BACKGROUND:Current decelularized methods have the certain damage to the extracelular matrix and reduce the biomechanical properties of acelular scaffolds. OBJECTIVE:To explore the biomechanical properties of decelularized scaffold of lyophilized bovine tendon. METHODS:Sixty lyophilized fiber bundles from fresh flexion tendon of calf toes were randomly divided into two groups: control group and experimental group. In the experimental group, serine protease inhibitors were placed asepticaly for 24 hours at room temperature, then the samples were rinsed with PBS and transferred to the low concentration of trypsin+ethanol mixed solution to remove the cel wal without destruction of the extracelular matrix at room temperature for 5 hours; after that, the fiber bundles were cultured in DNA enzyme solution for 5 hours, finaly the acelular scaffold was completed and rinsed with PBS for 48 hours and dried at room temperature in sterile room. No treatment was done in the control group. Modulus of elasticity, durability and maximum stress were determined in the two groups. RESULTS AND CONCLUSION:Similar elastic modulus and durability were found in the two groups, but the maximum stress in the experimental group was significantly lower than that in the control group (P < 0.01). These findings indicate that the lyophilized acelular tendon fibers can mimic the biological function of bovine tendon fibers to a certain extent.

BACKGROUND: Double gene transfection using bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF165) for bone marrow stromal stem cells (BMSCs) to induce osteogenesis provides experimental basis for the study on tissue engineering bone. OBJECTIVE: To investigate the ability of BMP2 and VEGF165 double gene modified rat BMSCs to induce osteogenesis. METHODS: BMSCs were isolated from the femur and tibia of four 4-week-old Sprague-Dawley rats by whole bone marrow adherent culture. Passage 3 BMSCs were randomized into five groups: non-transfection group, empty plasmid group, BMP2 transfection group, VEGF165 transfection group, BMP2 and BMP2/VEGF165 transfection group (co-transfection group). Then, western blot assay was used to detect expression of BMP2 and VEGF165 at 48 hours after transfection, and the activity of alkaline phosphatase was detected in each group at 7 days after transfection. RESULTS AND CONCLUSION: Highly expressed BMP2 in BMP2 and co-transfection groups and highly expressed VEGF165 in VEGF165 and co-transfection groups were found after transfection. The expression of BMP2 or VEGF165 in the co-transfection group was significantly higher than that in the BMP2 or VEGF165 transfection group after transfection, respectively (P < 0.05). Western blot results showed that the activity of alkaline phosphatase was ranked as: co-transfection group > BMP2 transfection group > VEGF165 transfection group > empty plasmid group and non-transfection group. There was a significant difference in the activity of alkaline phosphatase between co-transfection group and any of single gene transfection groups (P < 0.05). To conclude, BMP2/VEGF165 co-transfection promotes the ectopic osteogenesis of BMSCs.

BACKGROUND:The pathogenesis of ligamentum flavum hypertrophy remains poorly understood, and the expression of transforming growth factor beta1 (TGF-β1) is increased notably. Reactive oxygen species (ROS) accumulation is associated with tissue degeneration, which may accelerate the progression of ligamentum flavum hypertrophy by upregulating TGF-β1 expression. OBJECTIVE:To clarify the effect and significance of ROS H2O2-mediated up-regulation of TGF-β1 and collagen type Ⅰ in the progress of ligamentum flavum hypertrophy. METHODS:Ligamentum flavum was removed from a case of acquired lumbar disc herniation with normal ligamentum flavum during lumbar posterior decompression surgery, and then separated and cultured in vitro to the 4-6 generations, followed by exposure to H2O2 at various concentrations (0, 50, 100, 150, 200μmol/L) for 72 hours. The mRNA and protein expression levels of TGF-β1 and collagen type Ⅰ were detected by real-time PCR and western blot assay, respectively. RESULTS AND CONCLUSION:Real-time quantitative PCR showed that the mRNA expression level of TGF-β1 was significantly increased in the 150 and 200μmol/L groups (P<0.05). The mRNA expression level of collagen type Ⅰ was significantly higher in the experimental groups than that in the control group, especially in the 200μmol/L group (P<0.05). Western blot assay revealed that the protein expression levels of TGF-β1 and collagen type Ⅰ were significantly increased in a dose-dependent manner (P<0.05). These findings indicate that H2O2 may accelerate the progression of ligamentum flavum hypertrophy by up-regulating the expression levels of TGF-β1 and collagen type Ⅰ.

Objective To observe the effect of breviscapine on the open probability(Po)and the average open time(To) of L-type calcium channel in the hippocampal neurons of the whole brain ischemia-reperfusion rat model.Methods The whole brain ischemia rat model was established.At the reperfusion periods of 1.5 h,3.0 h,4.5 h and 6.0 h,the hippoeampal CA1 neurons were dissociated abruptly and the Ca~(2+)electricity of single channel on the neuron membrane were recorded with cell-attach model of patch clamp techniques.The Po and To of the L-type calcium channel were an- alyzed and the effect of the breviscapine was observed.Results Different concentrations of breviscapine can decrease the Po and To of the L-type calcium channel in hippoeampal neurons,particularly within 3 hours after ischemia-reperfu- sion.Conclusions Breviscapine has protective effect on the cerebral ischemia-reperfusion injury.

BACKGROUND: Demineralized bone matrix and bone morphogenetic protein have been shown to have good bone induction, but less studies concerned nanometer demineralized bone matrix. Its physical and chemical properties and biological security are not yet clear.
OBJECTIVE:On the basis of preparing the nanometer human demineralized bone matrix in previous experiment, we mixed the recombinant human bone morphogenetic protein-2 together to obtain the new bone graft substitute and to research its physical and chemical properties and biological security.
METHODS:The human demineralized bone matrixes were prepared by the method of modified Urist and nano-processed then mixed with the bone morphogenetic protein-2 in specific proportions in order to be lyophilized to complete the fol owing experiments. (1) Pyrogen experiment:the material extracts were injected in the rabbits by ear intravenous. (2) Toxicity experiments:material extracts and saline were separately injected via the tail vein of mice in vivo. (3) Implantation experiments:experimental materials andβ-tricalcium phosphate were implanted into rabbits on both sides of the hindlimb muscle.
RESULTS AND CONCLUSION:After lyophilized shaping, the nanometer demineralized bone matrix material had dense surface and it’s pore diameter was 100-400μm. The pore distribution was less uniform and the porosity was of less than 30%. The main elements were carbon, oxygen and nitrogen. Nanometer human demineralized bone matrix with recombinant human bone morphogenetic protein-2 did not have pyrogen effect and the rabbits’ body temperature had no significant fluctuations after injection. The acute systemic toxicity test results showed that the nanometer human demineralized bone matrix with recombinant human bone morphogenetic protein-2 complied with the relevant provisions of the State, without obvious toxic reaction. The inflammatory response of nanometer human demineralized bone matrix with recombinant human bone morphogenetic protein-2 was significantly lighter than the reaction ofβ-tricalcium phosphate. The results showed that the nanometer human demineralized bone matrix with recombinant human bone morphogenetic protein-2 is a nanometer al ogeneic bone graft substitutes with nontoxicity, good biocompatibility, high bioavailability, and less inflammatory reaction.

Objective To observe the clinical features of migraine based on out-patient clinic data and provide help for the diagnosis and treatment of migraine. Methods In a retrospective study of 309 patients with migraine, we investigated the clinical characteristics of migraine of both genders and different types, and the risk factors for MOH transformed from migraine. Results The female to male ratio was about 3:1,76.1% of the patients had triggering factors. The most common characteristics of headache were moderate to severe intensity of the pain (97.7%), aggravation by routine physical activity (75.1%), and association with nausea (90.9%) and/or vomiting (70.6%). There were significant differences in some clinical characteristics of migraine in females as compared with these in males and in patients with migraine without aura (MWOA) as compared with those with aura (MWA). The risk factors for MOH transformed from migraine were elder age of onset, high attack frequency and the analgesics frequently used (P < 0.05).Conclusion It is suggested that carefully collecting the characteristics of headache, triggering factors and therapeutic history is the foundation of correct diagnosis and effective treatment for migraine.

Objective To investigate MRI localization the accuracy and correct the deviation for gamma knife treatment.Methods With 25-point-matrix tank,the deviation of MRI localization and its regularities could be identified after the comparison between the coordinates of MRI localization and the ones which have already been verified by CT within the deviation of 0.5 mm.Then the original MRI coordinates will be corrected by the acquired mean deviation and the geometric distortion of images.Afterwards the corrected coordinates will be compared with the standard ones and finally validated by exposure film.Results There are no significant deviations on x-and z-axis after measurement in three hospitals,y-axis,however,bears deviation of (1.94 ±0.45) mm for hospital A,(-2.22 ±0.29) mm for hospital B,(-1.25 ±0.21) mm for hospital C,respectively.Furthermore there also exists geometric distortion of 1％ on y-axis in hospital A.The corrected coordinates on y-axis (Yc) will be calculated from the formula:Yc =(Y-M) + GD (Y0-Y) (Y:the original coordinates on y-axis,M:the mean of deviation on y-axis,GD:the geometric distortion,Y0:the coordinate on y-axis of the central point among the 25-point matrix).Once completed,the corrected coordinates of MRI localization is of no significant difference with the standard coordinates verified by CT.Even the deviation of focal spot on validation film is within 0.5 mm.Conclusions The 25-point-matrix tank in the multi-point measurement of the accuracy and the correction of deviation for gamma knife treatment is feasible to determine whether MRI can be utilized in the localization for head gamma knife treatment.

BACKGROUND:The exact pathogenesis of ossification of ligamentum flavum has not been elucidated yet. And osteopontin may be an important factor involved in the ossification of ligamentum flavum. OBJECTIVE:To clarify the expression and significance of osteopontin and its receptors, CD44 and integrin-β3, in ligamentum flavum cels between normal controls and patients with ossification of ligamentum flavum.METHODS:Ligamentum flavum tissues were obtained from normal adult controls and adult patients with ossification of ligamentum flavum (n=8 per group) who underwent thoracic/lumbar posterior decompression surgery. Ligmentum flavum cels were separated, cultured and identifiedin vitro, and osteopontin, CD44, integrin-β3 were stained using immunocytochemistry method and observed under inverted phase contrast microscope. And the mRNA expressions of osteopontin, CD44, integrin-β3 were measured by RT-PCR. RESULTS AND CONCLUSION: Immunocytochemistry results showed that the stronger positive staining for osteopontin, CD44, integrin-β3 was observed in the ossification of ligamentum flavum group than the control group (P < 0.01). The mRNA expressions of osteopontin, CD44 and integrin-β3 were also higher in the ossification of ligamentum flavum group than the control group (P < 0.05). These findings indicate that osteopontin and its receptors, CD44 and integrin-β3, in ligamentum flavum cels may play an important role in ossification of ligamentum flavum.

Objective To explore the association between PI3K polymorphisms in insulin signal transduction pathway and Alzheimer's disease (AD)risk.Methods There were three groups,including 112 cases for AD +T2D group,231 cases for only AD group,and 231 cases for healthy controls group.The polymorphisms in PI3K gene was sequenced by PCR and the concentration of PI3K in serum was tested by enzyme -linked immunosorbent assay (ELISA).Results Overall,there was significantly statistical difference in PI3K rs3730087 polymorphism among three groups (χ2 =20.99,P =0.000 3).The CC frequency of PI3K rs3730087 polymorphism in AD with T2D group and AD control group was significantly higher than that in the healthy control group.The PI3K protein level of differ-ent genotype was statistically significant (F =27.450,P <0.000 1).As for CC genotypes of PI3K rs3730087 poly-morphism,the PI3K protein level was statistically different among these three groups (F =8.096,P =0.000 6).Moreover,the PI3K protein level of the three groups was different (F =9.034,P =0.000 1),which in both AD group was lower as compared with healthy control group.Conclusion The study suggested that CC genotype of PI3K rs3730087 polymorphism in insulin signaling transduction pathway might be a risk factor for AD with T2D and it also affects the expression level of PI3K protein.However,the polymorphism is not shown to be exclusive in AD patients with T2D.

Objective To estimate the value of team-based learning ( TBL ) in the teaching of occupational health and occupational medicine for foreign students. Methods 42 foreign students from the majorofclinicalmedicineinHarbin Medical UniversitywereselectedtoformtheTBLdiscussiongroup. Before class, teachers assigned tasks, and the students were taught with the same teachers with TBL teaching method. The effect of learning was evaluated by questionnaire and classroom test. The t test was performed using SPSS 19.0 statistical software for comparison of the results of individual test and group test. Results The result of the questionnaire showed that students agreed that TBL teaching can improve students' interest, self-study ability and broaden their learning ideas. The classroom test results showed that after the TBL discussion, the test scores of occupational oncology and pneumoconiosis were significantly higher than those of individual test. The difference was statistically significant (P<0.05). Conclusion The TBL method can significantly improve the students' comprehension of knowledge and enhance their learning effect.