in nodule number compared with cultures treated only with BMP-7.

RGTA modulates the healing pattern of a defect in a monolayer of osteoblastic cells by acting on both proliferation and migration.

A family of heparan-like polymers, RGTAs, was shown to promote repair of various tissues. Like heparin and heparan-sulfates, RGTAs potentiate in vitro the biological activities of heparin-binding growth factors (HBGFs) and protect them against proteolytic degradation. It was postulated that RGTAs stimulate bone healing by interacting with HBGFs released in the wound site and, subsequently, by promoting the proliferation of cells implicated in this process. In a previous report, we examined how RGTA can modulate the proliferation of MC3T3-E1 osteoblastic cells. To further complete this study and to support this hypothesis, we developed an in vitro model of bone repair and examined the effects of RGTA alone or in association with FGF2, BMP-2, and TGF-beta1 which are representative of HBGFs known to stimulate bone repair. The model consisted of a 6-mm reproducible defect created on a MC3T3-E1 cell monolayer. In the presence of the different products added to the medium, the process of wound repair was measured through the filling of the acellular defect. We show that in 8 days, RGTA slightly inhibits repair alone compared to the control (2% FBS), that it inhibits the mitogenic effect of FGF2, and that it amplifies the inhibitory effect of BMP-2 and TGF-beta1. Repair was realized by an association of cell migration and cell proliferation mechanisms. To determine the part played by each process, DNA synthesis was evaluated for cell proliferation using an immunodetection technique [to measure incorporation of 5-bromo-2-deoxyuridine (BrdU)], coupled with a computer-assisted image analysis. The results show that the presence of RGTA (1) amplified the number of labeled nuclei compared to the control, (2) added to FGF2 or TGF-beta1, it reduced the number of labeled nuclei compared to FGF2 or TGF-beta1 alone, and (3) in the presence of BMP-2, it amplified the number of labeled nuclei compared to BMP-2 alone. Proper interpretation of these data requires a better understanding of the mechanism of action of RGTA on bone healing.

IGF‐I mediates the stimulatory effect of high phosphate concentration on osteoblastic cell proliferation

Although high concentrations of inorganic phosphate (Pi) are known to have a distinct anabolic effect on bone structure and metabolism, the precise mechanism by which phosphate possesses anabolic effect on bone formation has not been elucidated. The present study was performed to examine the effects of an increase in extracellular Pi concentration ([Pi](e)) on the proliferation of osteoblastic MC3T3-E1 cells. Increase in [Pi](e)(2-4 mM) dose-dependently stimulated DNA synthesis. Indomethacin, an inhibitor of prostaglandin synthesis, did not affect high [Pi](e)-induced DNA synthesis. DNA synthesis first increased affer a 3 h exposure to 4 mM [Pi](e) and its stimulatory effect was observed in a time-dependent manner up to 24 h. On the other hand, DNA synthesis was significantly but partially blocked by cycloheximide, suggesting that this stimulatory effect of high [Pi](e) was at least in part dependent on new protein synthesis. There is recent evidence that MG3T3-E1 cells constitutively produce and secrete insulin-like growth factor-I (IGF-I) and possess IGF-I receptors. IGF-I antiserum (1:10,000 to 1:100) significantly but partially blocked the stimulatory effect of [Pi](e) (4 mM) on DNA synthesis in a concentration-dependent manner. A neutralizing IGF-I antibody as well as IGF-I receptor antibody also significantly but partially blocked DNA synthesis stimulated by high [Pi](e) in a concentration-dependent manner, indicating that IGF-I at least in part mediated the high [Pi](e)-induced effect. Actually, high [Pi](e) significantly increased the secretion of immunoreactive IGF-I into the medium as well as the expression of IGF-I mRNA. Present findings indicate that an increase in [Pi](e) stimulated DNA synthesis partly via an increase in IGF-I action.