Objective: To investigate the effect of cobalt (Co) and calcium-phosphate (Ca/P) doped coating on titanium surfaces and their angiogenic effect. Methods : Microarc oxidation (MAO) was used to prepare Co-Ca/P-doped and Co-doped coatings. Titanium (Ti) sheet without MAO treatment was used as control. Scanning electron microscopy (SEM) was used to observe the surface micromorphology of the coatings. Energy dispersive spectrometry (EDS) was also applied to detect the doped chemicals and their contents. Standard soaking solutions of these coatings were prepared using an endothelial cell medium (ECM) solution for subsequent angiogenesis experiments. Human umbilical vein endothelial cells (HUVECs) were cultured on Matrigel with ECM soaking solutions for 4 h and 8 h. The microvessels were observed under a microscope, and the number of microtubules and their interconnecting nodes were analyzed with Image J software. Results: Co doped and Co-Ca/P-doped coatings were successfully prepared by MAO, which was demonstrated by both SEM observation and EDS analysis. SEM observation showed that irregular crystals of the above chemicals were present on both Co and Co-Ca/P-doped coatings, commonly with a diameter <2 μm. However, more crystals were observed on the Co-Ca/P coatings than on the Co coating, and the distribution of the crystals was more homogenous on the Co-Ca/P coatings. However, only polishing scratches were observed on the Ti sample surface. EDS analysis indicated that in contrast to only Co in the Co coating, Co, Ca and P were doped within the Co-Ca/P coating, and none of the three elements were observed on the Ti plate surface. The number of vascular rings and nodes formed by HUVECs in the extract of the Co-Ca/P group was significantly higher than that of the Co group (P<0.05), and the angiogenic effect of these two components was significantly better than that of the Ti group (P<0.05). Conclusion The Co-Ca/P coating exhibits good angiogenic properties in vitro and is valuable for the development of new titanium implants with high surface bioactivity.

Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Logistic Models ; Phenotype ; Risk Factors

OBJECTIVE: To investigate the effect of zoledronate (ZOL) on osteoclast differentiation and bone resorption under high glucose, and the regulation mechanism of p38 mitogen activated kinase (p38 MAPK) signaling pathway in this process. METHODS: RAW264.7 cells were divided into four groups: low group, high group, low+ZOL group and high+ZOL group after induced into osteoclasts. Cell proliferation activity was determined by MTT assay. The migration of RAW264.7 cells were examined Optical microscopy. Immunofluorescence microscopy was used to observe the cytoskeleton and sealing zones of osteoclasts. After adding group 5: high + ZOL + SB203580 group, trap staining was used to identify the number of positive osteoclasts in each group. The number and area of resorption lacunae were observed by SEM. The mRNA and protein expression of osteoclast related factors were detected by real-time PCR and Western blotting. RESULTS: The cells in the 5 groups showed similar proliferative activity. High glucose promoted the migration of RAW264.7 cells ( CONCLUSIONS High glucose inhibits osteoclast differentiation and bone resorption. ZOL inhibits osteoclast differentiation and bone resorption in high-glucose conditions by regulating p38 MAPK pathway, which can be a new pathway for ZOL to regulate diabetic osteoporosis.
Animals ; Bone Resorption ; Cell Differentiation ; Glucose ; MAP Kinase Signaling System ; Mice ; NFATC Transcription Factors ; Osteoclasts ; RANK Ligand ; Zoledronic Acid/pharmacology* ; p38 Mitogen-Activated Protein Kinases

Objective To investigate the effects of calmodulin-dependent kinase IIγ (GaMKIIγ) RNA interference on osteoclast differentiation and bone resorption. Methods Three CaMKIIγ recomninant RNA interference vectors were constructed using lentivirus. Negative vector was used to transfect RAW264.7 cells and the multiplicity of infection (MOI) with the optimal transfection efficiency was determined. Recombinant vectors were also used to transfect cells to determine the one with the best interference effect for following experiments.Then, the cells were divided into control group, negative vector group and interference vector group. Five days after virus transfection, osteoclastogenesis and bone resorption function were determined by TRAP staining and dentin resorption lacunae detection. Results Three CaMKIIγ recombinant interference vectors were constructed, and the optimal MOI was 30, under which transfection efficiency was about 81％. The #3 recombinant vector showed the best interference effect and the interference efficiency was up to 78.16％ at mRNA level and 67.02％ at protein level. When compared with control group, the number of multi-nucleated osteoclasts, the number and area of dentin resorption lacunaes in interference vector group decreased 59.99％、54.19％ and 57.94％ respectively (P < 0.01). No significant difference were observed between negative vector group and control group (P> 0.05).Conclusion CaMKIIγ RNA interference significantly inhibits osteoclastogenesis and bone resorption.

Objective The aim of this study was to investigate the effect of Ca2+ /calmodulin - dependent kinase II (CaMKII)γ RNA interference on the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1),tyrosine kinase(c-Src)and tartrate resistant acid phosphatase(TRAP)genes,and its role and molecular mechanism in osteoclast differentiation. Methods The CaMKII γ RNA interference vector was constructed by lentivirus and transfected into RAW264. 7 cells. The experiment was di-vided into three groups:A,B and C,which were the control group,negative vector group and interference vector group. After transfec-tion for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days. Real-time quanti-tative PCR,Western blot and immunofluorescence were used to detect the expression of NFATc1,TRAP and c-Src genes in three groups. Results The mRNA levels of NFATc1,TRAP and c-Src in the group C decreased by 49. 86% ,43. 65% and 53. 57% ,re-spectively(P<0. 001),and the protein levels decreased by 54. 22% ,46. 75% and 45. 86% ,respectively(P<0. 001). There was no significant difference between the A and the B groups(P>0. 05). The fluorescence intensity of the above genes in the group C was significantly weaker than that in the A and B groups,and the formation of osteoclasts was significantly less than that in the A and B groups. Conclusion CaMKIIγ RNA interference significantly inhibited the expression of NFATc1,TRAP and c-Src genes,sugges-ting that CaMKIIγ plays a key regulatory role in osteoclast differentiation.

Objective:To study the expression profiles and the role of Ca2+/calmodulin-dependent protein kinase Ⅱγ (CaMKⅡγ) during osteoclast differentiation.Methods:Mouse RAW264.7 cells were induced for osteoclastogenesis with 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and the cells were harvested at 0,1,3 and 5 days after induction.Tartrate-resistant acid phosphotase staining was performed to verify osteoclasts formation.RT-PCR,Western blot and immunofluorescent cytochemistry were used to detect the CaMKⅡγ gene expression during osteoclastogenesis.Results:The osteodasts were formed at day 3 under RANKL induction and more osteoclasts were observed at day 5.At day 0,1,3 and 5,the relative level of CaMKⅡγ mRNA were (1.067±0.179),(1.840±0.070),(9.493±0.453) and (30.767±0.573),respectively,and the relative protein level were (0.454±0.065),(0.613±0.021),(0.858±0.019) and (0.980±0.023),respectively.CaMKⅡγ expression was increased in a time-dependent manner except relative protein level at day 1 (P＜0.01),which showed no significant difference at day 0 (P＞0.05).Immunofluorescence assay showed that CaMKⅡγ protein was also increased with differentiation of osteoclasts.Conclusion:The CaMKⅡγ expression was increased in a time-depended manner during osteoclast differentiation and it might play a vital role during osteoclastogenesis.

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.

Objective: To investigate the effect of zoledronate on protein interaction between Ca²⁺/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation. Methods: Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis, and group B was treated with 1×10^-6 zoledronate for two days from day 2. Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMKⅡ and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc1 and TRAP in both groups. Osteoclast formation was also analyzed. Results: In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3±1.5, 8.7±2.1 and (5 034.4±775.4) μm² respevtively, which were significantly lower than those (37.7±5.7, 23.0±4.0 and [15 042.7±1 906.0] μm²) in group A (P<0.01). Co-IP and reverse Co-IP examination indicated that protein-binding between CaMKⅡ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A (P<0.01). The protein level of calmodulin and CaMKⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A (P<0.01). NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A (P<0.01), respectively. Conclusions Zoledronate could significantly inhibit protein-binding between CaMKⅡ and calmodulin and down-regulate protein level of NFATc1 and TRAP.

[ 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 .

OBJECTIVETo investigate the effect of thalidomide on the development of bisphosphonate-related osteonecrosis of the jaws (BRONJ).

METHODSThirty-six rats were randomly divided into groups A, B and C, and treated with saline, zoledronate and zoledronate plus thalidomide, respectively. Three weeks later, the left maxillary first molars of the rats were extracted. Four and eight weeks after tooth extraction, samples were harvested for evaluation of osteonecrosis of the jaws, microvessel density, and cell apoptosis.

RESULTSAt both of the time points, no exposed dead bone was observed at the extraction socket areas in the rats except for some small fistulas in groups B and C. Histological examination confirmed the absence of dead bone in group A, whereas small areas of dead bone were observed around the extraction socket in groups B and C. Compared with those in group A, the percentage of empty lacunae and the area of dead bone were significantly increased (P<0.01), whereas bone lacunae density was significantly decreased (P<0.01) in groups B and C at both time points. Microvessel density in groups B and C were also significantly decreased (P<0.01) by 25.87% and 55.27% at week 4, and by 45.62% and 72.84% at week 8, respectively; the apoptotic cells in groups B and C increased by 54.80% and 87.89% at week 4 (P<0.01), and by 208.08% and 250.58% at week 8 (P<0.01), respectively.

CONCLUSIONThalidomide can aggravate zoledronate-induced early-stage BRONJ, and their osteonecrosis-inducing effect of the jaw may be attributed, at least partly, to the inhibition of angiogenesis.

Animals ; Apoptosis ; Bisphosphonate-Associated Osteonecrosis of the Jaw ; pathology ; Bone Density ; Diphosphonates ; Disease Models, Animal ; Imidazoles ; Molar ; Neovascularization, Physiologic ; Rats ; Thalidomide ; adverse effects ; Tooth Extraction