Objective To evaluate the effects of adeno-associated virus-enhanced green fluorescent protein (AAV-EGFP)on the biologic behavior of rabbit's bone marrow stromal cells(BMSCs)by means of a simple method of culturing and osteogenic induction in vitro so as to find an ideal viral vector and cell tracing mark for tissue en- gineering.Methods Total bone marrow culture was conducted to obtain rabbit BMSCs which were then induced in the osteogenic direction.The morphology of the cells was observed continuously,and their surface antigen and ossification were detected by alkali phosphatase stain and Von Kossa stain.On the basis of the above results, AAV-EGFP was transfected into the induced cells.The morphologic changes of the cells,the expression time and intensity of fluorescent light were observed.The transfection efficiency was detected to find the best multiplicity of infection(MOI)value.The cell growth curves were drawn to evaluate the biologic effects of AAV-EGFP on the cyto-activity.Results The morphology and purity of the rabbit BMSCs obtained were good.The ossification of the cells was significant after osteogenic induction.The best MOI value was found to be 1?10~5.The expression intensity of fluorescent light was strong with the expression time more than eight weeks so that the fluorescent light could be observed after cell generations.The transfection efficiency of AAV was high without significant biologic effects on the cyto-activity.Conclusions The total bone marrow culture and in vitro cell induction can satisfy the requirements for seeding cells in tissue engineering.AAV is an ideal viral vector for tissue engineering.Transfection of AAV-EGFP to cells could be an ideal method for cell tracing mark.

AIMTo investigate the survival rate and the level of HaCaT cells damage with ultraviolet B (UVB) radiation at various doses, and observe the protective effects of ginsenoside Rg1 and Rb1 in vitro.

METHODSMTT assay was employed to analyze the cell survival rate after UVB radiation of 30, 60, 90 and 120 mJ x cm(-2). The damage of nucleolus and the protective effects of ginsenoside Rg1 and Rb1 were scanned by Hoechst 33258 staining and single cell gel electrophoresis assay (SCGE).

RESULTSIt was found that the cell survival rate decreased gradually and the damage of nucleolus aggravated as the radiation dose increased from 30 mJ x cm(-2) to 120 mJ x cm(-2). At the dose of 20 microg x mL(1-), obvious protective effect of ginsenoside Rg1 and Rb1 can be observed against UVB radiation-induced HaCaT cells growth inhibition and nucleolus damage.

CONCLUSIONUVB radiation inhibits HaCaT human keratinocytes growth and ginsenoside Rg1 and Rb1 can relief the damage.

Apoptosis ; drug effects ; radiation effects ; Cell Line ; Cell Survival ; drug effects ; radiation effects ; DNA Damage ; drug effects ; radiation effects ; Dose-Response Relationship, Radiation ; Ginsenosides ; isolation & purification ; pharmacology ; Humans ; Keratinocytes ; cytology ; drug effects ; radiation effects ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Protective Agents ; isolation & purification ; pharmacology ; Ultraviolet Rays ; adverse effects

Methods: In this study, 267 matched pairs of AIS and controls were recruited. The participants underwent EMG measurements at their first presentation and did not receive any treatment for 6 months at which point they underwent EMG and radiographs. Early curve progression was defined as >5° in Cobb angle at 6 months. The root mean square of the EMG (rms-EMG) signal was recorded with the participants in sitting and back extension. The rms-EMG ratio at the upper end vertebrae, apical vertebrae (AV), and lower end vertebrae (LEV) of the major curve was calculated. Results: The rms-EMG ratio in the scoliosis cohort was high compared with that in the controls (sitting: 1.2±0.3 vs. 1.0±0.1, p<0.01; back extension: 1.1±0.2 vs. 1.0±0.1, p<0.01). An AV rms-EMG ratio in back extension, with a cutoff threshold of ≥1.5 in the major thoracic curve and ≥1.3 in the major lumbar curve, was a risk factor for early curve progression after 6 months without treatment (odds ratio, 4.1; 95% confidence interval, 2.8–5.9; p<0.01). Increases in side deviation (SD) (distance between the AV and the central sacral line) were related to a higher rms-EMG ratio in LEV of the major thoracic curve (baseline: rs=0.2, p=0.03; 6 months: rs=0.3, p<0.01). Conclusions An EMG discrepancy was detected in the scoliosis cohort, which was related to increases in SD in the major thoracic curve. The AV rms-EMG ratio in back extension was correlated with curve progression after 6 months of no treatment.

OBJECTIVETo explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC).

METHODSmESC were cultured, and treated with the doses of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L respectively of BPA and DMSO (the solvent control group)for 24 hours, and three groups of cells were treated with the same method. The morphological changes of mESC in the control and exposure groups were observed through an inverted microscope. Cell counting kit 8 (CCK8) was used to detect the effects of BPA on proliferation of mESC, and based on the results, the half inhibitory concentration (IC50) was calculated. Real-time fluorescent quantitative polymerase chain reaction (RT-QPCR) and western blotting were used to detect the expression of OCT4 and SOX2.

RESULTSBPA had certain toxicity on mESC, the treatment of BPA significantly increased cell toxicity in a concentration-dependent manner, and the IC50 was 4.3×10(-4) mol/L, combined with the BPA exposure concentration of the environment and the related literature, eventually taking the five concentrations of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L as the experimental groups. The mESC morphology were effected after the treatment of BPA for 24 h, compared with the control group, the number of cells decreased, appearing some floating cells, and the cell cloning became irregular and differentiation in the higher concentration groups. The OCT4 mRNA expression level in the 10(-7) mol/L (1.146 ± 0.087), 10(-6) mol/L (1.156 ± 0.030), 10(-5) mol/L (1.158 ± 0.103) and the 10(-4) mol/L (1.374 ± 0.053) dose group were all significantly higher than the control group (1.000 ± 0.000) (t values were -2.384, -2.953, -3.203, -4.021 respectively, P value all < 0.05). Meanwhile, the SOX2 mRNA expression level in the 10(-4) mol/L (1.113 ± 0.052) were higher than the control group (1.000 ± 0.000) (t value was -2.765, P value < 0.05). Moreover, the OCT4 protein expression level in the 10(-5) mol/L (1.360 ± 0.168) and 10(-4) mol/L (1.602 ± 0.151) were all significantly higher than the control group (1.000 ± 0.000) (t values were -3.538, -4.002 respectively, P value all < 0.05), while no obvious change of the SOX2 protein expression level was detected in all treated groups.

CONCLUSIONBPA in a certain dose range could upregulate the expression of OCT4 gene in mouse embryonic stem cells while had no significant effect on the expression of SOX2 gene.

Animals ; Benzhydryl Compounds ; toxicity ; Cells, Cultured ; Embryonic Stem Cells ; drug effects ; metabolism ; Gene Expression ; Mice ; Octamer Transcription Factor-3 ; genetics ; Phenols ; toxicity ; SOXB1 Transcription Factors ; genetics ; Signal Transduction ; drug effects

OBJECTIVETo evaluate the efficacy of hBMP-4 gene modified tissue engineered bone graft in the enhancement of rabbit spinal fusion and find an ideal kind of substitute for the autograft bone.

METHODSRabbit BMSCs were cultured and transfected with AAV-hBMP-4 using different MOI value. The optimal MOI value were determined by observing cell's morphology change. BMSCs were then transfected with AAV-hBMP4 and AAV-EGFP respectively, following which the transfected cells were evenly suspended in a collagen sponge I, and implanted to either side of the L5,6 intertransverse spaces posterolateral in the New Zealand rabbits to induce spinal fusion. Fourteen rabbits were randomly divided into 2 groups. Group 1: AAV-hBMP-4 transfected BMSCs in the right side (hBMP-4 side) and autograft bone in the left side. Group 2: AAV-hBMP-4 transfected BMSCs in the right side (hBMP-4 side) and AAV-EGFP transfected BMSCs in the left side (EGFP side). Radiographs and three-dimensional CT of the spine, manual palpation, gross and histological examination of the fusion masses for all the animals were performed subsequent to animals having been sacrificed at 12 weeks after surgery.

RESULTSEvaluation has been taken in 12 New Zealand rabbits delivered into 2 groups which meet the criterion after operation. Eleven in 12 implemented sides involved hBMP-4 achieved bony fusion, to which 5 in 6 autografted sides was similar. But only 2 in 6 sides in EGFP-group achieved bony fusion meanwhile. Three-dimensional CT scan and palpation also evidenced the results. Bone formation was observed obviously on specimen both in hBMP4 sides and autografted ones. EGFP-group also got bony integration, but the quantity was small.

CONCLUSIONTissue-engineered bone graft constructed from application of hBMP4 is a fine substitute for autograft. Effective enhancement of bony integration in spinal fusion surgery has been evidenced in vivo.

Animals ; Bone Morphogenetic Protein 4 ; genetics ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; methods ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Myeloid Progenitor Cells ; Rabbits ; Random Allocation ; Spinal Fusion ; methods ; Stromal Cells ; Tissue Engineering ; Transfection

BACKGROUNDPercutaneous kyphoplasty (PKP) using balloon expander has been proved to be effective in the treatment of painful vertebral compression fractures. Recently, Sky-bone expander, an alternative bone expander for PKP has been developed. The purpose of this study was to show our preliminary clinical outcomes of PKP with Sky-bone expander.

METHODSPKP with Sky-bone expander was performed in 25 patients (30 vertebrae). The operation time, bleeding volume, cement volume injected were recorded. The pain and functional activities of the patients before and after the operation were compared using Wilcoxon signed-rank test. The cement distribution in the vertebrae, vertebral height restoration, and kyphosis correction after the procedure were evaluated by radiography. The pre- and post-operative absolute values of the vertebral height and kyphotic angle were compared by paired-sample t test. All the patients were followed up by telephone or clinic consulting after being discharged from our hospital.

RESULTSThe procedure was performed successfully in all the patients. Bipedicular injection was used in 2 of the patients, and unipedicular injection was made in the others. The operation time ranged from 25 to 120 minutes (45 minutes per vertebra on average). The average bleeding volume was about 20 ml. Polymethylmethacrylate 1.5-5.0 ml (mean, (3.15+/-0.78) ml) was injected through each pedicle into all the patients except one, who received calcium sulphate 3.5 ml instead. The patients were followed up for 12-15 months (13.5 months on average). The mean visual analogue scale (VAS) score, Oswestry Disability Index, anterior, midline, and posterior vertebral height, and kyphotic angle of the patients were improved significantly at the end of the follow-up compared with those before the operation. (2.5+/-1.3, 35.1%, (20.94+/-6.15) mm, (20.26+/-4.59) mm, (26.72+/-3.49) mm, and 8.2 degrees vs. 8.5+/-1.9, 61.2%, (19.11+/-6.72) mm, (15.88+/-5.73) mm, (25.78+/-3.67) mm, and 17.3 degrees; all P<0.05). The cement distribution with unipedicular injection was mostly limited within the injection site in the vertebral body. Cement extravasation was seen at ten levels (33.3%).

CONCLUSIONSPKP with Sky-bone expander is an effective and relatively safe alternative to the PKP using balloon expander. It can relieve pain, improve physical function, and restore the height of the collapsed vertebrae, but the cement extravasation is unsolved.

Adult ; Aged ; Bone Cements ; Female ; Fractures, Compression ; surgery ; Humans ; Male ; Middle Aged ; Prospective Studies ; Spinal Fractures ; surgery ; Spine ; surgery ; Tissue Expansion Devices ; Treatment Outcome

OBJECTIVETo compare the clinical results of balloon percutaneous kyphoplasty (PKP) and "Sky-bone expander" PKP.

METHODSFrom October 2004 to February 2006, 25 cases (57 vertebrae) balloon PKP and 24 cases (29 vertebrae) "Sky-bone expander" PKP procedures were performed. The operation time, bleeding volume, cement injected volume were recorded during operation. The patients' pain relief and functional activities recovery were evaluated after operation. The distribution of the cement and the restoration of vertebral height were also observed post-operation. All these patients were followed-up by telephone or clinic consults after discharged.

RESULTSAll cases were successfully experienced procedures. There are no significant differences in operative time, bleeding volume and cost of every vertebrae in these two group (P > 0.05). The balloon group had larger cement injected volume per pedicle than Sky group (4.27 +/- 1.08) ml vs. (3.15 +/- 0.78) ml (P < 0.05). The VAS and ODI scoring of these two groups were both decreased significantly after operation. The vertebral height were restored in both two groups with anterior height and midline height restored significantly in balloon group and midline height restored significantly in Sky group. The cement distribution of "Sky-bone expander" PKP with unipediclar injection mostly limited in the injective side of the vertebral body, but most of the balloon PKP vertebrae with unipediclar injection can be seen a cross-midline cement distribution in the anterior-posterior position X ray film.

CONCLUSIONSBoth balloon PKP and "Sky-bone expander" PKP are efficacious and safety in the treatment of vertebral compression fractures. "Sky-bone expander" PKP is more suitable for single level compressive fracture while balloon PKP is especially suitable for multiple level compressive fractures.

Aged ; Aged, 80 and over ; Catheterization ; instrumentation ; Female ; Follow-Up Studies ; Fractures, Compression ; etiology ; surgery ; Humans ; Male ; Middle Aged ; Osteoporosis ; complications ; Spinal Fractures ; etiology ; surgery ; Tissue Expansion Devices ; Vertebroplasty ; instrumentation ; methods