Animals ; Cytoplasm ; ultrastructure ; Fibroblasts ; ultrastructure ; Fractures, Bone ; pathology ; Microscopy, Electron ; Osteoblasts ; ultrastructure ; Rabbits ; Wound Healing

OBJECTIVETo explore the effects of cofilin on the actin cytoskeleton reorganization in osteoblasts induced by fluid shear stress.

METHODSFluid shear stress (1.2 Pa) was applied to osteoblasts for 0 (control group), 15, 30, 45, 60, 120 min in vitro. Cells were stained with fluorescein isothiocyanate (FITC)-phalloidin for fiber-actin, and confocal laser scanning microscope(CLSM) was used to observe the fluorescence of fiber-actin. Western blotting was used to detect the expression of the cofilin and the phospho-cofilin.

RESULTSActin filaments became organized into stress fibers that were thicker and more abundant than those in non-flowed cells. The fluorescence intensity (38.00 ± 6.88) of fiber-actin after 120 min (42.93 ± 6.41) loading it was 2.8 times as much as that in control group (15.41 ± 3.60, P < 0.05). Additionally, the level of phospho-cofilin protein was dramatically elevated after loading. Fluid shear stress induced an initial decrease of cofilin at 60 min. However, at 120 min cofilin (0.254 ± 0.026) increased to 1.5 times as much as that at 60 min (0.162 ± 0.004).

CONCLUSIONSThe results indicate that cofilin phosphorylation mediates fiber-actin reorganization in the osteoblasts induced by fluid shear stress.

Actin Cytoskeleton ; ultrastructure ; Actin Depolymerizing Factors ; biosynthesis ; Humans ; Osteoblasts ; ultrastructure ; Phosphorylation ; Stress, Mechanical

OBJECTIVETo study the effect of Ti-TiO2 nanotubes with different diameters on the adhesion of fibroblast and osteoblast, and to find which diameter was more favorable for cells' respective adhesion.

METHODSPure titanium sheets were polished and then anodized at different potentials for 1 h with Ti as anode and Pt as cathode. TiO2 nanotubes formed at 1, 5, 10 and 20 V potentials served as experimental groups and polished pure titanium served as control group. Field emission scanning electron microscopy (Fe-SEM) was used to analyze the surface topography. Stained nucleus with Hoechst33342 were used to measure the cell adhesion. The cell shape on the sample surface were analyzed with Fe-SEM.

RESULTSTiO2 nanotube array of different inner diameters from 15 nm to 100 nm were grown on titanium sheets by anodization at potentials from 1 to 20 V. At 30, 60 and 120 min, fibroblast adhesion at nanotubes anodized at 5 V was (141 ± 9), (388 ± 14) and (489 ± 15) respectively, significantly less than any other nanotube surface at the same time (P < 0.01). Nanotubes anodized at 20 V had the least inhibitory effect for fibroblast adhesion with a number of (579 ± 14) at 120 min, and the cell shape was also inhibited. At 30, 60 and 120 min, osteoblast had a significant better adhesion on nanotubes formed at 5 V than it did on any other surface at the same time (P < 0.01), except the control group at 30 min, with the adhesion number of (198 ± 10), (431 ± 10) and (501 ± 10) respectively, and osteoblast had a abundant spread on nanotubes formed at 5 V; while osteoblast adhesion on nanotubes anodized at 20 V was (152 ± 11), (403 ± 9) and (465 ± 12) respectively, less than on any other nanotube surface within the same time (P < 0.05), and the cell shape on the surface changed to be more elongate.

CONCLUSIONSFibroblast adhesion is inhabited more or less on Ti-TiO2 nanotubes of different diameters. Nanotubes formed at 5 V have the most osteoblast adhesion, and inhibit fibroblast adhesion.

Animals ; Cell Adhesion ; Fibroblasts ; cytology ; ultrastructure ; Mice ; Microscopy, Electron, Scanning ; Nanotubes ; chemistry ; Osteoblasts ; cytology ; ultrastructure ; Surface Properties ; Titanium ; chemistry

BACKGROUNDArticular cartilage injury is a common disease, and the incidence of articular wear, degeneration, trauma and sports injury is increasing, which often lead to disability and reduced quality of life. Unfortunately repair of articular cartilage defects do not always provide satisfactory outcomes.

METHODSChondrocyte and osteoblast composites were co-cultured using a bioreactor. The cartilage defects were treated with cell-β-tricalcium phosphate (β-TCP) composites implanted into osteochondral defects in dogs, in vivo, using mosaicplasty, by placing chondrocyte-β-TCP scaffold composites on top of the defect and osteoblast-β-TCP scaffold composites below the defect.

RESULTSElectron microscopy revealed that the induced chondrocytes and osteoblast showed fine adhesive progression and proliferation in the β-TCP scaffold. The repaired tissues in the experimental group maintained their thickness to the full depth of the original defects, as compared with the negative control group (q = 12.3370, P < 0.01; q = 31.5393, P < 0.01).

CONCLUSIONSPerfusion culture provided sustained nutrient supply and gas exchange into the center of the large scaffold. This perfusion bioreactor enables the chondrocytes and osteoblasts to survive and proliferate in a three-dimensional scaffold.

Animals ; Bioreactors ; Cartilage Diseases ; therapy ; Cells, Cultured ; Chondrocytes ; cytology ; ultrastructure ; Dogs ; Flow Cytometry ; Microscopy, Electron, Scanning ; Osteoblasts ; cytology ; ultrastructure

OBJECTIVETo investigate effects of the static magnetic field (SMF) generated by dental magnetic attachment on osteoblastic morphology and surface ultrastructure.

METHODSThe in vitro cultured rat osteoblasts were exposed continuously to 12.5 mT, 125 mT, and 250 mT static magnetic fields for 1, 3, 5, and 7 days. After exposed in SMF, osteoblasts were observed under a phase contrast microscope, and then HE stained and observed under a light microscope. In addition, the cells were observed under a scanning electron microscope (SEM).

RESULTSBy continuous exposure, the different intensities of SMF exposure did not change the vital osteoblast growth pattern or distribution. The SEM photos showed that there were certain changes in cellular microstructures for osteoblasts after exposed to 12.5 mT for 5 to 7 days, as well as 125 mT and 250 mT for 3 to 7 days. The more exposure time increased, the more microvesicles on the surfaces of cells were observed.

CONCLUSIONSContinuous SMF-stimulation could not affect the shape, distribution, and growth pattern of osteoblasts. The SMF of magnetic attachments could lead to certain changes in surface ultrastructures of osteoblasts in this study.

Animals ; Cells, Cultured ; Denture Precision Attachment ; Electromagnetic Fields ; Osteoblasts ; radiation effects ; ultrastructure ; Rats ; Rats, Sprague-Dawley

A 1.6-year-old male domestic short hair cat was brought to the Veterinary Medical Teaching Hospital, Kasetsart University, with signs of severe anemia, depression, and general lymph node enlargement. Complete blood count revealed leukocytosis and massive undifferentiated blasts. Testing for antibodies specific to feline leukemia virus (FeLV) was positive, and FeLV nucleic acid was confirmed by nested polymerase chain reaction. Base on cytochemistry and ultrastructure, the cat was diagnosed with acute monoblastic leukemia.
Animals ; Cat Diseases/*diagnosis/*virology ; Cats ; Leukemia Virus, Feline/*isolation & purification ; Leukemia, Monocytic, Acute/diagnosis/*veterinary/virology ; Male ; Osteoblasts/ultrastructure

OBJECTIVETo explore the growth of osteoblasts following culture on two absorbable collagen scaffolds.

METHODSMC3T3-E1 osteoblast cell line was inoculated on two collagen scaffolds (BME-10X and Bio-Gide) and co-cultured in vitro. The adhesion and migration of cells were detected by optical microscope. Cell proliferation was detected using CCK-8 reagent kit. The ultrastructure of the adhesion between cells and scaffolds were observed using electronic microscopy.

RESULTSMC3T3-E1 osteoblast cell line could adhere, migrate, and proliferate on both two membranes. The proliferation of cells showed no significant difference between porous layer and nonporous layer of BME-10X (P > 0.05), while the rate of proliferation was significantly different between loose layer and dense layer of Bio-Gide (P < 0.05).

CONCLUSIONBoth BME-10X and Bio-Gide have good biocompatibility with MC3T3-E1 osteoblast cell line, and the double-layer structure of Bio-Gide can prevent the cells to grow into the dense layer.

Biocompatible Materials ; Cell Adhesion ; Cell Line ; Cell Movement ; Cell Proliferation ; Collagen ; Humans ; Osteoblasts ; cytology ; ultrastructure ; Tissue Scaffolds

OBJECTIVETo evaluate the osteogenic ability of the three-dimensional interconnected porous titanium(TDIPT) coated with bonelike apatite, by the test of the culture of osteoblast in vitro.

METHODSThe pure TDIPT was prepared by the high temperature insostatic pressing (HIP) and was divided into two groups. One group of TDIPT was dipped into the 1.5 simulated body fluid (1.5 SBF) and developed the TDIPT coated with bone-like apatite (The test group). Another group was control group (pure TDIPT). The porous titanium of the two groups were all made into standard parts (5 mm x 5 mm x 4 mm) and placed into the 24-hole plates. The osteoblasts were extracted from SD rat. After the primary culture and subculture of the osteoblast in vitro, the osteoblasts were inoculated into the samples in the 24-pole plate and cultured continually. MTT Cell Proliferation Assay was done on the 1st, 3rd, 5th, 7th and 9th day respectively after inoculating. The ALP activity was tested on the 7th, 14th and 21st day, respectively. The collected data were analyzed by use of the student t test.

RESULTSThe MTT value and the ALP activity increased with the increasing of the culture time in the two group, but those of the test group was more significant than those of the control group (P < 0.05). Moreover, the osteoblast tachyauxesis and stable adhesion were observed in test group by SEM at the 7th day after culturing.

CONCLUSIONThe bioactivity of the TDIPT improves significantly after coating with the bone-like apatite onto the surface of the TDIPT.

Alkaline Phosphatase ; metabolism ; Animals ; Cells, Cultured ; Female ; Male ; Microscopy, Electron, Scanning ; Osteoblasts ; physiology ; ultrastructure ; Osteogenesis ; Porosity ; Rats ; Rats, Sprague-Dawley ; Titanium ; chemistry

OBJECTIVETo study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB).

METHODSThe bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation.

RESULTSThe results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones.

CONCLUSIONSThe developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D) interactions among cells and is suitable for osteopath expansion in vitro.

Animals ; Bioreactors ; Cell Culture Techniques ; instrumentation ; Cell Enlargement ; Culture Media ; Glucose ; metabolism ; Hydrogen-Ion Concentration ; Lactic Acid ; metabolism ; Osmolar Concentration ; Osteoblasts ; cytology ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

To study the biomechanical behaviors of the cells, reliable fluid shear stress loading system is needed. Compared to the traditional parallel plate flow chamber (PPFC) system, a rocking system presented by Zhou offers some advantages such as easier operation, lower cost and higher quantity of pocessing. But the feasibility of it has not been practically studied. To investigate the feasibility whether the rocking system can be used to apply quantified fluid shear stress loading, primary osteoblasts of mouse were loaded with fluid shear stress based on rocking system and traditional PPFC system, respectively. Another group of cells was unloaded as control. The cytoskeleton was observed with laser scanning confocal microscope (LSM) and average fluorescence of F-actin was recorded. Cell cycle was also measured by flow cytometry and percentage of S-phase cells was recorded. The result showed that average fluorescence of F-actin was enhanced after rocking system loading (46.8 +/- 4.5) compared to the control (20.4 +/- 1 8) and the percentage of S-phase cells was increased (10.6 +/- 1.04) after rocking system loading as well (which was 4.1 +/- 0.54 in control group). Furthermore, the fluid shear stress generated by rocking system could induce more significant biological effects compared to PPFC system. This study demonstrated that fluid shear stress generated by rocking system could induce biological effects of osteoblasts, and it could simulate the micro environment of cells in vioe better than PPFC. Rocking system is a convenient and feasible method for fluid shear stress loading.
Animals ; Animals, Newborn ; Cell Proliferation ; Cells, Cultured ; Cytoskeleton ; ultrastructure ; Feasibility Studies ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; cytology ; Shear Strength ; Skull ; chemistry ; Stress, Mechanical