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.

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.

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 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.

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 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.

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.

Objective To summarize the clinical experience of treating long bone defect with vascularized fibular graft.Methods From January,2008 to January,2015,31 cases of long bone defect were treated with vascularized fibula composite or not composite tissue flap graft.The length of transplanted fibula was 9-20 cm,and the flap area was 5 cm×3 cm to 21 cm×14 cm.All patients were followed up regularly.Limb function was assessed 12 months after surgery.Results Thirty-one cases of vascularized fibular flap survived after surgery.Thirty patients were followed up for 1.5 to 6 years (average,2.5 years).One patient was lost to follow-up.The bone defects of patients followed up were healed.There was one case fracture occurred for trauma,was treated with plaster cast for 6 months and healed.The transplanted fibular thickened for 1.3 to 2.5 years(average,1.6 years).Conclusion Vascularized fibular graft can reconstruct long bone defect for single use and shorten the duration of treatment with a good limb function.For cases combined soft tissue defect,vascularized fibula composite tissue flap can be applied to repair at the meantime.

Objective To investigate the choice of methods and time on the treatment of Gustilo type Ⅲ B and type ⅢC open tibia fractures by bone fixation and tissue flap repairing.Methods The clinic data of 136 cases of Gustilo Ⅲ B and Ⅲ C open tibia fractures were retrospectively studied.There were 102 males and 34 females,with average age of 34 years(range 14 to 68 years).Ninety-eight cases were classified as Gustilo type Ⅲ B and 38 cases were type Ⅲ C in degree.In all cases,one hundred and thirteen were treated with onestage external fixation,ninteen cases were treated with secondary bone fixation.Sixty-seven cases of all wounds were repaired by one-stage vascular pedicle skin flap.Seven wounds were repaired by one-stage free flaps with vascular anastomosis.Sixty-two cases were secondary repaired by different flaps after defect tissue under VSD (vacuum-sealing-drainage) treatment by 1 to several weeks,which contains 9 free skin graft,eleven muscle flaps based on posterior tibial artery perforator,thirteen flaps based on fibial artery perforator,fourteen gastrocnemius falps and 15 cross-leg flaps.Results Wound of all cases were healed wholly.Primary union occurred in 121 cases,twelve,cases healed after bone grafting for segmental bone defect.Three cases with delayed union healed after reoperation for infection that caused osteomyelitis and sinus tract.The period of bone healing was form 5 to 12 months.Conclusion The treatment of Gustilo Ⅲ B and Ⅲ C open tibia fractures by onestage or secondary bone fixation and tissue flap repairing get their advantages and disadvantages,but one-stage bone fixation and tissue flap repairing was better where appropriate.

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.