Differentiation Of Osteoblast-like Cells Research Articles

Effect of recombinant human bone morphogenetic protein‐7 (rhBMP‐7) on the viability, proliferation and differentiation of osteoblast‐like cells cultured on a chemically modified titanium surface

The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7). Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed. Cell viability (P=0.5516), cell number (P=0.3485), collagen content (P=0.1165) and mineralized matrix formation (P=0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio (P=0.00001) was affected by treatment and time. The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells

Lactoferrin (LF) has the ability to promote the proliferation and differentiation of osteoblasts, suggesting its potential utility as an osteogenic growth factor in bone tissue engineering. However, this type of application requires improved drug delivery system (DDS) technology at the target site. In this study, we report enhanced calcium deposition and alkaline phosphatase (ALP) activity using the type I collagen membrane during osteogenic differentiation of MG63 human osteoblast-like cells, indicating that type I collagen not only acts as a site for calcification but also promotes the expression of differentiated phenotypes. We also used this membrane as a drug delivery carrier for bovine LF. Approximately 27% of LF embedded on the type I collagen membrane was released within the first hour in cell-free condition. This initial burst release of LF was followed by a slower release from the collagen membrane. Bovine LF embedded in the type I collagen membrane promoted its calcification during osteogenic differentiation of MG63 cells without the loss of LF bioactivity. Taken together, ALP activity and osteocalcin production were enhanced in the MG63 cells plated on the LF-embedded collagen membrane, suggesting that LF incorporated in the collagen membrane promoted bone-like tissue formation by MG63 cells. These observations suggest that the type I collagen membrane is useful as a drug delivery carrier for LF in bone tissue engineering.

Advanced Oxidation Protein Products Inhibit Proliferation and Differentiation of Rat Osteoblast-like Cells via NF-κB Pathway

Background/Aims: Advanced oxidation protein products (AOPPs), a novel marker for oxidative protein damage, regulated cell behavior in many cell types. Although previous reports have revealed that oxidative stress resulted in an increased AOPPs content in cultured osteoblast-like cells, the effects of AOPPs on osteoblast-like cell function remain unclear. This study aimed to investigate the effect of AOPPs on the proliferation and differentiation of rat osteoblast-like (ROB) cells in vitro. Methods: ROB cells isolated from calvarias of newborn rats were incubated with AOPPs-modified rat serum albumin (AOPPs-RSA) prepared in vitro by incubation of RSA with hypochlorous acid. Cell proliferation was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Alkaline phosphatase activity was measured. Expression of osteocalcin mRNA and protein were examined using reverse transcriptase-polymerase chain reaction and Western blot, respectively. Reactive oxygen species (ROS) generation was detected by flow cytometry and fluorescent microscope. Phosphorylation of nuclear factor (NF)- κB p65 subunit was detected by Western blot. Results: Exposure of ROB cells to AOPPs-RSA significantly inhibited cell proliferation, decreased alkaline phosphatase activity, down-regulated the expression of osteocalcin mRNA and protein in both a concentration- and time-dependent manner. AOPPs challenge induced ROS generation and NF-κB p65 phosphorylation, which were inhibited by superoxide dismutase, catalase and NADPH oxidase inhibitors diphenyleneiodonium and apocynin. Furthermore, NF-κB inhibitor SN50 could reverse the AOPPs-induced inhibition of ROB cell proliferation and differentiation. Conclusion: These results suggest that AOPPs can inhibit proliferation and differentiation of ROB cells through ROS-dependent NF-κB pathway.

Influences of ionic dissolution products of dicalcium silicate coating on osteoblastic proliferation, differentiation and gene expression

This work aims to explore the influence of the ionic products of dicalcium silicate coating on osteoblastic proliferation and differentiation, as well as on the expression of BMP2 and its signal transducers Smad1, 6 and 7 in MG-63 osteoblast-like cells. Plasma-sprayed dicalcium silicate coatings were soaked in DMEM to obtain culture media containing the ionic dissolution products of dicalcium silicate coating (Ca 2SiO 4–DMEM). MG63 osteoblast-like cells were cultured in Ca 2SiO 4–DMEM (experimental group) for 3–12 days, while those cultured in normal DMEM served as control (control group). MTT assay was used to evaluate cell viability and proliferation. Alkaline phosphatase activity (ALP), osteocalcin (OC) and type I collagen (COLI) were investigated as differentiation markers. Gene expression of BMP2 and Smad1, 6, 7 was also detected. BMP2 protein was examined by ELISA assay. Alizarin Red-S (AR-S) assay was used to detect mineralization. The results demonstrated that Si concentration in Ca 2SiO 4–DMEM is markedly higher than that in normal DMEM. Compared to the control group, MG63 cells of the experimental group exhibited upregulated proliferation on day 3, and markedly upregulated gene expression of the differentiation markers, especially on days 9 and 12 for OC and on days 3, 6 and 9 for ALP. Gene expression of BMP2 and Smad1, as well as BMP2 protein secreted into culture media, was also upregulated in the experimental group, while gene expression of Smad6 and 7 was not influenced. AR-S assay indicated a higher calcium mineral content deposition in cells of the experimental group. In conclusion, the ionic products of plasma-sprayed dicalcium silicate coating are beneficial to the proliferation and differentiation of MG63 osteoblast-like cells.

IGF-I regulates tight-junction protein claudin-1 during differentiation of osteoblast-like MC3T3-E1 cells via a MAP-kinase pathway

Insulin-like growth factor I (IGF-I) is expressed in many tissues, including bone, and acts on the proliferation and differentiation of osteoblasts as an autocrine/paracrine regulator. Tight-junction proteins have been detected in osteoblasts, and direct cell-to-cell interactions may modulate osteoblast function with respect, for example, to gap junctions. In order to investigate the regulation of expression of tight-junction molecules and of function during bone differentiation, osteoblast-like MC3T3-E1 cells and osteocyte-like MLO-Y4 cells were treated with IGF-I. In both MC3T3-E1 cells and MLO-Y4 cells, the tight-junction molecules occludin, claudin-1, -2, and -6, and the gap-junction molecule connexin 43 (Cx43) were detected by reverse transcription with polymerase chain reaction. In MC3T3-E1 cells but not MLO-Y4 cells, mRNAs of claudin-1, -2, and -6, Cx43, and type I collagen, and proteins of claudin-1 and Cx43 were increased after treatment with IGF-I. Such treatment significantly decreased paracellular permeability in MC3T3-E1 cells. The expression of claudin-1 in MC3T3-E1 cells after IGF-I treatment was mainly upregulated via a mitogen-activated protein (MAP)-kinase pathway and, in part, modulated by a PI3-kinase pathway, whereas Cx43 expression and the mediated gap-junctional intercellular communication protein did not contribute to the upregulation. Furthermore, in MC3T3-E1 cells during wound healing, upregulation of claudin-1 was observed together with an increase of IGF-I and type I collagen. These findings suggest that the induction of tight-junction protein claudin-1 and paracellular permeability during the differentiation of osteoblast-like MC3T3-E1 cells after treatment with IGF-I is regulated via a MAP-kinase pathway, but not with respect to gap junctions.

Novel Textile Chitosan Scaffolds Promote Spreading, Proliferation, and Differentiation of Osteoblasts

Two novel scaffold models made of chitosan fibers were designed, fabricated, and investigated. Raw chitosan fibers were either tightened between plastic rings or were processed into stand-alone scaffolds. Chitosan fiber scaffolds were further modified by coating with a thin layer of fibrillar collagen type I to biologize the surface. Cell culture experiments were carried out using murine osteoblast-like cells (7F2). Confocal laser scanning microscopy (cLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were cultivated for up to 4 weeks on the materials and proliferation as well as osteogenic differentiation was quantitatively analyzed in terms of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. We found a 14-16-fold increase of cell number and the typical pattern of ALP activity, whereas the collagen coating does not remarkably influence these parameters. The maintenance of osteogenic phenotype on the novel materials was furthermore confirmed by immunostaining of osteocalcin and study of matrix mineralization. The feature of the collagen-coated but also the raw chitosan fiber scaffolds to support the attachment, proliferation, and differentiation of osteoblast-like cells suggest a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.

Novel carbon fiber composite for hip replacement with improved in vitro and in vivo osseointegration

A novel composite femoral stem has been developed to match cortical stiffness and achieve fixation by osseointegration with the primary goal to reduce cortical bone loss associated with stress shielding. The femoral stem consists of three distinct material layers: the first is a long carbon fiber (CF) in a polyamide 12 (PA12) polymer matrix (PA12/CF); the second is a PA12/HA (hydroxyapatite) interface; and the third is a plasma-sprayed coating of HA. In vitro studies with MG63 cells indicated that the HA surface supported improved proliferation and differentiation of osteoblast-like cells as determined by alkaline phosphatase activity and osteocalcin production when compared with Ti-6Al-4V (Ti64). In vivo studies comparing the composite and Ti64 rods in the rabbit femur demonstrated significantly higher bone apposition to the composite than Ti64 rods. The results of this study indicate that the invasion of surrounding bone cells and thus osteointegration together with its bone-matching mechanical properties make the PA12/CF/HA stem a promising hip replacement candidate.

Attachment, proliferation and osteogenic response of osteoblast‐like cells cultured on titanium treated by a novel multiphase anodic spark deposition process

A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using the electrochemical anodic spark deposition (ASD) technique and results in a thickened titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by means of evaluation of the attachment, morphology, proliferation, metabolic activity, differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium (Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation, and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase (alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way. Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a good surface for osteoblast attachment and proliferation, also promoting the maintenance of cell differentiation and matrix mineralization, a fundamental requirement for sustain the osseointegration and the clinical success of dental implants.

Effects of alkylphenols on bone metabolism in vivo and in vitro

Alkylphenols are endocrine disruptors that show estrogen-like effects in various wildlife species. However, little information is available about the action of these chemicals on bone metabolism. We investigated the effects of alkylphenols, such as nonylphenol (NP) and octylphenol (OP), on the formation of bone using several culture systems for osteoclasts and osteoblasts, as well as in vivo experiments. NP and OP dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) in cocultures of mouse spleen cells or mouse bone marrow cells with ST2 cells. However, β-estradiol at 10 −9 M to 10 −6 M did not affect this process. In contrast, neither compound affected the proliferation and differentiation of rat calvarial osteoblast-like cells (ROB cells). When NP or OP (0.1 mg/kg body weight) was administered subcutaneously to pregnant mice at 10 days, 12 days and 14 days post-coitus, fetuses at 17.5 days post-coitus showed stimulation of sternebrae bone calcification. Our findings suggest that alkylphenols have critical effects on the formation of bone by non-estrogenic effects.

Proliferation and differentiation of osteoblast‐like MC3T3‐E1 cells on biomimetically and electrolytically deposited calcium phosphate coatings

Biomimetic and electrolytic deposition are versatile methods to prepare calcium phosphate coatings. In this article, we compared the effects of biomimetically deposited octacalcium phosphate and carbonate apatite coatings as well as electrolytically deposited carbonate apatite coating on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. It was found that MC3T3-E1 cells cultured on the biomimetically deposited carbonate apatite coating demonstrated the greatest proliferation rate and the highest differentiation potential. Cells on the biomimetically deposited octacalcium phosphate coating had lower proliferation rate before day 7, but higher after that, than those on the electrolytically deposited carbonate apatite coating. There was no difference on the expression of early differentiation markers, that is, alkaline phosphatase activity and collagen content, between biomimetically deposited octacalcium phosphate and electrolytically deposited carbonate apatite coatings. However, higher expression of late differentiation markers, that is, osteocalcin and bone sialoprotein mRNA, was found on the biomimetically deposited octacalcium phosphate coating on day 14. These results suggest that the difference in in vitro osteoblast cell performance of calcium phosphate coatings might relate to their physicochemical properties. Biomimetic carbonate apatite coating is the most favorable surface for the proliferation and differentiation of MC3T3-E1 cells.

Beads of collagen–nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells

The aim of this work was to develop a novel method for preparing a three-dimensional bone-like matrix comprising nanohydroxyapatite crystals and fibrous collagen and to apply it for bone tissue engineering. Hydroxyapatite and collagen are the major components of natural hard bone. Therefore, they have been used extensively in orthopedic surgery as bone-filling materials. According to the principle of complex coacervation, three-dimensional collagen beads can be formed by extruding collagen solution into chondroitin sulfate A (CSA) solution. Subsequently, the collagen beads thus formed are soaked in simulated body-fluid solution to biomimic the formation process of natural bone matrix via the fabrication of collagen–nanohydroxyapatite beads. We also investigate the effect of the collagen–nanohydroxyapatite matrix on the proliferation and differentiation of MG63 cells. The presence of crystalline hydroxyapatite structure on the surface of fibrous collagen was confirmed by X-ray diffraction. MG63 cells cultured on the collagen–nanohydroxyapatite beads proliferate at the normal rate. Moreover, alkaline phosphatase (ALP) activity and the expression levels of three osteogenic genes, namely, type I collagen osteopontin and osteocalcin, in MG63 cells were significantly higher when the cells were cultured on collagen–nanohydroxyapatite beads than when they were cultured on collagen alone. The results of this study reveal that, in the presence of nanohydroxyapatite, the three-dimensional cell beads not only provide a substrate for cell growth but could also enhance the osteoblast-like cell differentiation of MG63 cells.

Improvement of bone properties and enhancement of mineralization by ethanol extract of Fructus Ligustri Lucidi

Fructus Ligustri Lucidi (FLL), a kidney-tonifying Chinese herb, was shown to regulate Ca balance in ovariectomized (OVX) rats in our previous study. This study investigated whether it could improve bone properties in aged normal and OVX rats and increase osteoblastic differentiation in rat osteoblast-like UMR-106 cells. Ten-month-old aged rats underwent sham-operation or ovariectomy, were orally administered with FLL extracts or its vehicle and fed with diets containing different levels of Ca (LCD, 0.1 % Ca; MCD, 0.6 % Ca; HCD, 1.2 % Ca) for 12 weeks. Ovariectomy induced bone loss at multiple-sites of both tibia and femur in all rats being studied. FLL extract increased bone mineral density and bone mineral content at both tibial and femoral diaphysis as well as the lumbar vertebra (LV-2) in rats fed either LCD or MCD. In addition, FLL increased biomechanical strength of the tibial diaphysis in these rats. Combination of FLL and high-Ca diet significantly improved bone mass of cortical and trabecular bone at appendicular bones and LV-2 and decreased bone loss associated with ovarietomy and low-Ca feeding. Treatment of UMR-106 cells with FLL extracts accelerated the formation of calcified matrix and increased extracellular Ca and P depositions in time- and dose-dependent manner. The level of mineralization reached a maximum by 6 d incubation at the dosage of 10 mug FLL extract/ml. Our study indicated that FLL extract could improve bone properties in aged rats possibly via its direct action on osteoblastic cells by enhancement of the mineralization process.

Initial effects of low-level laser therapy on growth and differentiation of human osteoblast-like cells

Low-level laser therapy is a clinically well established tool for enhancement of wound healing. In vitro studies have also shown that low level laser therapy has a biostimulatory effect on cells of different origin. The aim of this in vitro study was to investigate the initial effect of low-level laser therapy on growth and differentiation of human osteoblast-like cells. SaOS-2 cells were irradiated with laser doses of 1 J/cm2 and 2 J/cm2 using a diode laser with 670 nm wave length and an output power of 400 mW. Untreated cells were used as controls. At 24 h, 48 h and 72 h post irradiation, cells were collected and assayed for viability of attached cells and alkaline phosphatase specific activity. In addition, mRNA expression levels of osteopontin and collagen type I were assessed using semi-quantitative RT-PCR. Over the observation period, cell viability, alkaline phosphatase activity and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm2 compared with untreated control cells. Increasing the laser dose to 2 J/cm2 reduced cell viability during the first 48 h and resulted in persistently lower alkaline phosphatase activity compared with the other two groups. The expression of osteopontin and collagen type I mRNA slightly decreased with time in untreated controls and cells irradiated with 1 J/cm2, but their expression was increased by treatment with 2 J/cm2 after 72 h. These results indicate that low-level laser therapy has a biostimulatory effect on human osteoblast-like cells during the first 72 h after irradiation. Further studies are needed to determine the potential of low-level laser therapy as new treatment concept in bone regeneration.

Effect of polyphosphoric acid pre‐treatment of titanium on attachment, proliferation, and differentiation of osteoblast‐like cells (MC3T3‐E1)

To evaluate the effect of polyphosphoric acid (PPA) pre-treatment of titanium (Ti) on the initial attachment, proliferation, and differentiation of mouse osteoblast-like cells (MC3T3-E1). Adsorption of PPA to Ti was achieved by immersing Ti disks (15 mm in diameter) into 0, 1, and 10 wt% PPA and 10 wt% orthophosphoric acid (OPA) for 24 h. On each pre-treated Ti disk, 5.0 x 10(4) MC3T3-E1 cells were seeded, and 1, 3, and 5 h later cell attachment was evaluated. Cell proliferation was also determined 1, 3, and 5 days after cell seed. Cell differentiation was evaluated 5, 10, and 15 days after cell seed using osteoblast marker gene expression. Total RNA was purified from each culture and Type-I collagen, alkaline phosphatase, and osteocalcin mRNA expression levels were measured by real-time reverse transcription polymerase chain reaction. Adsorption of PPA or OPA to Ti significantly accelerated initial cell attachment at every time point (P<0.0001). As with cell attachment, cell proliferation was also accelerated on the PPA-treated Ti disks in a dose-dependent manner at every time point (P<0.0001). However, OPA-treated Ti disks did not enhance the cell proliferation. Regarding osteoblastic differentiation, PPA-treated Ti significantly accelerated the Type-I collagen gene expression at 5 and 10 days after cell seed. Regarding alkaline phosphatase and osteocalcin gene expression, no significant difference was found among the different Ti surface conditions. The accelerated cell behavior following Ti pre-treatment with PPA is a promising new strategy to fabricate bioactive Ti implants.

Effect of exposure to an extremely low frequency-electromagnetic field on the cellular collagen with respect to signaling pathways in osteoblast-like cells

The effect of exposure to extremely low frequency-electromagnetic field (ELF-EMF: 3 mT, 60 Hz) on differentiation of mouse osteoblast-like MC3T3-E1 cells was examined together with addition of insulin-like growth factor I (IGF-I). As a marker of the differentiation, the cellular collagen content was determined by the absorbance of Sirius red-stained cells measured at the wavelength of 510-520 nm with an imaging microspectroscopy. Exposure to ELF-EMF increased significantly the collagen in the cells. Treatment with PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) activation, reduced the collagen in all of the cells examined on control, IGF-I addition and ELF-EMF exposure, however, PD98059 did not prevent the increase in the collagen caused by ELF-EMF exposure, and IGF-I also increased the collagen in the presence of the inhibitor. When phosphatidylinositol 3-kinase (PI3K) pathway was inhibited by LY294002, the increase in collagen induced by ELF-EMF exposure was accelerated, however, the increase in collagen observed by IGF-I addition was suppressed. Treatment with SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), suppressed the increase in the collagen induced by ELF-EMF exposure, whereas IGF-I addition increased the collagen in the presence of the inhibitor. These results suggested that collagen synthesis stimulated by ELF-EMF exposure was carried out by the participation of p38 MAPK pathway, and that PI3K pathway may have the role to suppress the collagen synthesis induced by ELF-EMF exposure, and that the suppression of the PI3K pathway may allow the acceleration of the collagen synthesis.

Cytobiocompatibility of collagen and ePTFE membranes on osteoblast-like cellsin vitro

In guided bone regeneration (GBR), a semipermeable membrane is placed over an osseous defect to create a secluded environment in which bone formation can proceed without ingrowth of connective tissue cells from the overlaying soft tissue. Although the cell-occlusive property of GBR membranes appear to be essential to new bone formation, the role of transmembrane tissue fluid diffusion is not known. The objective of this study was to evaluate the degree to which diffusion across commonly used GBR membranes can support functional properties of osteoblast-like cells in vitro. Cells from an established osteoblast-like line (SAOS-2) were cultured on membranes of cross-linked collagen, noncross linked collagen, and ePTFE. The membranes rested on metal grids which allowed the membranes to lightly contact the surface of the culture medium. As a control, cells were directly plated and cultured in control wells. At days 7 and 21, cells were harvested by scraping the membranes or culture wells and analyzed for expression of alkaline phosphatase (ALP), core binding factor 1 (cbfa-1), bone sialoprotein-2 (BSP-2), and osteocalcin (OC). Expression was determined by quantitative real-time PCR. Glucose-6-phosphate dehydrogenase (G6PD) served as a reference gene. The membranes were examined by transmission light microscopy. RT-PCR revealed up-regulation of ALP of up to 60-fold and of cbfa-1 and BSP of up to threefold relative to G6PDH. Expression of OC was less then onefold. The expression profile for each of the four genes tested demonstrated small variations among cells grown on different membranes. Microscopic observations revealed remnants of undisrupted osteoblast-like cells attached to both collagen membranes. Cell morphology and spatial arrangement indicated that vitality was maintained. Diffusion through the three membranes evaluated in this study was sufficient to support osteoblast-like cell differentiation.

In vitro behavior of osteoblast-like cells on PLLA films with a biomimetic apatite or apatite/collagen composite coating

To investigate the methods to improve the cell-material interaction of devices or tissue engineering scaffolds made of poly(L-lactic acid) (PLLA) polymer, apatite and apatite/collagen composite coatings were formed on PLLA films within 24 h through accelerated biomimetic processes. In vitro investigation using Saos-2 osteoblast-like cells through cell culture was conducted to assess the biological performance of these biomimetic coatings. The cell morphology on three types of surfaces, viz., PLLA film, PLLA film with the apatite coating, and PLLA film with the apatite/collagen composite coating, was studied using scanning electron microscopy (SEM). Cell viability was estimated using the MTT assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. The results obtained indicated that the biomimetic apatite and apatite/collagen composite coatings could significantly enhance the proliferation and differentiation of osteoblast-like cells. The apatite/collagen composite coating appears to be promising for the surface modification of PLLA-based devices with much improved interactions with osteoblastic cells.

Effects of extremely low frequency pulsed electromagnetic field on different-derived osteoblast-like cells

To investigate the effect of the extremely low frequency pulsed electromagnetic field (PEMF) on the proliferation and differentiation of osteoblast-like cells. The MC3T3-E1 cell and the primary osteoblast cell derived from 2-day-old Sprague Dawley (SD) rat calvaria were exposed to PEMF with a magnetic flux density of 1.55 mT at 48 Hz for 24 or 48 h. MTS was applied to analyze cell proliferation and flow cytometry to detect cell cycle. The intracellular alkaline phosphatase (ALP) activity was measured by colorimetry. PEMF of 1.55 mT at 48 Hz decreased significantly the cell percentage of S or G(2)M phase (P < 0.05), but did not affect cell number of MC3T3-E1 cells. Although the number of the primary osteoblast cells did not alter by MTS assay after exposure to PEMF for 24 h continuously, the cell percentage of G(2)M phase increased significantly (P < 0.01). When the culture time extended to 48 h, the cell number increased greatly (P < 0.01) and the cell percentage of G(2)M phase decreased significantly despite of the exposure type (P < 0.01). After the primary osteoblast cells were exposed to PEMF for 24 h continuously, the ALP activity decreased significantly (P < 0.05), whereas it increased significantly after exposure to PEMF for 48 h continuously (P < 0.05). PEMF of 1.55 mT at 48 Hz does not affect proliferation and differentiation of MC3T3-E1 cell, but it promotes proliferation of primary osteoblast cell, inhibits differentiation at proliferation stage and promotes differentiation at differentiation stage of primary osteoblast cell.

Novel production method and in‐vitro cell compatibility of porous Ti‐6Al‐4V alloy disk for hard tissue engineering

Porous metals are attractive due to its unique physical, mechanical, and new bone tissue ingrowth properties. In the present study, the production of highly porous Ti-6Al-4V parts by powder metallurgical technology and subsequently it's uses in in vitro bone tissue engineering is described. A space-holder method using carbamide with different particle size to produce parts with porosities between 35 and 70% were applied. The compressive strength and Young's modulus of porous Ti-6Al-4V were determined. Results indicated that stress and Young's modulus decrease with increasing porosity and pore size. The porous parts are characterized by scanning electron microscopy. Furthermore, study was to investigate the effects of three different porosities of porous Ti-6Al-4V (35, 50, and 70%) on proliferation, differentiation, and cell-matrix interaction of mouse osteoblast-like cells, MC-3T3. Results showed that the cell proliferation was significantly (p < 0.05) higher on 70% porous Ti-6Al-4V. However, synthesis of different types of extra cellular matrix proteins was also more abundant on 70% porous Ti-6Al-4V than 35 and 50% porous Ti-6Al-4V disk except some specific proteins. An increase in alkaline phosphate activity was significantly (p < 0.05) higher on 70 and 50% porous Ti-6Al-4V disk after 12 days of MC-3T3 cells incubation. Above all, results indicated that porosity (nearly 70%) of porous Ti-6Al-4V topography affects proliferation and differentiation of osteoblast-like MC-3T3 cells. The results showed that this novel process is a promise to fabricate porous biomaterials for bone implants.

Oxidized NiTi surfaces enhance differentiation of osteoblast‐like cells

A new oxidation treatment (OT) on NiTi shape memory alloys was developed in a previous work. This OT treatment significantly decreases Ni ion release into the exterior medium, and therefore is thought to be beneficial for NiTi cytocompatibility. As to confirm this expectation, the in vitro response of MG63 osteoblast-like cells cultured on untreated and oxidized NiTi surfaces was studied. An adhesion test at 1, 4, and 8 h of incubation was performed. Statistical differences were evidenced at 1 h of adhesion depending on the surface treatment and chemical composition of the substrate. However, at larger times of study, there were no statistically significant differences between untreated and oxidized surfaces. The proliferation test (until 9 days) showed that untreated and oxidized NiTi surfaces are not cytotoxic for MG63 cells. The differences of adhesion at short times did not affect the proliferation of MG63 cells. However, after 48 h of stimulation with ascorbic acid and dexamethasone, the MG63 cells cultured on oxidized surfaces showed higher alkaline phosphatase activity and osteocalcin levels. The improvement of osteoblast differentiation due to OT treatment could accelerate bone formation, and, therefore, could allow earlier loading of NiTi devices used in dental and orthopedic applications.