BACKGROUND: The application of adipose derived stromal cells to tissue engineering has been more and more popular around the world. Compatibility of scaffold material is the key point for its further research.OBJECTIVE: To determine the growth rules of rabbit adipose-derived stromal cells with polylactic-co-glycolic acid (PLGA).DESIGN, TIME AND SETTING: An in vitro study was performed at the Institute of Ophthalmology, Otolaryngology Hospital,Fudan University and Shanghai Tissue Engineering Center from September 2007 to March 2009.MATERIALS: Six female New Zealand rabbits aged six months were used for extraction of adipose-derived stromal cells. PLGA was provided by Sigma, USA.METHODS: Adipose tissue was harvested from the nape fat pad of the rabbits following anesthesia. Primary cultured cells were established using type I collagenase and cell cultures were maintained with DMEM containing 10% volume fraction of fetal bovine serum. Cells were passaged when 80% was confluent. The fourth passages of cells were utilized for the study. PLGA consisted of polylactic acid and polyglycolic acid as the ratio of 7:3, and the relative molecular mass was 104900.MAIN OUTCOME MEASURES: Initially, the adherent rate of cells to scaffold was detected. After one week co-culture, the scaffold bearing adipose-derived stromal cells labeled with Dio agent were investigated by fluorescence inverse microscope,scanning electron microscopy and laser scanning microscope.RESULTS: The best adherent rate of adipose-derived stromal cells with PLGA reached 99%. After one-week-incubation in vitro,cells of fiber-shaped or ovule-shaped proliferated well and exhibited stratified growth on the surface of PLGA scaffold. In addition,it also secreted visible extracellular matrices, which could be examined by scanning electron microscopic examination.Meanwhile, the adipose-derived stromal cells grew well and distributed equably inside the PLGA in terms of the investigation with laser scanning microscope.CONCLUSION: The compatibility of adipose-derived stromal cells to PLGA in vitro was well.

Objective To investigate the effect of pregnancy on the potency of bupivacaine for spinal anesthesia in rats. Methods Female non-pregnant SD rats weighing 180-220 g and 17 day pregnant SD rats weighing 350-400 g were used in this study. The rats ( 18 non-pregnant, 18 pregnant) in which PE-10 catheter were successfully placed without complications were selected. The 18 non-pregnant rats were randomly divided into 3 groups (n =6 each): control group (group C), 2% bupivacaine group (group B2 ) and 4% bupivacaine group (group B4). The 18 pregnant rats were also randomly divided into 3 groups (n = 6 each): control group (group PC),2% bupivacaine group (group PB2 ) and 4% bupivacaine group (group PB4 ). Group C and PC received intrathecal (IT) normal saline 30 μl, and the other 4 groups received 2% or 4% bupivacaine 30 μl intrathecally. Analgesia was determined using the taifllick latency (TFL) before IT administration (baseline), and at 10 min, 20 min,30 min, 1 h, 2 h, 4 h, 1 d, 2 d, 3 d and 4 d after IT administtation. The percentage of the maximal possible effect (MPE) was calculated. Hind-limb motor function (MF) was also assessed. Results Compared with the baseline value, MPE at 10 min-2 h after administration and MF scores at 10 min-1 h after administration were significantly increased in group B2, MPE at 10 min-4 h after administration and MF scores at 10 min-1 h after administration were significantly increased in group B4;MPE at 10 min-1 d after administration and MF scores at 10 min2 h were significantly increased in group PB2 and MPE at 10 min-1 d after administration and MF scores at 10 min4 h were significantly increased in group PB4 ( P ＜ 0.05 ). Conclusion Pregnancy can enhance the potency of bupivacaine for spinal anesthesia in rats.

Objective To evaluate the efficacy of decortication by video-assisted thoracic surgery (VATS) in pa﹣tients with tuberculous empyema, and discuss its indications. Methods 60 patients with tuberculous empyema who underwent decortication by VATS for surgical management from December 2010 to December 2015 were included. Under a thoracoscope, we cleaned up the pus, separated adhesions, scraped granulation tissues and caseous necrosis on the inner wall of the abscess cavity, and stripped the thickened fiberboard of the parietal and visceral pleurae. Af﹣ter the procedure, sufficient drainage and antituberculosis therapy were carried out. Results All the patients in this group were operated successfully. All the patients were cured without perioperative death and complications. No re﹣currence of empyema was observed at the follow-up examination from 2 months to 5 years, and suffered pulmonary reexpansions were better. Conclusions The decortication by VATS for tuberculous empyema is safe, effective, mini﹣mally invasive. The imaging manifestations of pleural thickening in 1 cm, no obvious calcification, no serious lesions in the lungs are the indications for the operation.

BACKGROUND:Bladder acellular matrix graft (BAMG) is frequently used for domains of tissue engineering scaffold due to its great biocompatibility and cell adhesion. OBJECTIVE:To verify the biological characteristics of BAMG after frozen and lyophilized processing. DESIGN,TIME AND SETTING:A biocompatibility experiment was performed at Shanghai Tissue Engineering Research and Development Center and Experimental Animal Department of the Sixth People's Hospital of Shanghai between May and November 2008. MATERIALS:Two New Zealand rabbits were used in this study for BAMG preparation. METHODS:After midsection of rabbit bladder,mucous membrane of urinary bladder was isolated and dipped in three-distilled water for 24 hours. Thereafter,the samples were incubated with acellular solution containing 0.1% Triton X-100 and 0.15% aqueous ammonia for 14 days. The culture medium was changed regularly. The samples in the control group were stored in 75% ethanol,while samples in the experimental group were frozen for 24 hours at -80 ℃,vacuum-dried for 24 hours,and stored in 75% ethanol. MAIN OUTCOME MEASURES:Biological characteristics of BAMG were detected using hematoxylin and eosin staining,Masson staining,and scanning electron microscopy; biological characteristics were compared between the two groups using cell adhesion test,MTT assay,and subcutaneously embedding test. RESULTS:Hematoxylin and eosin staining and Masson staining revealed that no residual cells were detected in the BAMG,and collagen was intact. Scanning electron microscopy demonstrated that cells exhibited a slit-shaped structure mainly containing collagen which was beneficial for cell adhesion. Mechanical test revealed that the BAMG after frozen and lyophilized processing not only reserved the mechanical properties of the raw BAMG,but also had a great elongation. MTT assay confirmed that cytotoxicity was grade 0,and BAMG had a good compatibility to smooth muscle cells. After subcutaneously embedding for one month,BAMGs had good adhesions to subcutaneous tissues,and muscular adhesion and vascular proliferation were observed. CONCLUSION:BAMG after frozen and lyophilized processing reserves original biocompatibility and has great elongation; therefore,it will become a useful and ideal biomaterial for tissue engineering scaffold.

Objective To explore the targeted nursing care for patients with severe traumatic brain injury complicated with intracranial infection. Methods 35 patients of severe traumatic brain injury complicated with intracranial infection from January, 2009 to December, 2011 were reviewed. Results There were 10 cases cured, 12 cases improved very well, 4 cases improved, 5 cases invalid and 4 cases died. The total effective rate was 62.86%and the fatality rate was 11.43%. Conclusion Intracranial infection can be controlled effectively by the venous and intrathecal injection of antibiotics and targeted care in the patients with severe traumatic brain injury.

Objective To explore the method of culture of rat adipose-derived stem cells(ADSCs) and differentiation induction into myoblasts. Methods Adipose tissues were obtained from SD rats,and were isolated by enzyme digestion and cultured into ADSCs.The expression of surface antigen CD90,CD105 and CD34 was detected by immunofluorescence and flow cytometry.ADSCs of the second passage with logarithmic growth were obtained,and culture media containing 5-azacytidine(5-aza) and basic culture media were employed for cells in induction group and control group,respectively.The induction lasted for 7 d,14 d,21 d,28 d and 35 d,respectively.Cell growth and cell morphology were observed by inverted phase contrast microscope,and immunofluorescence and flow cytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin. Results ADSCs were successfully isolated and cultured,and were identified to be stem cells.On the 28th day of induction,cells in induction group displayed "swirl" morpholgy,and multinucleation was observed.It was revealed by immunofluorescence and flow cytometry that the highest expression rates of desmin and myosin were 52.57% and 50.04%,respectively on the 28th day of induction,while there was no expression before induction and in control group. Conclusion ADSCs can be isolated and cultured from rat adipose tissues,and can further differentiate into myoblasts after induction by culture media with 5-aza.The expression of myoblast specific antigen is the highest on the 28th day of induction.

Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.

Objective The existing Quality Standards for Qianliean Capsules lack the quantitation control item and therefore cannot completely reflect the quality of the preparation .This study aimed to establish the methods for qualitation and quantitation of Qianliean Capsules . Methods Thin-layer chromatography ( TLC) was used for the qualitative identification of Radix Salviae miltior-rhizae, Rhizoma curcumae, and Glycyrrhiza uralensis in Qianliean Capsules .The content of hesperidin was determined by high-per-formance liquid chromatography ( HPLC) , and the method of determination was systematically evaluated . Results TLC spots were clear with a strong specificity .The content of hesperidin showed a good linear relationship within the range of 11 .56-310 .00μg/mL (r=0.999 5), with a mean recovery rate of 99.55% and relative standard deviation of 1.93%. Conclusion The qualitation and quantitation methods we established were simple , accurate and reliable , with a good reproducibility , and can be used for the quality control of Qianliean Capsules .

BACKGROUND:Fibrin glue introduced into calcium phosphate cement has not been confirmed whether this way could overcome the compressive limits and the low degradation of calcium phosphate cement and to modify the biological properties of calcium phosphate cement. OBJECTIVE:To explore the mechanical and biological properties of calcium phosphate cement/fibrin glue at different powder/liquid ratio for bone regeneration in vitro. METHODS:Calcium phosphate cement and fibrin glue were mixed at ratios of 1:1, 3:1, 5:1 (mL/g), and the pure calcium phosphate cement served as controls. Setting time, scanning electron microscope and the biomechanical test were used to analyze the composite scaffold structure, physical performance and the mechanical properties. Passage 3 osteoblasts were respectively inoculated on the material surface of the four groups, and pure cells served as blank controls. celladhesion, proliferation and alkaline phosphatase activity were observed. RESULTS AND CONCLUSION:The initial and final setting time of calcium phosphate cement/fibrin glue at 1:1 and 3:1 (mL/g) was higher than that in the control group (P<0.05), while the initial and final setting time of calcium phosphate cement/fibrin glue at 5:1 (mL/g) was lower than that of the control group (P<0.05). Scanning electron microscope showed smoother and denser surface of composite scaffolds compared with the pure calcium phosphate cement. The aperture of the composite scaffolds was decreased with the increasing concentration of fibrin glue. The compressive strength of composite scaffolds at 3:1 and 5:1 was higher than that of the control group (P<0.05), while the modulus of the composite scaffolds at 1:1, 3:1, 5:1 was higher than that of the control group (P<0.05). celladhesion, proliferation and alkaline phosphatase activity showed no difference among the three composite scaffold and control groups, but al higher than the blank control group (P<0.05). These findings indicate that fibrin glue introduced into calcium phosphate cement can overcome the low-strength limits of calcium phosphate cement, and maintain the good biological properties of calcium phosphate cement for bone regeneration.

Objective To investigate the feasibility of constructing tissue engineering urethral substituted graft using human lingual keratinocytes and natural derived scaffold.Methods From Oct.2009to Jan.2010,ten patients with anterior urethral stricture were enrolled in this study.A 0.5 × 0.8 cm lingual mucosa was harvested during the operation.Lingual keratinocytes were then isolated and cultured from the mucosa.AE1/AE3 antibody was used to identify the lingual keratinocytes.Keratinocytes were collected and seeded onto three types of scaffold including dehydrated BAMG,liquid stored BAMG and 4-layer SIS product,with the density of 1 × 107/ml at passage three.After being cultured for seven days in vitro,H&E staining and Electronic Scan Microscopy were used to evaluate the compound matrixes.Results No complication occurred in the patients after operation.The primary passage of confluence lingual keratinocytes appeared in a typical cobblestone shape after being cultured for 14 days in vitro.The proliferation rate of these cells increased rapidly during the three passages.However,it decreased significantly in the 4th passage.H&E and Electronic Scan Microscopy examinations showed that few cells grew on the surface of the liquid stored BAMG.Nevertheless,multiple keratinocytes layers could be seen in dehydrated BAMG and 4-layer SIS.Meanwhile,cellular infiltration could be observed in SIS sections.Conclusions Human lingual keratinocytes could be the alternative of seeding cells for constructing tissues for engineering the urethra.These cells exhibited good compatibility and are adhesive to the SIS or BAMG.The compound matrix,which used human lingual keratinocytes and natural derived scaffold,may meet the clinical need of urethral disease in the future.