OBJECTIVETo observe the variations of intracellular localization and expression of importin 8 (IPO8) during osteoblast differentiation.

METHODSAlizarin red staining, immunocytochemistry and real-time PCR were employed to examine the changes in the intracellular localization and expression of IPO8 mRNA during induced osteogenic differentiation of human osteoblast-like SaOS-2 cells.

RESULTSNumerous red mineralized nodules were observed on day 10 in the induced cells with alizarin red staining. Immunocytochemical staining showed that IPO8 immunoreactivity was the strongest in the perinuclear cytoplasm of the cells. On day 3 of osteoblast differentiation, IPO8 immunoreactivity in the cell nuclei became stronger. On day 7, IPO8 was located mainly in the nuclei, and on day 10 the cells were osteocyte-like and IPO8 was distributed in the cytoplasm. Real-time PCR showed a significantly increased expression of OPN mRNA during osteoblast differentiation, and the expression level of IPO8 mRNA was the highest on day 3 and declined on days 7 and 10.

CONCLUSIONThe intracellular localization and expression level of IPO8 undergo significant changes during osteogenesis, indicating its role in regulating osteoblast differentiation.

Cell Differentiation ; Cell Line ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteogenesis ; beta Karyopherins ; metabolism

The purpose of this study was to investigate the effect on the expression of NF-kappaBp65 of osteoblast-like cell under stretch load with the same amplitude but different daily loading times. The osteoblast-like cells MG-63 were passage cultured and stretched by the four-point-bend loading device; based on the daily loading times, the osteoblast-like cells were randomly divided into four groups. The first was the control, the others were stretched with mechanical tension with the same amplitude of 2,000 mu strain and at the same frequency of 0.5 Hz., but the daily loading times were 1 time/d, 2 times/d, 4 times/d differently for each group, the periods of mechanical tension applied to the cells of the three groups were all 60 min/d and lasted for 2d total. After the cells being streteched, the expression levels of NF-kappaBp65 of the osteoblast-like cells of the three groups and control group were investigated by using the techniques of immunohistochemistry, and were compared with each other. The results showed that the positive expression ratios of the four groups were different significantly; the positive expression ratio of the control was lower than those of the other three groups; the positive expression ratio of the 4 times/d group was higher than those of the other two stretched groups; the positive expression ratio of the 2 times/d group was higher than that of the 1 time/d group. The results suggested that when the osteoblast-like cell was under the stretch load with different daily loading times but the same amplitude, the expression ratio of NF-kappaBp65 in the cell increased with the rising of the stimulating times. It means that the mechanical strain with high daily loading times could promote the transcriptional level of osteoblast-like cell more effectively.
Cell Line ; Humans ; Mechanotransduction, Cellular ; Osteoblasts ; cytology ; metabolism ; Stress, Mechanical ; Time Factors ; Transcription Factor RelA ; biosynthesis

To establish an experimental model of osteoblasts to easily cause calcification of bone matrix in vitro, we took cranium of a newborn rabbit out under an aseptic condition, removed the connective tissue of the bony suture, and cut the cranium freely into the fragments of not more than 1 mm2. The we isolated and cultured the osteoblasts using tissue explant method. We observed growth status of primary osteoblasts and subcultured osteoblasts using inverted microscope. Then we conducted enzyme staining and alizarin red staining for the third generation of osteoblasts to detect the alkaline phosphatase (ALP) expression and calcified nodules. The result showed that there were calcified nodules or calcification formed after the primary osteoblasts climbing out from the bone for 1 week, and each generation of osteoblasts had the similar calcification with the primary osteoblasts, and there was an increase in calcified nodules after the continuous culture. There was a strong expression of ALP in the plasma membrane of osteoblasts. The calcified nodules were red with alizarin red staining. It is well concluded that osteoblasts isolated with this method easily cause calcification, and can be used as a new experimental model.
Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Cell Separation ; methods ; Osteoblasts ; cytology ; Primary Cell Culture ; methods ; Rabbits ; Skull ; cytology

BACKGROUNDSeveral studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro.

METHODSADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay.

RESULTSSecondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05).

CONCLUSIONSSecondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs.

Adipocytes ; cytology ; metabolism ; Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Chondroitin ABC Lyase ; metabolism ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteoblasts ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

MicroRNA(miRNA) is a class of non-coding RNA that plays an important role in gene expression and controlling. In recent years,the role of miRNA in the development of the disease has aroused great interest. Abnormal osteoclastogenesis and persistent inflammatory response induced by wear particles or osteoblast differentiation and maturation is the main cause of aseptic loosening in joint replacements. New researches shows that persistent inflammatory response, osteoclastogenesis and osteoblast differentiation are closely associated with miRNA, suggesting that there are certain relations between miRNA and aseptic loosening of prostheses. Additionally, the alteration of the expression levels of some miRNA may be curative for aseptic loosening. With the findings of the new miRNA targets, the important role of miRNA is further confirmed.
Animals ; Arthroplasty, Replacement ; Cell Differentiation ; Humans ; Joint Diseases ; genetics ; metabolism ; surgery ; MicroRNAs ; genetics ; metabolism ; Osteoblasts ; cytology ; metabolism ; Prostheses and Implants

OBJECTIVETo explore the effect of MicroRNA-146a (miR-146a) on the ability of BM-MSC to differentiate into adipocytes and osteoblasts.

METHODSBM-MSC were isolated from the bone marrow of healthy donors. The differentiation of BM-MSC into adipocytes and osteoblasts cells were done in vitro. After transfection with miR-146a inhibitor or mimics, the expression of miR-146a in BM-MSC was detected by real time quantitative PCR. The effect of MicroRNA-146a on the differentiation potential of BM-MSC was evaluated after transfection.

RESULTSBM-MSC possessed the ability to differentiate into adipocytes and osteoblasts cells when cultured in the induction medium. The expression of miR-146a was correspondingly down-regulated and up-regulated in BM-MSC after transfection. Compared with the control group, the expression of miR-146a was down-regulated (P < 0.01) after transfection with miR-146a inhibitor, while after transfection with miR-146a mimics it was significantly up-regulated. This study proved that the transfection with miR-146a inhibitor can inhibit BM-MSC differentiate into adipocytes (P < 0.01), while transfection with miR-146a mimics can promote differentiation of BM-MSC into adipocytes (P < 0.01). No effect of miR-146a inhibitor or miR-146a mimics on osteogenic differentiation of BM-MSC was observed (P > 0.05).

CONCLUSIONBM-MSC possess the ability to differentiate into adipocytes and osteoblasts. The miR-146a can promote BM-MSC to differentiate into adipocytes.

Adipocytes ; cytology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; metabolism ; Osteoblasts ; cytology ; Osteogenesis ; Transfection

OBJECTIVEIn order to determine the effect of factors pertaining to the development and metabolism of periodontal ligament, the author investigated the interaction between osteoblasts and fibroblasts.

METHODSThe cell co-culture model was established by cell culture inserts. The expression difference of protein related to mineralization (ALP, Type I collagen) between these two kinds of cells cultured individually and co-cultured were evaluated.

RESULTSCo-culture changed the ALPase activity of the two kinds of cells greatly .The ALP expression of osteoblasts decreased, but the ALP expression of periodontal ligament fibroblasts increased. The Col-I expression of these two kinds of cells did not change significantly when they were co-cultured.

CONCLUSIONPeriodontal ligament fibroblast could restrain the osteogenesis function of osteoblast, while osteoblast could induce the differentiation of fibroblast when both of them existed in periodontal tissue.

Alkaline Phosphatase ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Coculture Techniques ; Collagen Type I ; metabolism ; Fibroblasts ; cytology ; Humans ; Osteoblasts ; cytology ; Osteogenesis ; Periodontal Ligament ; cytology

OBJECTIVE: To establish an in vitro method to culture mesenchymal stem cells(MSCs) derived from human nasal mucosa, and explore their stemness and differentiation potential. METHOD: Based on the observation of distribution of MSCs in human nasal mucosa, we cultured and proliferated MSCs in vitro and identified the expression of stem cell markers on them including Nestin, CD133, Vimentin and Sa114 with immunofluorescence. The MSCs were induced to differentiate to osteoblasts with medium containing dexamethasone, ascorbic acid and beta sodium glycerophosphate, and to neurons with Neurobasal medium containing B27, ATRA and TSA. Histochemistry and immunofluorescence were applied to evaluate the differentiation. RESULT: The nestin and vimentin immunofluorescence-positive MSCs existed extensively in human nasal mucosa. While the MSCs were cultured in the osteogenic-inducing medium, activities of alkaline phosphatase were increased significantly, and bone nodules were found on the surface of the osteoblasts by alizarin red staining. After the induction by neural-inducing medium, the MSCs adopted neuron like appearance with many slim protrusions interconnected as a network. The induced cells expressed neural markers NF-200 and BM88 strongly. CONCLUSION The MSCs derived from human nasal mucosa are multipotent stem cells and can be utilized as seed cells to repair bone or neural injury.
Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; cytology ; metabolism ; Multipotent Stem Cells ; Nasal Mucosa ; cytology ; Neurons ; Osteoblasts ; cytology

OBJECTIVETo investigate the effects of static magnetic fields (SMFs) with different exposure time on the maturation of rat osteoblasts in vitro and the expression of the estrogen receptor (ER) gene.

METHODSThe calvarial osteoblasts were isolated from newborn rats by enzyme digestion and randomly divided into 9 groups after one passage based on the exposure time of the SMFs[0 (control), 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, and 4.0 h]. The intensity was 3.9 mT in all SMFs. Those without SMFs exposure were used as the controls. The oeteoblasts were observed under the contrast phase microscope on a daily basis. After 48 h, cell proliferation was assayed by MTT method. The osteocalcin contents were measured after exposure to SMFs for 3 d, 6 d, 9 d, and 12 d. ERΑ and ERΒ mRNA expressions were measured by real-time PCR after SMFs treatment for 0 h, 24 h, 48 h, and 72 h.

RESULTSCompared with the controls, the cell proliferation was significantly enhanced in the 2.0-h, 2.5-h, and 3.0-h groups (P<0.05). After SMFs treatment for 6 d, 9 d and 12 d, the 2.5-h group had significantly higher osteocalcin content than the control group did (P<0.05). After SMFs treatment for 0 h and 72 h, elevated ERΑ mRNA expression and reduced ERΒ mRNA expression were observed.

CONCLUSIONExposure to SMFs, regardless of exposure time, is associated with enhanced cell proliferation, increased osteocalcin contents, and altered ERΑ and ERΒ mRNA expressions in opposite directions.

Animals ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Magnetic Fields ; Osteoblasts ; cytology ; metabolism ; Rats ; Receptors, Estrogen ; genetics ; metabolism

OBJECTIVETo observe the intake of minocycline and its amount in mature rat mandibular osteoblasts (MRMOB) in vitro, and to identify the feasibility of intracellular anti-bacterial activity of minocycline.

METHODSFour groups of MRMOB were incubated in 100 mg/L minocycline for 15, 30, 45 and 60 minutes respectively, and the accumulation of minocycline within MRMOB was measured using a fluorescence spectrophotometer.

RESULTSThe intracellular accumulation amount of minocycline in the four groups of MRMOB was (17.29 +/- 1.49), (16.87 +/- 1.57), (16.96 +/- 1.67) and (17.94 +/- 1.63) mg/g respectively after osteoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained minocycline for 15, 30, 45 and 60 min. There was no significant difference in amount of minocycline among the four groups of MRMOB.

CONCLUSIONSThe mature rat mandibular osteoblasts can ingest minocycline, and the accumulation amount of minocycline in MRMOB is irrelevant with the exposure time of MRMOB to minocycline.

Animals ; Cells, Cultured ; Mandible ; cytology ; drug effects ; metabolism ; Minocycline ; pharmacokinetics ; Osteoblasts ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley