Alkaline Phosphatase Activity Test Research Articles

Biochemical Responses of Anodized Titanium Implants with a Poly(lactide-co-glycolide)/Bone Morphogenic Protein-2 Submicron Particle Coating. Part 1: An In Vitro Study.

This study was conducted to examine the effects of coating poly(D,L-lactide-co-glycolide) (PLGA)/recombinant human bone morphogenetic protein-2 (rhBMP-2) submicron particles by electrospray onto titanium (Ti) implants on the proliferation and differentiation capacity of mesenchymal stem cells. In addition, the duration of rhBMP-2 release was investigated. Ti disks were fabricated and divided into four groups as follows. Group C (control) was anodized at 300 V, group P was anodized and then coated with 0.2% PLGA, group B1 was anodized and then coated with PLGA/rhBMP-2 submicron particles at 200 ng per disk, and group B2 was anodized and then coated with PLGA/rhBMP-2 submicron particles at 600 ng per disk. The topography of the PLGA/rhBMP-2-coated Ti surfaces was examined, and proliferation assays, alkaline phosphatase activity tests, and rhBMP-2 releasing tests were conducted in vitro. There was no difference in the roughness of control Ti disks and Ti disks coated with PLGA/rhBMP-2 submicron particles by electrospray. The proliferation of mesenchymal stem cells increased over time; at 7 days, cell proliferation on the Ti disks coated with rhBMP-2 was significantly higher than the other groups. Anodized Ti disks coated with PLGA/rhBMP-2 (groups B1 and B2) released rhBMP-2 for approximately 21 days. In accordance with the amount of rhBMP-2 released from the PLGA/rhBMP-2 construct, the differentiation capacity of mesenchymal stem cells on the PLGA/rhBMP-2-coated disks was increased significantly. Submicron PLGA/rhBMP-2 coating on Ti implants by electrospray facilitated cell proliferation and differentiation, which is important for early healing and integration of implants.

Isolation and enrichment of mouse female germ line stem cells.

The existence of female germ-line stem cells (FGSCs) has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS) and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH) and stage-specific embryonic antigen-1 (SSEA1) markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR) (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3), alkaline phosphatase (AP) activity test and immunocytochemistry. Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV) positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl) whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

The effect of Emdogain and Simvastatin on periodontal ligament stem cells differentiation to osteoblasts: An in vitro study

Context: Regeneration of periodontium is one of the main goals of periodontal treatment. This may be achieved by differentiation of stem cells to periodontal tissue cells. Aims: This study sought to assess the differentiation of human periodontal ligament stem cells (PDLSCs) cultured in osteogenic medium supplemented with Emdogain or Simvastatin. Settings and Design: This is an experimental study. Materials and Methods: After teeth extraction, the PDL attached to the middle third of the root surface was removed. Cells were expanded in culture medium and used for osteogenic differentiation and added to the test materials (different combinations of the basic medium, dexamethasone [DEX], Simvastatin and Emdogain). Osteogenic differentiation was evaluated using Alizarin Red staining (ARS), alkaline phosphatase (ALP) activity and calcium content tests. Statistical Analysis Used: Statistical analysis was performed using SPSS version 17 software. Deliberation was done with 6 × 3 factorial (six groups × three periods). Then, the data were submitted to ANOVA, and the differences between the groups were compared using Tukey post-hoc test. Results: Quantitative analysis of ARS demonstrated that the frequency of mineralized nodule formation was the highest in the enamel matrix protein derivatives (EMD) + DEX group. The highest ALP activity on day 7 was in DEX + Simvastatin group. Calcium content was the lowest in the control group and the highest in EMD + DEX group at all tested time points. Conclusions: This study showed that 100 μg/ml Emdogain had a significant effect on osteogenic differentiation of human PDLSCs to osteoblasts and 10−8 M Simvastatin had an effect comparable to that of DEX.

Two approaches to form antibacterial surface: Doping with bactericidal element and drug loading

Two approaches (surface doping with bactericidal element and loading of antibiotic into specially formed surface microcontainers) to the fabrication of antibacterial yet biocompatible and bioactive surfaces are described. A network structure with square-shaped blind pores of 2.6±0.6×10−3mm3 for drug loading was obtained by selective laser sintering (SLS). The SLS-fabricated samples were loaded with 0.03, 0.3, 2.4, and 4mg/cm2 of co-amoxiclav (amoxicillin and clavulanic acid). Ag-doped TiCaPCON films with 0.4, 1.2, and 4.0at.% of Ag were obtained by co-sputtering of composite TiC0.5-Ca3(PO4)2 and metallic Ag targets. The surface structure of SLS-prepared samples and cross-sectional morphology of TiCaPCON-Ag films were studied by scanning electron microscopy. The through-thickness of Ag distribution in the TiCaPCON-Ag films was obtained by glow discharge optical emission spectroscopy. The kinetics of Ag ion release in normal saline solution was studied using inductively coupled plasma mass spectrometry. Bacterial activity of the samples was evaluated against S. epidermidis, S. aureus, and K. pneum. ozaenae using the agar diffusion test and photometric method by controlling the variation of optical density of the bacterial suspension over time. Cytocompatibility of the Ag-doped TiCaPCON films was observed in vitro using chondrocytic and MC3T3-E1 osteoblastic cells. The viability and proliferation of chondrocytic cells were determined using the MTS assay and PicoGreen assay tests, respectively. The alkaline phosphatase (ALP) activity of the SLS-fabricated samples loaded with co-amoxiclav was also studied. The obtained results showed that the moderate bacteriostatic effect of the Ag-doped TiCaPCON films is mainly manifested in the change of bacterial colony morphology and optical densities of bacteria suspensions. In contrast, the SLS-prepared samples showed a very rapid initial drug release resulting in strong bactericidal effect just from the start of the test and for as long as several days. Cytocompatibility and ALP activity tests demonstrated that it is possible to achieve a pronounced antibacterial effect compatible or even higher than that in the control sample (standard disk loaded with the amoxicillin/clavulanic acid mixture (30μg)) without compromising the material biocompatibility and bioactivity.

An in situ synthesis of mesoporous SBA-16/hydroxyapatite for ciprofloxacin release: in vitro stability and cytocompatibility studies.

The present work developed a biomaterial (HA/SBA-16) based on the growth of calcium phosphate (HA) particles within an organized silica structure (SBA-16) to evaluate its application as a drug delivery system. The samples were charged with ciprofloxacin as a model drug and in vitro release assays were carried out. The samples were characterized by elemental analysis (CHN), Fourier transform infrared spectroscopy, nitrogen adsorption, scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray scattering (SAXS) and X-ray diffraction. The results obtained by TEM, SEM and SAXS reveal a well-defined cubic arrangement of a uniform spherical mesoporous structure, an intrinsic characteristic of these materials, which indicated that SBA-16 and HA/SBA-16 could potentially encapsulate bioactive molecules by means of ordered mesopores. It was found that both surface interaction and pore volume affect the rate and amount of ciprofloxacin released from the mesoporous materials. In vitro assays were performed to evaluate the adhesion, viability, and growth behavior of human adipose tissue-derived stem cells (hADSC) on SBA-16 and HA/SBA-16 nanocomposites to verify their potential as a scaffold for application in bone-tissue engineering using MTT assay and alkaline phosphatase activity tests. The results showed that the materials are promising systems for bone repair, providing a good environment for the adhesion and proliferation of rat mesenchymal stem cells and hADSC in vitro.

Evaluation of the Effect of Platelet-Rich Plasma on Proliferation and Differentiation of Human Dental Pulp Stem Cells with or without Ga-Al-As Laser

Background Recently, the clinical use of low power lasers has increased, and it is said that wound healing is accelerated by their irradiation. The aim of this study was evaluation of the effect of platelet-rich plasma on proliferation and differentiation of human dental pulp stem cells with or without Ga-Al-As laser. Methods: In this experimental study, human lower third molar dental pulp cells prepared from torabinejad research center located in isfahan. When cells reached to adequate extent, divided in 4 groups including PRP, PRP+Laser, Laser, and control for implementation of MTT test and alkaline phosphatase activity test. In Laser and PRP+Laser groups, each well irradiated 45 seconds for MTT test and 135 seconds for alkaline phosphatase activity test. Results: Results demonstrated that PRP and PRP+Laser increased cell proliferation and viability up to 3 days but decreased cell proliferation and viability up to 5 days. Alkaline phosphatase activity was more in PRP+Laser, PRP and Laser, respectively, which all of them were less than control group. Alkaline phosphatase activity up-regulated in control group whereas in other groups down regulated. Conclusion: Laser irradiation can induce cell proliferation and in this field better acted than PRP. However, for assessment of stimulatory effect of Laser and PRP more studies are warranted. Keywords:Dental pulp stem cells, Ga-Al-As laser, Platelet-rich plasma.

Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells

Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8–40mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca2+]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10mM of Ca2+. Several experiments were conducted to demonstrate the effect of [Ca2+]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca2+]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10mM Ca2+ stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca2+. Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca2+]o, revealing that the CaSR is a key modulator of these cellular responses.

TiNi shape memory alloy coated with tungsten: a novel approach for biomedical applications

This study explores the use of DC magnetron sputtering tungsten thin films for surface modification of TiNi shape memory alloy (SMA) targeting for biomedical applications. SEM, AFM and automatic contact angle meter instrument were used to determine the surface characteristics of the tungsten thin films. The hardness of the TiNi SMA with and without tungsten thin films was measured by nanoindentation tests. It is demonstrated that the tungsten thin films deposited at different magnetron sputtering conditions are characterized by a columnar microstructure and exhibit different surface morphology and roughness. The hardness of the TiNi SMA was improved significantly by tungsten thin films. The ion release, hemolysis rate, cell adhesion and cell proliferation have been investigated by inductively coupled plasma atomic emission spectrometry, CCK-8 assay and alkaline phosphatase activity test. The experimental findings indicate that TiNi SMA coated with tungsten thin film shows a substantial reduction in the release of nickel. Therefore, it has a better in vitro biocompatibility, in particular, reduced hemolysis rate, enhanced cell adhesion and differentiation due to the hydrophilic properties of the tungsten films.

Effects of the combination of fibroblast growth factor-2 and bone morphogenetic protein-2 on the proliferation and differentiation of osteoprecursor cells.

Fibroblast growth factor (FGF) plays a critical role in bone growth; FGF-2 is known to be an important regulator of osteoblast activity because it stimulates osteoblast replication and decreases differentiation markers. Bone morphogenetic protein-2 (BMP-2) has been shown to be an active inducer of osteoblast differentiation and stimulates expression of mineralization-associated genes. The dose-dependent impact of FGF-2 and BMP-2 on the cellular proliferation and differentiation of osteoprecursor cells was evaluated. The alkaline phosphatase activity (ALP) test was performed to assess differentiation, and protein expressions related to bone formation were measured using the Western blot analysis. Cultures grown in the presence of FGF at 20 ng/mL showed significantly increased value when compared with control group and cultures loaded with FGF-2 at 20 ng/mL, and BMP-2 at 100 ng/mL showed significant decrease in cellular proliferation when compared with cultures loaded with FGF-2 at 20 ng/mL. The ALP activity increased when cells were treated with 10 and 100 ng/mL BMP-2, with relative ALP activity of 213.1% and 312.5%, respectively, when ALP activity of the uncontrolled control was considered 100%. However, when 100 ng/mL BMP-2 was combined with 20 ng/mL FGF-2, the relative increase reached up to 392.2%, but this did not reach a statistically significant increase when compared with 100 ng/mL BMP-2 alone. Within the limits of this study, BMP-2 significantly enhanced osteoblast differentiation but combined delivery of FGF-2 and BMP-2 did not produce synergistic effects on osteoblast differentiation under the current experimental condition.

Effects of the combination of dexamethasone and fibroblast growth factor-2 on differentiation of osteoprecursor cells

Dexamethasone is a potent modulator of osteogenic differentiation and previous studies have shown that dexamethasone increases the bone formation capacity of osteoprogenitor cells in vitro. Fibroblast growth factor‑2 (FGF‑2) is reported to have numerous biological activities, including stimulation of cell growth, migration, angiogenesis, wound healing, tissue repair, differentiation and morphogenesis. In the present study, the dose‑dependent effect of dexamethasone and FGF‑2 on the differentiation of osteoprecursor cells was evaluated. The alkaline phosphatase (ALP) activity test was performed to assess differentiation and protein expression associated with bone formation was measured by western blot analysis. The results showed that the protein content of the cultures grown with the osteogenic differentiation media in the presence of 2 ng/ml FGF‑2 was increased compared with that of the untreated control. ALP activity was increased when cells were treated with dexamethasone, with the highest value at 100 nM. The addition of 20 ng/ml FGF‑2 to 100 nM dexamethasone showed an increase in ALP activity, however, this was not statistically significant. The combination of 100 nM dexamethasone and 20 ng/ml FGF‑2 produced the highest expression of bone morphogenetic protein receptor‑IA and bone morphogenetic protein receptor‑II, leading to the highest value of pSmad1/5/8 expression. Within the limits of this study, dexamethasone significantly enhanced osteoblast differentiation, however, the combined delivery of dexamethasone and FGF‑2 did not produce synergistic effects on osteoblast differentiation under the current experimental conditions.

Combined effects of simvastatin and fibroblast growth factor-2 on the proliferation and differentiation of preosteoblasts

Simvastatin reportedly promotes osteoblastic and inhibits osteoclastic activity. It increases bone formation when injected subcutaneously over the calvaria in mice. It also increases cancellous bone volume in rats following oral administration. Fibroblast growth factor-2 (FGF-2), a member of the FGF family, is expressed by cells of the osteoblastic lineage. FGF-2 promotes osteoblast proliferation and it is secreted during the healing process of fractures or at bone surgery sites. FGF-2 reportedly regulates bone formation and osteoblast differentiation. In this study, the combined effects of simvastatin and FGF-2 on the proliferation and differentiation of preosteoblasts were investigated and an alkaline phosphatase (ALP) activity test was performed to assess the differentiation. Moreover, the expression of proteins associated with bone formation were measured using western blot analysis. The results demonstrated that the protein content of the cultures grown in osteogenic differentiation media in the presence of FGF-2 at a concentration of 20 ng/ml was higher compared to that of the untreated control cultures. ALP activity was decreased when cells were treated with FGF-2 (2 and 20 ng/ml) and increased when cells were treated with simvastatin. The cultures grown in the presence of 1 μM of simvastatin and 2 ng/ml of FGF-2 exhibited increased ALP activity when compared to that in the 2 ng/ml FGF-2-only group. The combination of 1.0 μM simvastatin and 2 ng/ml FGF-2 achieved a higher estrogen receptor-α expression compared to the 2 ng/ml FGF-2-only group. Within the limits of this study, simvastatin enhanced osteoblast differentiation. However, the combined treatment with simvastatin and FGF-2 did not exert synergistic effects on osteoblast differentiation under the current experimental conditions. Future studies are required to evaluate divergent conditions and determine the selective timing and optimal dosage for the delivery of the agents.

A comparative study of proliferation and osteogenic differentiation of rat adipose-derived stem cells in β-tricalcium phosphate (β-TCP), forsterite (Mg2SiO4) and clinoenstatite (MgSiO3)

In this study, the effects of forsterite and clinoenstatite powder extracts on the proliferation and osteogenic differentiation of rat adipose-derived stem cells (ASCs) were investigated and compared with the β-tricalcium phosphate (β-TCP) powder extracts. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and live-dead staining were performed to evaluate the viability and proliferation of rat ASCs. Osteogenic differentiation of rat ASCs were assayed by alkaline phosphatase (ALP) staining and ALP activity test. The expression of osteogenic marker genes (alkaline phosphatase (ALP), runt related transcription factor 2 (Runx2), collagen type Iα1 (Col1α1), secreted phosphoprotein1 (Spp1, osteopontin), integrin binding sialoprotein (Ibsp), bone gla protein (Bglap)) were detected by real-time polymerase chain reaction (PCR). The MTT assay and the live-dead staining showed that all the three ceramics possessed good cytocompatibility with rat ASCs. Furthermore, forsterite and clinoenstatite promoted the proliferation of rat ASCs compared with β-TCP. The results of the ALP activity test and the real-time PCR demonstrated that forsterite and clinoenstatite promoted the osteogenic differentiation of rat ASCs. These results suggested that forsterite and clinoenstatite are bioactive ceramics that may be used for preparation of bone tissue engineering (BTE) scaffolds. forsterite, clinoenstatite, adipose-derived stem cells, osteogenesis, bone, bone tissue engineering

In vitro degradation and biocompatibility of Ca-P coated magnesium alloy

Calcium-phosphate compounds(Ca-P) coating was prepared on an Mg-Al alloy(AZ60). Biodegradation of Ca-P coated magnesium alloy was evaluated in simulated body fluid(SBF) by examining the changes in magnesium ion concentration and pH value, which indicated that the Ca-P coating on magnesium alloy strongly affected the corrosion of magnesium alloy. Osteoblast MC3T3-E1 cells were utilized to investigate the cellular cytocompatibility. The cytocompatibility was measured by carrying out a series of tests, such as cholecystokinin-octapeptide(CCK-8) test, alkaline phosphatase activity(ALP) test, cellular morphology of hematoxylin-eosin(HE) staining and the induction of apoptosis. It was found that the cell function showed better in the Ca-P coated Mg-alloy extract than in the uncoated magnesium alloy extract. In summary, the results indicate that the Ca-P coating can improve the corrosion resistance of magnesium alloy and elevate cellular proliferation and differentiation of osteoblast MC3T3-E1 cells.

Evaluation of the proliferation and differentiation behaviors of mesenchymal stem cells with partially converted borate glass containing different amounts of strontium in vitro

The objective of this study was to examine the proliferation and differentiation behaviors of different compositions of strontium-containing (from 0-12 mol%) borate glasses with mesenchymal stem cells (MSCs). The Cell Counting Kit-8 (CCK-8) assay revealed that after three days of culturing, the 6Sr group had the highest cell growth rate. Analysis of cell morphology revealed that cells proliferated well near the particles of the samples in all the groups on day 3. On day 7, cells in the 6Sr group demonstrated a higher proliferation rate than other 4 groups under the microscope. When performing the Live-Dead staining experiment, the 6Sr group had the least number of dead cells. Total DNA qualification indicated that the 6Sr group had a statistically higher concentration compared with the remaining groups. It was found that on day 7, compared with the 0Sr group, the core binding factor α1 (Cbfa1) mRNA expression level was significantly higher in the 6Sr, 9Sr and 12Sr groups. On day 14, compared with the 0Sr group, the bone sialoprotein (BSP) mRNA level was significantly higher in the 6Sr group. Additionally, on day 21, the 6Sr and 9Sr groups demonstrated higher osteocalcin (OCN) mRNA expression levels compared with the 0Sr group. In the alkaline phosphatase (ALP) activity test, on day 21, the 6Sr group presented a higher activity than the 0Sr group. Further, the number of mineralized nodules per unit in MSCs was measured by Alizarin Red S staining. The results showed that the 6Sr and 9Sr groups had the greatest number of mineralized nodules. Therefore, it could be concluded that borate glasses containing strontium oxide of 0, 3, 6, 9 and 12 mol% demonstrate a significant level of proliferation when interacting with MSCs. The borate glass containing 6 mol% strontium oxide had the greatest level of proliferation when cultured with MSCs. The borate glass containing 6 and 9 mol% strontium oxide facilitated an improved bone formation ability compared with the remaining two compositions.

Adhesion, proliferation and differentiation of osteoblasts on zirconia films prepared by cathodic arc deposition

Zirconia films were prepared on titanium by cathodic arc deposition technique. The surface topography and element composition of the films were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Osteoblast-like MG63 cells were cultured on the surface of the zirconia films in vitro, and cell behaviour was investigated, with titanium as control. The results obtained from scanning electron microscopy and immunofluorescence studies showed that the MG63 cells on ZrO2 films spread better than those on Ti. The CCK8 assay indicated that the zirconia films promoted the proliferation of MG63 cells. The results of alkaline phosphatase (ALP) activity test and the expression of osteogenic marker genes, such as ALP, collagen I and osteocalcin, demonstrated that the differentiation of MG63 cells might be enhanced by zirconia films. In addition, the zirconia films possibly regulated osteoclastogenic gene expression by stimulating the expression of osteoprotegerin and reducing the expression of receptor activator of nuclear factor-kappaB ligand. The present work suggests that the ZrO2 film is worth further consideration for orthopedic implant applications.

The effects of fulvestrant, an estrogen receptor antagonist, on the proliferation, differentiation and mineralization of osteoprecursor cells

Fulvestrant is a novel type of endocrine treatment and is considered to be a potent inhibitor of breast cancer cell proliferation. Fulvestrant is reported to work by downregulating as well as degrading the estrogen receptor, leading to an inhibition of estrogen signaling through the estrogen receptor. The effects of various doses of fulvestrant for bone cells have not yet been fully investigated. In the present study, the effects of fulvestrant on osteoprecursor cells were evaluated. The effect on cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and protein measurement. Differentiation and mineralization were examined using an alkaline phosphatase activity (ALP) test and Alizarin red S staining. The protein expression of osteocalcin was evaluated using western blot analysis. Cultures grown in the presence of fulvestrant at concentrations of 0.1-10 µM did not show any significant change in cell proliferation. Cultures grown in the presence of fulvestrant showed a dose-dependent reduction in ALP activity, however, statistically significant differences were not achieved. Cultures grown in the presence of fulvestrant presented with a dose-dependent reduction in mineralization with a statistically significant difference at the 10 µM concentration. The use of fulvestrant may produce negative effects on the mineralization of osteoprecursor cells, while long-term use of fulvestrant may have detrimental effects on osteoblastic activity.

Biological Behavior of Osteoblast-like Cells on Titania and Zirconia Films Deposited by Cathodic Arc Deposition

Cathodic arc deposition technique was used to deposit zirconia (ZrO(2)) films and titania (TiO(2)) films on titanium (Ti) disks respectively. The surface topography was characterized by scanning electron microscopy and atomic force microscopy. The element composition of the films was detected by X-ray photoelectron spectroscopy. The phase of films was identified by thin film X-ray diffraction. The biological behavior of osteoblast-like MG63 cells cultured on Ti, TiO(2) and ZrO(2) was investigated and the possible signaling molecules involved was studied by the gene expressions of integrin β1, extracellular related kinase 1/2 (ERK1/2), and c-fos. The results indicated that both the TiO(2) and ZrO(2) films were amorphous. Scanning electron microscopy study showed that the adhesion of MG63 cells on TiO(2) and ZrO(2) films was significantly enhanced compared to Ti. The CCK8 assay indicated that the TiO(2) and ZrO(2) films promoted the proliferation of MG-63 cells. The alkaline phosphatase (ALP) activity test and the production of type collagen I (COLI) by immunofluorescence showed that both the TiO(2) and ZrO(2) films can enhance ALP activity and COLI expression of MG-63 cells. In addition, the ALP activity on ZrO(2) films was higher than on TiO(2) films at day 4, which indicate ZrO(2) films may lead to promotion of a more osteoblastic phenotype of MG-63 cells than TiO(2) films. Real-time polymerase chain reaction analysis demonstrated that The gene expression of integrin β1, ERK1/2, and c-fos was higher on TiO(2) and ZrO(2) films than on Ti. The present work suggests that the amorphous ZrO(2) films produced by cathodic arc deposition may be favorable for orthopedic implant applications and worth further study.

Effect of polyethylene terephthalates coated with bioactive glass on osteoblastic proliferation and cytoactivity in vitro

Objective To search for a novel method to promote the compabibility of materials and bone.The anterior cruciate ligament (ACL) reconstruction using artificial ligament [polyethylene terephthalates (PET)],which has good biocompatibility and mechanical properties,is prevalent.However,the low tendon-to-bone healing rate due to the PET' s hydrophobicity limits the clinical application.Methods This study aimed to observe the effects of the coating bioactive glass (58S) synthetic anterior cruciate ligament (ACL) on osteoblastic proliferation and cytoactivity in vitro.By using coating technique,bioactive glass (58S) was coated on the polyethylene terephthalates (PET) fibers.The scanning electron micrographs showed that bioactive glass (58S) were spindle-shaped and well-orientated on the PET fibers.Then,MC3T3-E1 osteoblasts were seeded onto the PET or the coating PET.Results The number of osteoblasts was significantly increased in the coating PET (8.92 ± 0.17 ) as compared with the PET (6.25±0.27) by using automated cell counter at the 5th day (P ＜ 0.05 ).Alkaline phosphatase activity and methyl thiazol tetrazolium ( MTT) test also showed that the coating fibrous scaffold induced signifienat osteogenesis and cell proliferation as compared to PET without coating.Conclusion The coating bioactive glass (58S) provides an instructive microenvironment for osteoblast proliferation. Key words: Anterior cruciate ligament; Polyethylene terephthalates; Artificial ligament; Bioactive glass

Osteoblast Activity on Anodized Titania Nanotubes: Effect of Simulated Body Fluid Soaking Time

Early phase osseointegration is crucial for orthopedic implants. For the improvement of osseointegrative properties of orthopedic implants several surface modification methods such as acid etching, hydroxyapatite (HA) coating and sandblasting can be applied. In this article titanium implants were anodized to possess nanotubular titania structures on the surface. Titania nanotube structures with a 45-50 nm of average inner diameter were obtained and to enhance bioactivity, samples were soaked in 10X simulated body fluid (SBF) for apatite deposition on surface for different time periods (1, 2, 3, 5, 8 hours). Apatitic calcium phosphate deposited surfaces were analyzed with infrared spectrometry and wettability studies. Effect of soaking time on osteoblast cell was investigated by cell viability, alkaline phosphatase activity tests and morphological evaluations. As a result, 3 hours of soaking time was found as the optimum time period (p < 0.005). This in vitro study indicated that soaking in 10X SBF can be a rapid and economical technique to enhance osseointegration of anodized titanium implants however excess and/or uncontrolled HA coating of titania layer limits the bioactive potential of the implant.

Combination of simvastatin and bone morphogenetic protein-2 enhances the differentiation of osteoblasts by regulating the expression of phospho-Smad1/5/8

Statins inhibit 3-hydroxy-3-methylglutarylcoen zyme A reductase, which catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate, a rate-limiting step in cholesterol synthesis. A number of studies have demonstrated bone-promoting effects when simvastatin is applied locally with different carriers in various animal models. In the prsent study, the dose-dependent impact of simvastatin and bone morphogenetic protein-2 (BMP-2) on the cellular proliferation and differentiation of osteopre-cursor cells was evaluated. The alkaline phosphatase activity (ALP) test was performed to assess differentiation, and protein expression related to bone formation, including that of phospho-Smad1/5/8 (pSmad1/5/8), was measured using western blot analysis to evaluate the underlying mechanism(s). Cultures grown in the presence of 0.1 μM simvastatin with 60 ng/ml BMP-2 exhibited the highest value for ALP activity. The results of the western blot analysis indicated that the addition of simvastatin upregulated pSmad1/5/8 expression and the combination of 0.1 μM simvastatin and 60 ng/ml BMP-2 produced a significant increase in protein expression. Based on these findings, it was concluded that the combination of simvastatin and BMP-2 produced positive effects on the differentiation of osteoprecursor cells. The results also suggest that the combination of simvastatin and BMP-2 has synergistic effects that are achieved through the BMP pathway by enhancing the expression of pSmad1/5/8 expression.