Objective:To study the effects of BMP-7 gene transfected bone marrow mesenchymal stem cells(MSCs)on callus acceleration in rat mandibular distraction osteogenesis.Methods:Forty-eight adult male SD rats were randomly divided into experimental and control groups.MSCs were obtained from individual rat and transfected by pEGF-P-BMP7 for the 24 experimental rats and by the empty vector pEGFP-N1 for the 24 control rats.The rats were underwent right mandibular distraction.1?106/100 ?l BMP-7 gene transfected cells in 10 ?l of normal saline were injected into the distraction gap in each experimental rat,while the same number of empty vector transfected cells in each control rat.The distracted mandibles were harvested 2,4,and 8 weeks respectively after cell injection and evaluated by radiological,histological and histomorphometric analysis.Results:Radiological and histological examinations showed that more new bone was formed in experimental group than in control.Histomorphometric analysis also demonstrated that both new bone volume(NBV1 and NBV2)and the thickness of new trabeculae(TNT)were significantly higher in experimental rats than those in control(P

OBJECTIVETo explore the effect of zoledronate (ZOL) on the osteoclast adhesion and expression of integrin α(v) and β3 in vitro.

METHODSMice RAW264.7 cells were used for osteoclast differentiation in vitro, and osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining and dentin resorption lacunae examination. The cells were then divided into 2 groups, the control group and ZOL treatment group (treated with 1 x 10(-6) mol · L(-1) ZOL for 2 d). The adhesion ability of osteoclasts and mRNA and the protein expressions of integrin α(v) and β3 were examined by crystal violet staining, real-time fluorescence quantitative polymerase chain reaction, Western blot analysis, and immunofluorescent chemistry.

RESULTSTRAP staining and dentin resorption lacunae examination revealed the formation of multi-nuclear osteoclasts. ZOL treatment significantly decreased the adhesion ability of osteoclasts (P < 0.01). In the ZOL-treated group, the mRNA levels of integrin α(v) and β3 were 0.66 ± 0.05 and 0.59 ± 0.08, respectively. In the control group, the mRNA levels of integrin α(v) and β3, were 1.01 ± 0.01 and 1.01 ± 0.02, respectively; these values were higher than those in the ZOL-treated group (P < 0.01). The protein level of integrin α(v) and β3 in the ZOL-treated group (31,934.84 ± 112.91 and 18,812.79 ± 194.13) was downregulated by approximately 39.19% and 40.17%, respectively, compared with those in the control group (52,517.81 ± 211.72 and 31,441.93 ± 456.87) (P < 0.01). Immunofluorescent examination showed that the fluorescent intensities of integrin α(v) and β3 in the ZOL-treated group (9.491 ± 0.748 and 4.744 ± 0.759) were also significantly decreased compared with those in the control group (15.159 ± 1.143 and 11.418 ± 1.095) (P < 0.01).

CONCLUSIONZOL significantly inhibits osteoclast adhesion and downregulates integrin α(v) and β3, expression, thus contributing to the ZOL-induced inhibition of osteoclast- mediated bone resorption.

Animals ; Bone Resorption ; Diphosphonates ; Gene Expression ; Imidazoles ; Integrin alphaV ; Mice ; Osteoclasts ; RNA, Messenger

[ ABSTRACT] AIM:To study the expression profiles and the role of Ca 2+/calmodulin-dependent kinase II delta ( CaMKIIδ) during osteoclast differentiation .METHODS:Mouse RAW264.7 cells were induced by receptor activator of nuclear factor κB ligand ( RANKL) at 50μg/L for osteoclastogenesis .Tartrate-resistant acid phosphatase ( TRAP) staining and bone resorption lacunae examination were performed to verify osteoclast formation .The expression of CaMKIIδat mR-NA and protein levels was also determined by immunofluorescent cytochemistry , RT-qPCR and Western blot at days 0, 1, 3 and 5.RESULTS:TRAP positive multinuclear cells with bone resorption function were formed after 5 d of induction. The mRNA levels of CaMKIIδdetected by RT-qPCR were 1.028 ±0.041, 2.478 ±0.087, 10.524 ±1.284 and 42.914 ± 2.667 at days 0, 1, 3 and 5, respectively, while the protein levels of CaMKIIδ detected by Western blot were 0.762, 0.963, 1.802 and 3.136, respectively.The changes of protein level were also verified by immunofluorescence cytochemis -try, in which the fluorescence intensity increased in a time-dependent manner (P<0.05).CONCLUSION:The expres-sion of CaMKIIδincreases with the differentiation of osteoclasts .CaMKIIδmay play a key role in the osteoclastogenesis .

BACKGROUND:Studies have shown that bisphosphonates inhibit osteoclast resorption, but whether cathepsin K, a key cytokine of bone resorption, plays an effect has rarely been reported.
OBJECTIVE:To study the effect of bisphosphonate on capthesin K and bone resorption function during osteoclast differentiation.
METHODS:Osteoclasts were cultured by mouse monocyte-macrophage cellline-RAW264.7. The cells were divided into two groups:control group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor;alendronate group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor+10-7 mol/L alendronate. Osteoclastogenesis and resorption function of osteoclasts were examined at 7 days of culture and gene expression of capthesin K was detected by immunofluorescence method at 72 hours of culture. Western blot assay was used to detect capthesin K protein expression at 72 hours of culture.
RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase positive multinuclear cells were observed and resorption lacunae formed in two groups. Control group showed the higher number of tartrate-resistant acid phosphatase positive multinuclear cells and larger size of resorption lacunae than the alendronate group (P<0.01). Immunofluorescence showed expression of capthesin K was higher in the control group than the alendronate group (P<0.01);furthermore, the protein expression of capthesin K was also lower in the alendronate group than the control group (P<0.01). These findings indicate that bisphosphonates could strongly inhibit osteoclastogenesis and its resorption function by inhibiting gene expression of capthesin K.

BACKGROUND:Tartrate-resistant acid phosphatase is a specific marker for osteoclast differentiation and bone resorption, which is a sign of osteoclast maturity.
OBJECTIVE:To study the effect of alendronate on tartrate-resistant acid phosphatase related to osteoclast differentiation and bone resorption.
METHODOsteoclasts were cultured by mouse monocyte-macrophage cellline-RAW264.7. The cells were divided into two groupcontrol group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor;alendronate group, treated with 100μg/L receptor activator of nuclear factorκB ligand factor+10-7 mol/L alendronate. Osteoclastogenesis and resorption function of osteoclasts were examined at 7 days of culture. Gene expression of tartrate-resistant acid phosphatase was detected by immunofluorescence method. Western blot assay was used to detect protein expression of tartrate-resistant acid phosphatase.
RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase positive multinuclear cells were observed and resorption lacunae formed in two groups. Control group showed the higher number of tartrate-resistant acid phosphatase positive multinuclear cells and larger size of resorption lacunae than the alendronate group (P<0.01). Immunofluorescence showed expression of tartrate-resistant acid phosphatase was higher in the control group than the alendronate group (P<0.01);furthermore, the protein expression of tartrate-resistant acid phosphatase was also lower in the alendronate group than the control group (P<0.01). These findings indicate that bisphosphonates could strongly inhibit osteoclastogenesis and its resorption function by inhibiting protein expression of tartrate-resistant acid phosphatase.

In this study, the rescue effect of receptor activator for nuclear factor-kappaB ligand (RANKL) on zoledronate acid (ZOL) induced inhibition of osteoclastogenesis and gene expression of NF-kappaB p50 and c-Jun was investigated. Mice calvarial osteoblasts (OBs) were harvested and co-cultured with RAW264.7 cells and the cells were divided into 4 groups and received treatment with ZOL and RANKL, either single or combined. The formation of multi-nucleated osteoclast (OC) was examined and gene expression of NF-kappaB p50 and c-Jun was detected. Group B (ZOL) showed least multi-nucleated OC and resorption lacunae among the 4 groups (P < 0.05 or P < 0.01) and it was followed by group C (ZOL+RANKL). Group D (RANKL) showed highest OC and resorption lacunae while it was similar to Group A (control) (P > 0.05). Gene expression of NF-kappaB p50 and c-Jun was the lowest in group B (P < 0.05 or P < 0.01) among the four groups and was significantly increased in group C when compared with group B (P < 0.05). Group A and D showed highest gene expression and they were similar to each other (P > 0.05). This study suggest that RANKL might partly rescue ZOL induced inhibition of osteoclastogenesis, and the effect of RANKL and ZOL on osteoclastogenesis may be mediated by NF-kappaB p50 and c-Jun.
Animals ; Bone Resorption ; drug therapy ; Cell Line ; Diphosphonates ; pharmacology ; Gene Expression ; Imidazoles ; pharmacology ; Mice ; NF-kappa B p50 Subunit ; metabolism ; Osteoblasts ; drug effects ; Osteoclasts ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; RANK Ligand ; pharmacology

Objective To study the effect of zoledronate (ZOL) on Ca2+/calmodulin-dependent kinase Ⅱ δ (CaMK Ⅱ δ) and down-stream gene expressions during osteoclast differentiation.Methods Mouse osteoclast precursors RAW264.7 cells were divided into the control group and ZOL group.The cells in both groups were induced with 50μg/L receptor activator of nuclear factor kappa B ligand (RANKL) and were harvested on 5 d,while the cells in ZOL group were also simultaneously treated with 1 × 10-6 mol/L ZOL for 2 d.Five days later,the cells were harvested and examined osteoclastogenesis,as well as gene expressions of CaMK Ⅱ δ,nuclear factor of activated T-cells cytoplasmic 1 (NFATc1),tartrate-resistant acid phosphatase (TRAP) and cell-sarcoma receptor coactivator (c-Src).Results The number of TRAP positive multinuclear osteoclasts,number and size of dentin absorption lacunae and area in the ZOL group were (20.0±3.2),(18.0±4.2) and (6 335.3± 1 043.2)μm2 respectively,which were significantly lower than (36.0 ± 8.4),(37.2 ± 5.0) and (11 636.2 ± 3 661.1) μm2 in the control group and decreased by 44.4 ％,51.6 ％ and 45.6 ％ respectively (P＜0.01).ZOL also significantly inhibited the gene expressions of CaMK Ⅱ δ,NFATc1,TRAP and c-Src,and the mRNA levels of these genes were decreased by 44.1％,49.0％,53.8％ and 49.6％ respectively,the protein level were decreased by 43.5 ％,32.2 ％,45.5 ％ and 48.0 ％ respectively.The immunofluorescent cytochemistry detection results showed the fluorescence intensity of CaMK Ⅱ δ,NFATc1,TRAP and c-Srcin in the ZOL group was significantly weakened when compared with the control group.Conclusion ZOL could significantly inhibit the osteoclast formation and bone absorption function,and down-regulates gene expressions of CaMK Ⅱ δ,NFATc1,TRAP and c-Src in osteoclast differentiation.

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.
Animals ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; Mice ; RNA Interference ; Recombinant Proteins ; biosynthesis ; genetics ; Smad6 Protein ; biosynthesis ; genetics ; Transfection

Chronic periapical periodontitis is characterized by destruction of periapical tissue and demonstrates translucent feature under X-ray examination. In this article, a localized mineralized structure, which showed high density under X-ray examination, was reported in a patient with chronic periapical periodontitis of left maxillary first premolar. Possible causes of the structure were analyzed and relevant literatures were reviewed.
Bicuspid ; Humans ; Maxilla ; Periapical Periodontitis ; Periodontitis

OBJECTIVETo investigate the effect of alendronate on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in mouse osteoblasts.

METHODSMouse calvarial osteoblasts cultured in vitro were identified by alkaline phosphatase (ALP) staining and immunofluorescence assay of OPG and RANKL expressions. The second passage of the osteoblasts were treated with different concentrations of alendronate (10(-4) to 10(-7) mol/L) for 48 h, and the changes in OPG and RANKL mRNA and protein expressions were examined using real-time PCR and Western blotting, respectively.

RESULTSThe isolated osteoblasts were positive for ALP and expressed OPG and RANKL. Real-time PCR and Western blotting showed that at the concentration of 1×10(-4) mol/L, alendronate caused an obvious down-regulation of OPG and RANKL expressions in the cells, whereas at lower concentrations, alendronate increased the expressions of both genes with the highest expressions occurring after treatment with 1×10(-5) mol/L.

CONCLUSIONHigh concentrations of alendronate (>1×10(-4) mol/L) decrease the expressions of OPG and RANKL, whereas low concentrations (1×10(-5) to 1×10(-7) mol/L) increase their expressions in mouse osteoblasts cultured in vitro.

Alendronate ; pharmacology ; Animals ; Cells, Cultured ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; drug effects ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism