Animals ; Bone Matrix ; pathology ; Bone and Bones ; pathology ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Disease Models, Animal ; Humans ; Muscle, Skeletal ; metabolism ; pathology ; Osteoblasts ; pathology ; ultrastructure ; Osteoclasts ; pathology ; Osteocytes ; pathology ; Osteogenesis Imperfecta ; classification ; metabolism ; pathology ; Skin ; pathology

OBJECTIVETo find an ideal method inducing dental pulp stem cells (DPSC) osteogenic differentiation. To compare the effect of co-culture method and that of mineralizing culture medium.

METHODSDPSC were co-cultured with osteoblasts using cell culture inserts system as experiment group, and DPSC were cultured in mineralizing culture medium as control group. The cell morphology and ultrastructure and mineralized nodes were analyzed under phase contrast microscope, transmission electron microscope, and alizarin red S staning. Bone sialoprotein (BSP), Runx-2, osteocalcin, and collagen-1 (Col-1) osteoblastic genes expressions of DPSC cultivated in special niche of osteoblasts were assayed by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe mineralization nudoles of experiment group were more than control group. Fifteen days later, BSP and Col-1 genes in the DPSC of co-cultures were 9.807 ± 1.135 and 2.913 ± 0.310, respectively. And those in the DPSC of mineralizing culture medium were 6.478 ± 0.781 and 1.703 ± 0.184, respectively. Co-cultures and mineralizing were significantly different (P < 0.05).

CONCLUSIONSAs osteoblasts can secret lots of osteogenic cell cytokines, they have more significant effect than mineralizing culture medium on osteogenesis of DPSC.

Cell Differentiation ; Coculture Techniques ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Gene Expression Regulation, Developmental ; Humans ; Integrin-Binding Sialoprotein ; metabolism ; Microscopy, Electron, Transmission ; Microscopy, Phase-Contrast ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Osteogenesis ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; ultrastructure

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

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

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 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 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

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 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

OBJECTIVETo investigate the influence of the viability and new bone formation of osteoblasts by the super high molecular weight poly D,L-lactic acid (SHMW-PDLLA).

METHODS1. The osteoblasts derived from neonatal rat were grown and maintained at steep of SHMW-PDLLA and normal culture medium. The viability and function of the osteoblasts were measured with MTT array. 2. The plate and screws made of SHMW-PDLLA were implanted and fixed at the artificial fractured mandible of dogs. Specimens were gained at 3 and 6 months and examined with macroscopy and SEM.

RESULTS1. There is no significant difference of OD values between the experimental group and the control group (P > 0.05). The SHMW-PDLLA isn't toxic to osteoblast at 1 week and 2 weeks, and the toxicity is 3% at 3 days. 2. There were a lot of new bone formed between the implanted SHMW-PDLLA plate and bone tissues under SEM.

CONCLUSIONSHMW-PDLLA hasn't pathological influence on the viability and new bone formation of osteoblasts and it is feasible in tissue engineering of bone.

Animals ; Animals, Newborn ; Bone Plates ; Bone Screws ; Cell Survival ; drug effects ; Cells, Cultured ; Dogs ; Lactic Acid ; chemistry ; pharmacology ; Mandibular Fractures ; surgery ; Microscopy, Electron, Scanning ; Molecular Weight ; Oral Surgical Procedures ; instrumentation ; methods ; Osteoblasts ; cytology ; drug effects ; ultrastructure ; Osteogenesis ; drug effects ; Polyesters ; Polymers ; chemistry ; pharmacology ; Rats