Objective To explore indications for replantation and amputation of complex severed lower legs. Methods Fifteen lower legs in 25 cases with complex severed lower limbs were replanted including four finally amputated again, and other 10 legs were amputated directly after trauma. Those with successful replantation were repaired with microsurgical technology and limb lengthening procedure for complications such as soft tissue defect, bone defect and limb shortening. The amputated cases were fitted with prosthetic limb after wound healing. Results All cases were followed-up for three to 10 years (6.4 years in average), and 11 lower legs were survived in 15 replanted cases and other four were finally amputated and fitted with prosthetic replacement. The limbs of those with successfully replantation shortened to varied extent, and one can be categorized as grade Ⅰ, four as grade Ⅱ, four as grade Ⅲ and two as grade Ⅳ, according to Chen's classification of function evaluation for replanted limbs. Appearance of prosthetic limbs in 14 cases had no significant difference from the normal, and 12 of them could walk with load and two had slight claudication. Those with prosthetic limbs were more satisfactory than those with replantation of the limbs. Conclusion Replantation for complex severed lower legs should be comprehensively considered based on mangled extremity severity score (MESS), their local condition and function prognosis.

Objective To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells. Methods Bone marrow mesenchymal stem cells were isolated and cultured in vitro. The positive rate of transfection was defected by cell immunohistochemistry methods after Ad-hTGF-β1 transfected for 1 week. Twenty-four adult New Zealand white rabbits with full articular cartilage defects were randomly divided into 4 groups, each group had 6 animals, both hind limbs were used in the experiment. Group A: hydrogel combined with transfected cells; Group B: hydrogel combined with untransfected cells; Group C: hydrogel group; Group D: blank control group. Specimens and histological observation were used to evaluate the repair effect after 16 weeks according to Pineda's score. Results The positive rate of hTGF-β1 expression in BMSCs was about 85.4% after transfection. After 16 weeks the defects of group A were repaired by cartilage-like tissue, the cell arrangement and densities of regenerated cartilage were similar to normal cartilage, type Ⅱ collagen immunohistochemistry were positive. There was a significant difference in Pineda's score compaired with other groups (P ＜ 0.05). Conclusion Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1-transfected bone marrow mesenchymal stem cells.

ObjectiveTo evaluate the physicochemical properties and bicompatibility of carbon-nanotubes/hydroxypatite/chitosan scafflod for bone tissue engineering. MethodsMWCNT/n-HA/CS scaffolds wre generated by solution blending and freeze-drying technology.The morphology and composition of the scaffolds were analyzed by scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy, after this, the results of which mixed CNTS in scaffolds were evaluated. The effects of MWCNT/n-HA/CS scaffolds on adherence and proliferation of rabbit bone marrow stroma cells were assessed by scaffolds surface seeding methods, and using scanning electron microscopy, MTT assay to observe their adhesion and proliferation on scaffolds.Results MWCNT/n-HA/CS scaffolds showed abundant homogeneous pores with (87.26％) porosity. 66％ fracture strength of the scafflod was improved by MWCNT,and porosity decreased by 3％. Conclusion MWCNT/n-HA/CS scaffold can be prepared with solution blending and freeze drying process, which has fair poriness, good mechanical strength and tissue compatibility and can be applied as a bone graft material.

Objective To investigate the composite of chitosan(CS) and polyvinyl alcohol (PVA) as scaffold carrier for rabbit chondrocytes nurture and growth.Methods The third passage of chondrocytes were seeded in CS/PVA gel scaffold and 24,48 and 72 h after which cytoactive and toxicity were determined by MTT respectively.After one,two and three weeks,the growing status and morphology of chondrocytes in CS/PVA gel were observed with scaning electron microscope (SEM) and laser confocal scanning fluorescence microscope (LCSM).Results The third passage of chondrocytes in CS/PVA gel scaffold remained high proliferation ability.MTT measuring cell activity and virulence,the result showed that the number of cells obviously increased with the time,with statistical significance of difference between each groups (P＜0.05),without side effect to cells by the material.Observation of scaning electron microscope and confocal laser scanning fluorescence microscope showed that chondrocytes grew well with the scaffold of CS/PVA gel.Conclusion CS/PVA mixed gel material can be used as scaffold for rabbit chondrocytes growing for repairing cartilages defect in tissue engineering.

In order to investigate the strength, structure and cell cytocompatibility of injectable thermosensitive chitosan (CS)/poly(vinyl alcohol) (PVA) composite hydrogel, chitosan hydrochloride solution was transferred to a neutral pH and mixed with different proportions of PVA, then the gelation time and strength of these different hydrogels were tested and spatial structures were observed under a scanning electron microscopy (SEM) after freeze-drying. The cytocompatibility of the hydrogels was evaluated through cytotoxicity test and three-dimensional culture with bone marrow mesenchymal stem cells. The results showed that the CS/PVA solution kept in liquid state at low temperature (0-4 degrees C) and turned into transparent elastomer about 15-20 min at 37 degrees C. Gelation time was prolonged, the strength increased and porous structure became dense with the PVA content increased in the mixed hydrogel. The cytotoxicity grades of these gels were from 0 to 1. Rabbit bone marrow mesenchymal stem cells could survive and proliferate in the gel within 3 weeks, and the gel had good cytocompatibility. It was concluded that thermosensitive CS/PVA composite hydrogel not only has interpenetrating network structure and better mechanical strength, but also has good cytocompatibility, and may be used as an injectable scaffold for tissue engineering.

Objective To investigate the feasibility of Ad-hTGF-β1 transfected bone marrow mesenchymal stem cell(BMSCs) into chondrocytes differentiation and the change of TAZ mRNA. Methods Rats BMSCs were obtained and cultured by whole bone marrow method, and then the third-generation cells were seeded into cell culture plate, and divided into three groups:Ad-hTGF-β1 transfected group,Ad-EGFP transfected group and the control group. The control group was added in common medium without any treatment while the other two groups were respectively added in serum-free medium containing Ad-hTGF-β1 or that containing Ad-EGFP. Seven days later, real-time fluorescent quantitation PCR and Western blot were employed for detecting the expression of TGF-β1 ,while immunohistochemical and Western blot for the expression of collagen Ⅱ , and real-time fluorescent quantitation PCR for the expression of TAZ mRNA. Results Seven days after the transfection, real-time fluorescent quantitation PCR revealed that the average relative expression of TGF-β1 was:Ad-hTGF-β1 group 0.863, Ad-EGFP group 0.183, and the control group 0.180; The average relative expression of TAZ was:Ad-hTGF-β1 group 0.810, Ad-EGFP group 0.416, and the control group 0.366.The expression difference of TGF-β1 and TAZ were statistically significant (P ＜ 0.05). Western blot and immunohistochemical proved strong collagen Ⅱ expression in Ad-hTGF-β1 group while it was detected a little in the other two groups. Conclusion BMSCs could be successfully and stably induced into chondrocytes differentiation by Ad-hTGF-β1. Meanwhile, the mRNA of TAZ is up regulate during the differentiation,so it is suppose that TGF-β1 improve BMSCs into chondrocytes differentiation by TAZ.

Objective To observe the change of invasion and migration of the pancreatic carcinoma cell line SW1990 transfeeted with EEF1A2 gene.Methods Pancreatic carcinoma cell line SW1990 was transfected with EEF1A2 by recombinant adenovirus vector.The alteration of motility、invasion and adhesion property of SW1990 was evaluated by wound healing assay,transwell With or without Matrigel basement membrane and adhesion assay.Results Wound healing assay revealed that EEF1A2 enhanced cell motility and transwell assay with Matrigel indicated that the average numbers of transwell cells with EEFlA2 was increased from 23.25±5.23 to 65.42±8.24(P＜0.05).The adhesive rate was substantially increased in EEF1A2 transfected SW1990 cells compared with control cells.Conclusions EEF1A2 gene can promote the migration.invasion and adhesion ability of pancreatic cancer cell in vitro.It is indicated that EEF1A2 may involve in the development of human pancreatic cancer by influencing cell biological characteristics.

Objective To investigate the expression of the ternary complex factor Net in human pancreatic carcinoma cell line BxPC3 and its effect on cell proliferation and the expression of c-fos.Methods pEGFP-Net prokaryotic expression plasmid and empty vector pEGFP were transfed into BxPC3 cens by using lipofectamine 2000,then monoclonal cell which stably expressing Net was established.Human pancreatic carcinoma cells proliferation was detected by MTT and flow cytometry.The tuRNA and protein expression of Net and c-fos in BxPC3 cells were detected by real.time PCR and Western blot.Results Net was low expressed in BxPC3 cells.After pEGFP-Net transfection,Net wag stably expressed and the expression of c-fos was inhibited,cell proliferation was also inhibited after pEGFP-Net transfection,the inhibitory rates at the 3rd, 5th,7th day was 38.81%,55.34%and 56.92%respectively,which were significantly higher than those in the empty vector group(5.09%,12.42%,8.6%,P<0.05).G_0/G_1 phase cell was(61.79±5.67)%,which were significantly higher than(45.14±3.37)%in the empty vector group(P<0.05).Conclusions The ternary complex factor Net could inhibit pancreatic carcinoma cell line BxPC3 proliferation.Its mechanism was possibly repressing expression of oncogene c-fos.

BACKGROUND:Bioactive glass, a multi-phase composite material, has good biological activity, bone conductivity and biocompatibility, but as a bone repair material it cannot be completely degraded, and has low mechanical strength that is insufficient.
OBJECTIVE:To design a kind of bioactive glasses/chitosan composite scaffold, and to investigate its physicochemical properties and cellcompatibility.
METHODS:Hydrochloric acid solution containing 2.0%chitosan was mixed withβ-glycerophosphate at a radio of 7:1 to prepare chitosan solution. Bioactive glasses of 0.5, 1.0, 1.5 g were added into the prepared chitosan solution, and the mass ratios of chitosan and bioactive glass were 2:1, 1:1, and 1:1.5 respectively. The composite materials were immersed and mineralized in simulated body fluid for 7 days.
RESULTS AND CONCLUSION:Scanning electron microscopy showed that the composite scaffold had an interconnected porous structure with the porosity of 89%and the pore size of 100-300μm;bioactive glasses dispersed in a needle shape between the chitosan scaffolds, arranged evenly, and were ful y wrapped tightly by the scaffolds. With the increase in mass of bioactive glass, the porosity of the composites decreased, but the fracture strength gradual y increased. There was a positive correlation between the composite porosity and fracture strength. X-ray diffraction and Fourier transform infrared spectroscopy confirmed that the composite scaffold appeared to have no changes in the nature of single materials, and differential scanning calorimetry analysis showed no mass loss at normal body temperature. After 3 days of mineralization, hydroxyapatite forming on the material surface gradual y grew up as a vil ous shape, and also significantly increased in number. After 7 days of mineralization, hydroxyapatite changed from a vil ous shape to a needle shape, the amount of hydroxyapatite was increased further, and many mineralized products were in a spherical shape.

ObjectiveTo investigate the feasibility of hTGF-β1 transfected bone mrrow mesenchymal stem cell (BMSCs) combined with calcium alginate gel in three dimensional condition to construct tissue engineering cartilage.MethodsRats BMSCs were obtained and cultured by whole bone marrow method,and then the third-generation cells were seeded into cell culture plate,and were divided into 3 groups:AdhTGF-β1 transfected group, Ad-EGFP transfected group and control group. The control group was added in common medium without any treatment while the other 2 groups were respectively added in serum-free medium containing Ad-hTGF-β1 or that containing Ad-EGFP.Seven days later,real-time fluorescent quantitation PCR and Western blot were employed for detecting the expression of TGF-β1.Then,the BMSCs which successfully transfected by Ad-hTGF-β1,were continually cultured in vitro.Andthe confound of cells-calcium alginate gel,the cell density was 1.0 × l07 per ml,were prepared and cultuered in constant temperature incubator.Ten days later,examine the morphous and proliferation of cell.Last,paraffin slice of the cell-gel confound was stained by HE,toluidine blue and Masson staining,while immunohistochemical for the secretion of collagen Ⅱ.ResultsSeven days after the transfection, real-time fluorescent quantitation PCR revealed that the average relative expression of TGF-β1 was:Ad-hTGF-β1 group 0.863,and Ad-EGFP group 0.183, the control group 0.180, and the expression difference of TGF-β1 was statistically diffence(P ＜0.05). Western blot proved strong TGF-β1 expression in Ad-hTGF-β1 group while it was detected a little in the other two groups. Globose cells were observed through inverted microscope in the calcium alginate gel.MTT proved the amount of cells were not statistically diffence (P ＞ 0.05) at different time point.HE staining proved amount cartilage lacuna formation in the gel, while the secretion of cartilage matrix were proved by toluidine blue and Masson,and immunohistochemical proved the expression of collagen Ⅱ.Conclusion BMSC trnsfected by hTGF-β1 could be successfully induced to chondrocyte, as the cells morphous maintained.This three dimensional condition could preferably mimicry cell growth pattern as in vivo.