OBJECTIVETo study the role of osteocyte in bone remodeling due to mechanical loading in vitro.

METHODSMLO-Y4 osteocyte-like cells were exposed to fluid flow-induced shear stress(12dyn/cm(2))for 0, 1, 2, 4, 6, 12, and 24 hours. Osteocyte exposed to shear stress at different time points were used in co-culture system for 9 days, and then the cells were stained with tartrate-resistant acid phosphatase on the 9(th) day and the amount of positively stained osteoclasts were counted and compared. The expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa (RANKL) were detected by semi-quantitative reverse transcription polymerase chain reaction (semi-quantitative RT-PCR).

RESULTSCompared with bone cells without stimulation with fluid flow-induced shear stress, the amount of osteocytes significantly decreased at all time points after the application of fluid flow-induced shear stress (all P<0.05). The OPG expression at mRNA levels was significantly up-regulated in the first 12 hours (P<0.001), the RANKL mRNA expression was significantly down-regulated in the first 4 hours (P<0.05), and the RANKL/OPG ratio significantly decreased within 12 hours (P<0.01). However, all these indicators showed no significant difference at 24 hours when compared with the pre-stimulation level.

CONCLUSIONOsteocytes may act as mechanosensors that are able to inhibit bone resorption after mechanical loading; however, such effect shows certain adaptation ability to shear stress as time goes.

Animals ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Male ; Mice ; Osteoclasts ; cytology ; Osteocytes ; cytology ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; Stress, Mechanical

OBJECTIVETo investigate the expression of calcitonin receptor mRNA in the osteoclasts of the resorbing deciduous teeth.

METHODSAfter fixing the collected deciduous teeth, toluidine blue was performed and tartrate-resistant acid phosphatase (TRAP) staining was used to identify the osteoclasts on the resorbing surface of human deciduous teeth and in situ hybridization of calcitonin receptor mRNA to show its existence.

RESULTSThere were a number of TRAP positive osteoclasts on the root surface which showed the expression of calcitonin receptor mRNA.

CONCLUSIONOn the resorbing surface of human deciduous teeth there are osteoclasts that express calcitonin receptor mRNA, so it is feasible to use this kind of osteoclast to test the effect of external factors on the expression of CTR mRNA.

Humans ; In Situ Hybridization ; In Vitro Techniques ; Osteoclasts ; metabolism ; RNA, Messenger ; biosynthesis ; Receptors, Calcitonin ; biosynthesis ; genetics ; Tooth, Deciduous ; cytology ; metabolism

This study was aimed to explore the influence of brucine on the early differentiation of osteoblasts and the metabolic pathway of osteoclast in multiple myeloma (MM) and to compare the effects of brucine and bortezomib on MM. The half inhibitory concentration (IC(50)) of brucine and bortezomib on MM cell line U266 was determined by MTT method; the mRNA levels of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and osteoprotegerin ligand (RANKL) were detected by RT-PCR after the supernatant of cultured U266 cells was added into the culture system for inducing the differentiation of osteoblast line MC3T3-E1 and culturing. The results showed that the IC(50)of bortezomib and brucine on U266 cells for 48 hours were 22.4 nmol/L and 0.16 mg/ml respectively. As compared with osteoblasts treated by supernatant of cultured MM cells alone, the mRNA levels of ALP, OC and OPG in osteoblasts treated by brucine combined with supernatant of cultured MM cells were enhanced (p < 0.05), while the RANKL mRNA level was lowered (p < 0.05), moreover the enhanced and lowered degree also was large (p < 0.05). It is concluded that the influence of brucine on metabolism of osteoblasts and osteoclasts in MM may be realized through the regulation of osteoclasts by osteoblasts. The therapeutic efficacy of brucine on MM is superior to bortezomib.
Animals ; Cell Line ; Cell Line, Tumor ; Humans ; Inhibitory Concentration 50 ; Mice ; Multiple Myeloma ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteoclasts ; cytology ; drug effects ; metabolism ; Strychnine ; analogs & derivatives ; pharmacology

OBJECTIVETo compare the osteogenic differentiation potential and osteoclast capacity between stem cells from human exfoliated deciduous teeth (SHED) in the physiological root resorption period and dental pulp stem cells (DPSCs).

METHODSSHED and DPSCs were isolated, purified and cultured in vitro. The two stem cells were examined with ALP staining at 14 days and with alizarin red staining at 21 days of osteogenic induction, and the expressions of the genes associated with osteogenesis and osteoclastogenesis were detected using real-time PCR.

RESULTSThe isolated SHED and DPSCs both showed an elongate spindle-shaped morphology. After osteogenic induction of the cells, Alizarin red staining visualized a greater number of mineralized nodules in SHED than in DPSCs (P<0.05), and SHED also exhibited a stronger ALP activity than DPSCs (P<0.05). RT-PCR test results showed that the two stem cells expressed RANKL,OCN, ALP, OPG and Runx2 mRNA after osteogenic induction, but the expression levels of Runx2, OCN and ALP were lower in DPSCs than in SHED (P<0.05), and the ratio of RANKL/OPG was significantly higher in SHED (P<0.05).

CONCLUSIONSCompared with DPSCs, SHED has not only the ability of osteogenic differentiation but also an osteoclast capacity, which sheds light on the regulatory role of SHED in physiological root resorption bone remodeling.

Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Humans ; Osteoclasts ; cytology ; Osteogenesis ; Osteopontin ; metabolism ; RANK Ligand ; metabolism ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo isolate, culture and identify odontoclasts in vitro and to establish a method of culturing human odontoclasts.

METHODSHealthy and retentive deciduous teeth were extracted, and then placed in α-minimum essential medium containing 0.1% collagenase and 0.2% dispase for 1 h.Odontoclasts were obtained and incubated from the absorbing root surfaces of deciduous teeth.Isolated cells were viewed by inverted phase contrast microscope firstly. Then, the isolated odontoclasts were morphologically observed by hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) staining. The prepared teeth slices were cocultured with the isolated odontoclasts and scanning electronic microscope(SEM) was used to demonstrate the presence of resorption lacunae.

RESULTSThe isolated odontoclasts appeared as multinucleated giant cell with many vacuolus in cytoplasm. TRAP staining demonstrated that the cytoplasm of the odontoclasts was full of claret-red positive particles.Resorption lacunae on teeth slices which cocultured with odontoclasts were seen under SEM.

CONCLUSIONSEnzyme digestion is an effective method to isolate odontoclasts from absorbing root surface of deciduous teeth.

Acid Phosphatase ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Giant Cells ; cytology ; metabolism ; ultrastructure ; Humans ; Isoenzymes ; metabolism ; Microscopy, Phase-Contrast ; Osteoclasts ; cytology ; metabolism ; ultrastructure ; Root Resorption ; Staining and Labeling ; methods ; Tartrate-Resistant Acid Phosphatase ; Tooth Root ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo investigate the osteoblast-like functional characteristics exhibited by human periodontal ligament cells (hPDLCs) under mechanical force.

METHODSHuman PDLCs cultured in vitro were stretched by mechanical force. Radioimmunoassay (RIA) was used to measure the expression of secreting alkaline phosphotase (ALP) and osteocalcin (OCN). The non-secreting ALP, OCN and osteopontin (OPN) in cells were determined by immunohistochemistry.

RESULTSIt exhibited increasing of ALP secreted into conditional media, and in the 24 hour period there were two peaks which appeared at the 2nd and 4th hour and the 24th hour (P < 0.01). While in the late of the 24 hours, expression of OCN in conditional media increased (P < 0.05).

CONCLUSIONMechanical force induces hPDLCs to differentiate into functional osteoblast-like cells and plays a role in bone remodeling.

Alkaline Phosphatase ; metabolism ; Cells, Cultured ; Humans ; Osteocalcin ; analysis ; Osteoclasts ; physiology ; Osteopontin ; Periodontal Ligament ; cytology ; Sialoglycoproteins ; analysis ; Stress, Mechanical

SG600, SG900 and SG1100 were synthesized by the sol-gel method. Further treatments with increasing temperatures influenced and determined the crystallization degree of the material. Primary cultured osteoclasts were incubated for 4h and 48h on samples. Osteoclast actin labeling was examined by cytochemical staining. The concentrations of Ca and P in culture medium were quantified by colorimetric methods. SEM examined osteoclast morphology and resorption lacuna. Actin staining revealed on all three materials the typical adhesion contact ring. The Ca concentration in the culture medium of SG600 was significantly higher than that in control medium, SG900 and SG1100. Ca and P concentrations were always higher in culture media with the presence of osteoclasts. Morphological studies by scanning electron microsopy(SEM) showed a good adhesion behavior of osteoclasts on all three samples. Well-developed and deep resorption lacunae appearing after the osteoclastic resorption action were detected on all three samples. The synthetic bioglasses with different crystallizations caused different solubility, which seemed to have little effect on the osteoclast resorption behavior. The results of morphological studies on osteoclasts and resorption lacunae clearly demonstrate that the synthetic bioglasses are easily resorbed in vitro by osteoclasts.
Absorbable Implants ; Biocompatible Materials ; Biodegradation, Environmental ; Bone Substitutes ; Cells, Cultured ; Ceramics ; Crystallization ; Osteoclasts ; cytology ; metabolism ; ultrastructure ; Phase Transition

We hypothesized that the formation and differentialtion of osteoclasts are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow in the early stage of stroke. We randomly divided white female Sprague-Dawley (SD) rats (n = 30) into two groups, stroke (n = 15) and sham group (n = 15). On the 7th day after stroke, after cutting away the epiphyses of the femurs and tibias, diaphyseal channels were flushed using alpha-minimum essential medium (alpha-MEM) and bone marrow cells were collected. Bone marrow stem cells, which were extracted from the femur and tibia, were cultured on the 7th day after middle cerebral artery occlusion. We then estimated the ratio of non-adherent cells to total bone marrow cells that included osteoclast precursor cells. After culturing these cells separately, cells that tested positive on the tartrate resistant acid phosphatase (TRAP) were counted and bone resorption was evaluated by using the OAAS(TM) plate. In comparison to the control group, the stroke group showed a higher increase of non-adherent cells in the hemiplegic side bone marrow. In addition, after the primary culture, the stroke group showed an increased number of TRAP positive cells and a higher degree of bone resorption estimated by OAAS(TM) plate. As a result, osteoclastogenesis and osteoclast differentiation are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow and these changes are detected as early as within the first week after middle cerebral artery occlusion in SD rats.
Animals ; Bone Marrow Cells/cytology/drug effects ; Bone Resorption/*physiopathology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Female ; Femur/cytology ; Osteoclasts/cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells/cytology/metabolism ; Stroke/*metabolism/pathology ; Tartrates/pharmacology ; Tibia/cytology

OBJECTIVETo detect the changes in the expression of apoptosis signals: Fas, FasL and NF-kappa B in the process of osteoclast-like cell (OLC) apoptosis effected by sodium fluoride.

METHODSAfter co-culture of osteoclast-like cells with 0, 5, 10 and 15 mg/L sodium fluoride, Fas, FasL and NF-kappa B antibody expressions were detected by immune-histochemistry.

RESULTSThe expression of Fas and FasL increased with the concentration of the sodium fluoride, however the expression of NF-kappa B decreased with the concentration of sodium fluoride.

CONCLUSIONIn the process of OLC apoptosis induced by sodium fluoride, the expression of Fas and FasL increased, and that of NF-kappa B decreased with the concentration of sodium fluoride respectively, and the changes of the expression present a dose-dependent pattern.

Animals ; Apoptosis ; Fas Ligand Protein ; Female ; Fluorides ; pharmacology ; Ligands ; Membrane Glycoproteins ; metabolism ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; metabolism ; Osteoclasts ; cytology ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the degradation of electrodeposited calcium phosphate (ECP) coating and calcium phosphate/chitosan (ECPC) coating in vitro.

METHODSOsteoclasts were isolated from neonatal rabbit long bone cavities and incubated with ECP and ECPC coatings. Calcium ion concentrations in the culture medium were analyzed at 3 days and 6 days. The osteoclastic resorption was observed with scanning electron microscope.

RESULTSBoth coatings demonstrated osteoclastic resorption lacunae. The calcium ion concentrations of the culture mediums were decreased when incubated with calcium phosphate coatings (P < 0.05). Compared with coatings cultured with osteoclasts, the calcium ion concentrations of those cultured without osteoclasts were higher on day 3 (P > 0.05) but lower on day 6 (P < 0.05).

CONCLUSIONSBoth ECP and ECPC coatings can be resorbed by osteoclasts in vitro and can dissolve in the culture medium.

Animals ; Calcium ; metabolism ; Calcium Phosphates ; administration & dosage ; metabolism ; Cells, Cultured ; Chitosan ; administration & dosage ; metabolism ; Dental Alloys ; Electrochemistry ; Osteoclasts ; cytology ; drug effects ; metabolism ; Rabbits