This study was aimed at exploring the viability of osteoblasts on conductive tissue engineering material. Conductive biodegradable Polyprrole/Polylactide (PPy/PLA) was prepared by emulsion polymerization. Scanning electron microscope (SEM) and spectroscopy showed evenly dispersed PPy in PLA. PPy/PLA membrane was found being able to keep conductive stability for more than one month to provide electric circumstances (ECs) for osteoblasts. SEM displayed that osteoblasts adhered and spread well on PPy/PLA. ECs of 12.5, 25, 50, 75, 100, 125, 150, 175, 200 microA/cm2 were separately used to stimulate osteoblasts for 24h, 48h, 72h and 96h. Methyl-thiazole-tetrazolium (MTT) assay after 24h revealed that 50 microA/cm2 evidently accelerated osteoblasts proliferation. Alkaline phosphatase (ALP) activity assay revealed that, 48h later, 50 microA/cm2, and 75 microA/cm2 promoted osteoblasts differentiation. 50 microA/cm2 enhanced osteoblasts mineration. Conclusively, 50 microA/cm2 can strengthen osteoblasts' function and promote their viability.
Absorbable Implants ; Biocompatible Materials ; chemistry ; metabolism ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Electric Conductivity ; Electric Stimulation ; Humans ; Osteoblasts ; cytology ; Polyesters ; chemistry ; Polymers ; chemistry ; Pyrroles ; chemistry ; Tissue Scaffolds ; chemistry

OBJECTIVETo investigate the growth activity of osteoblast on a novel strontium incorporated calcium sulfate and make comparison with normal calcium sulfate material.

METHODSOsteoblast was inoculated on samples and cell proliferation was measured on the 1st, 3rd, 5th days, and the activities of ALP and osteocalcin were observed on the 5th day. And microcosmic morphology of osteoblast was observed by scanning electron microscopy(SEM).

RESULTSOsteoblast grows robustly on tested material. Cell quantity on the surface of novel material was obviously higher than normal calcium sulfate material (P < 0.05). The activity of ALP and osteocalcin on novel material was 57.8% and 40.2% higher than on normal calcium sulfate material respectively (P < 0.05). On strontium incorporated surface, osteoblast spread well. Cells were polygonal with abundant cytoplasm and the morphology was active.

CONCLUSIONStrontium incorporated calcium sulfate can sustain robust growth activity of osteoblast, which is promising to be used for bone substitute materials.

3T3 Cells ; Alkaline Phosphatase ; metabolism ; Animals ; Bone Substitutes ; chemistry ; pharmacology ; Calcium Sulfate ; chemistry ; pharmacology ; Cell Proliferation ; drug effects ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; metabolism ; Strontium ; chemistry

In order to study the cytocompatibility of nanophase hydroxyapatite ceramic in vitro, we prepared hydroxyapatite by use of the wet chemistry techniques. The grain size of hydroxyapatite of interest to the present study was determined by scanning electron microscopy and atomic force microscopy with image analysis software. Primary culture of osteoblast from rat calvaria was established. Protein content, synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase hydroxyapatite ceramics and on conventional hydroxyapatite ceramics for 7, 14, 21 and 28 days were examined. The results showed that the average surface grain size of the nanophase and that of the conventional HA compact formulations was 55 (nanophase) and 780 (conventional) nm, respectively. More importantly, compared to the synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase was significantly greater than that on conventional ceramics after 21 and 28 days. The cytocompatibility was significantly greater on nanophase HA than on conventional formulations of the same ceramic.
Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; Cells, Cultured ; Ceramics ; chemistry ; Durapatite ; chemistry ; Microscopy, Atomic Force ; Microscopy, Electron, Scanning ; Nanostructures ; chemistry ; Osteoblasts ; cytology ; Rats

OBJECTIVETo investigate the influence of surface modification of titanium on OPG/RANKL mRNA expression in human osteoblast-like cells.

METHODSMG-63 osteoblast-like cells were seeded on the titanium plates with surface polishing and with surface modification by sandblasting plus acid-base treatment, with the cells on glass slides as the control. On days 2, 4, 6, and 8 following cell seeding, the cells were harvested for examination of OPG/RANKL mRNA expression using RT-PCR and real-time PCR.

RESULTSThe expression of OPG/RANKL mRNA was sensitive to the surface microphotography. Compared with the other groups, the cells on the titanium plates with sandblasting plus acid-base treatment, which resulted in a porous micro-structure and high roughness, showed significantly up-regulated expression of OPG mRNA. OPG mRNA expression also showed a time-dependent up-regulation, and was the highest on day 8. The expression of the RANKL mRNA in cells on both of the titanium plates was higher than that in the control cells. The peak level of RANKL mRNA expression occurred on day 6 followed by a gradual decrease.

CONCLUSIONA rough and porous surface of the culture plates and prolonged culture time can synergistically up-regulate the ratio of OPG/RANKL mRNA.

Cell Culture Techniques ; Cell Line ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Porosity ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Surface Properties ; Tissue Scaffolds ; Titanium ; chemistry ; pharmacology

OBJECTIVETo evaluate the adhesion, proliferation and differentiation of osteoblast-like cells on the ultraviolet (UV)-treated titanium in different storing environment, and to find a way to enhance the bioactivity of titanium and to prevent its age-related degradation.

METHODSAcid-etched titanium disks stored under ambient conditions for 4 weeks and treated with UV light for 48 h.Then disks were divided into three groups and placed in a sealed container for 0 h (no-stored,NO group) , 4 weeks (air-stored, AS group) or in a sealed container filled with nitrogen for 4 weeks (nitrogen-stored,NS group) respectively. A group of UV-untreated titanium served as negative control (NC group).The surface morphology was evaluated using scanning electron microscopy (SEM), and hydrophilicity of disks were measured using contact angle measuring device. Cell counting kit-8 was used to measure the cell adhesion and proliferation. Cell differentiation was evaluated by testing alkaline phosphatase (ALP) activity using ALP reagent kit.

RESULTSThere was no difference in surface topography among groups.Contact angels in NS group [(67.70 ± 3.59)°] and NO group [(0.70 ± 0.28)°] were smaller than the others (P < 0.05). Cell adhesion in NS group at 2 h and 4 h point was (0.237 ± 0.006) and (0.578 ± 0.039), respectively, and proliferation at 3 d and 5 d point was (0.743 ± 0.026) and (1.548 ± 0.046) respectively, which were significantly higher than those in AS group [(0.158 ± 0.036), (0.400 ± 0.010), (0.499 ± 0.019) and (1.174 ± 0.062)] and in NC group [(0.161 ± 0.024), (0.390 ± 0.011), (0.508 ± 0.015) and (1.209 ± 0.025)] at the same time point (P < 0.05). How ever the results mention above in the NS group were lower than those in the NO group (P < 0.05). No difference were found between data from the AS group and NS group (P > 0.05). Osteoblast-like cells had an abundant spread on NS and NO group during 2 h incubation, but did not exactly spread on AS and NC group after incubation for 4 h. No difference were found in ALP among groups.

CONCLUSIONSUV treatment can enhance bioactivity of titanium, and nitrogen storage can slow down its biological aging.

Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; radiation effects ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Cells, Cultured ; Mice ; Microscopy, Electron, Scanning ; Nitrogen ; chemistry ; Osteoblasts ; cytology ; metabolism ; Surface Properties ; Titanium ; chemistry ; radiation effects ; Ultraviolet Rays

We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation.
Animals ; Bioreactors ; *Bone Regeneration ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Lactic Acid/chemistry ; *Mechanical Processes ; Mechanotransduction, Cellular/physiology ; Mice ; Osteoblasts/cytology/*metabolism ; Polyesters/chemistry ; Polymers/chemistry ; Tissue Engineering/methods ; Tissue Scaffolds/chemistry/utilization

The Wnt/beta-catenin pathway has a role in osteoblast differentiation and bone formation. We screened 100 plant extracts and identified an extract from Euodia sutchuenensis Dode (ESD) leaf and young branch as an effective activator of the Wnt/beta-catenin pathway. ESD extract increased beta-catenin levels and beta-catenin nuclear accumulation in murine primary osteoblasts. The ESD extract also increased mRNA levels of osteoblast markers, including RUNX2, BMP2 and COL1A1, and enhanced alkaline phosphatase (ALP) activity in murine primary osteoblasts. Both ESD extract-induced beta-catenin increment and ALP activation were abolished by beta-catenin knockdown, confirming that the Wnt/beta-catenin pathway functions in osteoblast differentiation. ESD extract enhanced terminal osteoblast differentiation as shown by staining with Alizarin Red S and significantly increased murine calvarial bone thickness. This study shows that ESD extract stimulates osteoblast differentiation via the Wnt/beta-catenin pathway and enhances murine calvarial bone formation ex vivo.
Animals ; Cell Differentiation/*drug effects ; Evodia/*chemistry ; HEK293 Cells ; Humans ; Mice ; Osteoblasts/cytology/*drug effects/*metabolism ; Osteogenesis/drug effects ; Plant Extracts/chemistry/*pharmacology ; Skull/anatomy & histology/drug effects/metabolism ; Wnt Signaling Pathway/*drug effects ; beta Catenin/genetics/metabolism

In this experimental study, the RGD-containing peptide was used to modify the surface of biomimetic PLGA-(ASP-PEG) matrix, and bone marrow stromal cells (BMSCs) were seeded onto these modified surfaces for three weeks. The effects of modified surfaces of matrix on the adhesion, proliferation and differentiation of BMSCs were explored. BMSCs were harvested from whole bone marrow of Sprague-Dawley (SD) rats in vitro, then were seeded onto peptide surface-modified matrix (Experiment group, EG) and matrices without modification (Control group, CG) respectively for three weeks. The number of adhesive cells was counted by using precipitation method after 4 h and 12 h incubation; the cells cytoskeletons were stained with FITC-conjugated phalloidin after 24h incubation; the cell density was investigated after 1 d, 2 d and 3 d of incubation; ALP activity of BMSCs was measured after 7 d, 14 d and 21 d of incubation with osteogenic medium. The cells from bone marrow were BMSCs and their purity was beyond 90% using flow cytometry (FCM) analysis. Sulphur binding energy in EG was shown by XPS to be 164 eV. BMSCs adhered on peptide surface-modified matrix were observed with SEM. Cell adhesion efficiency and quality in EG was better than that in CG, and cell cytoskeleton was more robust in EG. ALP activity was higher in EG than in CG. Peptide surface-modified PLGA-(ASP-PEG) was noted to have good compatibility with BMSCs and to promote cell adhesion and differentiation.
Animals ; Biocompatible Materials ; chemistry ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Adhesion ; drug effects ; Cell Differentiation ; drug effects ; Lactic Acid ; chemistry ; Oligopeptides ; chemistry ; Osteoblasts ; cytology ; Peptides ; chemistry ; Polyglycolic Acid ; chemistry ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; cytology ; drug effects ; metabolism ; Surface Properties ; Tissue Engineering ; Tissue Scaffolds

The Wnt/β-catenin and bone morphogenetic proteins (BMPs) pathways play important roles in controlling osteogenesis. Using a cell-based kinase inhibitor screening assay, we identified the compound bisindoylmaleimide I (BIM) as a potent agonist of the cytosolic β-catenin accumulation in preosteoblast cells. Through suppressing glycogen synthase kinase 3β enzyme activities, BIM upregulated β-catenin responsive transcription and extended duration of BMP initiated signal. Functional analysis revealed that BIM promoted osteoblast differentiation and bone formation. The treatment of human mesenchymal stem cells with BIM promoted osteoblastogenesis. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by integration of the cellular signaling pathways.
Animals ; Bone Morphogenetic Proteins ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Indoles ; chemistry ; pharmacology ; Maleimides ; chemistry ; pharmacology ; Mesenchymal Stem Cells ; cytology ; metabolism ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Signal Transduction ; drug effects ; Wnt Proteins ; metabolism ; beta Catenin ; antagonists & inhibitors ; genetics ; metabolism

Natural products especially flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. The present study is to investigate the effects of isoquercitrin from Craibiodendron yunnanense with different concentrations at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) on proliferation, differentiation and mineralization of MC3T3-E1. Cell proliferation was assessed by CCK-8 kit at 1, 3, 5 and 7 days of culture. Alkaline phosphatase (ALP) activity were performed qualitatively and quantitatively on day 7, and alizarin red S staining was employed to access the mineralization of cells on day 21. The osteogenic markers ALP, collagen type I (COL 1A1), runt-related transcription factor 2 (Runx-2) and Osterix were detected to analysis early osteogenic differentiation of cells on day 3 by RT-PCR. The results showed that isoquercitrin had a dose-dependent effect on the proliferation, osteogenic differentiation, mineralization and gene expression of MC3T3-E1 in the range from 1 x 10(-7) to 1 x 10(-5) mol x L(-1). At concentrations above 1 x 10(-4) mol x L(-1) isoquercitrin showed cytotoxicity, while 1 x 10(-6) mol x L(-1) is the optimal concentration of isoquercitrin to improve the osteoblastic activity. All these results implied that isoquercitrin might be the major composition of traditional Chinese medicine C. yunnanense to treat bone fractures.
Alkaline Phosphatase ; metabolism ; Animals ; Cell Line ; Collagen Type I ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ericaceae ; chemistry ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteogenesis ; drug effects ; Quercetin ; analogs & derivatives ; pharmacology