Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were counted following Dopa staining under a microscope. The trypsinized skin sheets were scraped with a scalpel to harvest melanocytes which were subjected to a primary culture. Then, the melanocytes were inoculated onto fresh or cryopreserved AM followed by a culture for various durations (4, 8 and 12 days). The morphology and dendrite development of melanocytes were visualized under an inverted microscope after dopa-staining, cell viability evaluated by MTT assay, the adhesion to AM examined by hematoxylin and eosin (HE) staining protocol. Results The density of melanocytes was 1543.1±13.3 cells per mm2 and 857.4±101.7 cells per mm2 in skin sheets obtained from the forearm flexure and lower abdomen of the volunteer, respectively. A skin sheet of about 25.1 mm2 from approximately two blister roof was required to ensure the success of primary culture of melanocytes within 1 month. After culture on fresh or cryopreserved AM for 4, 8, and 12 days, most melanocytes were bi-polar with extended slender dendrites compared with those cultured in common cell culture medium. HE staining showed that melancytes adhered and were evenly distributed on the basement membrane of AM. MTT assay showed that the AM inhibited the proliferation of melanocytes, and no statistical difference was observed in the inhibitory effect between fresh AM and cryopreserved AM (P＞ 0.05). Conclusions Enriched with melanocyes, flexural forearm is a preferable donor site to offer skin sheets for primary culture of melanocytes. Human AM could improve the adhesive growth and dendrite development of melanocytes, and may serve as a promising bioscaffold for in vitro expansion of melanocytes.

Poly (N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer, can be potentially applied to replace enzymes or cell scrapers to recover attached cells. Taking full advantage of this unique function of PNIPAAm, cells can be protected from enzymatic hydrolysis and mechanical treatment, thereby to provide ideal seed cells with high quality for biomedical fields. In this review we describe the method to facilitate cell effective adhesion and rapid detachment on thermoresponsive two dimensional surfaces, including selecting special substrate, introducing hydrophilic group, adjusting reactant ratio, controlling polymer thickness/density, providing appropriate external force, so as to effectively improve adherent cell adaptability to thermoresponsive surfaces, depress the risk of bacterial contamination and reduce the effect of low-temperature treatment on the cells. The three dimensional cell culture systems involved in temperature-sensitive microcarriers, scaffolds and gels were briefly discussed. The application based on the platforms for cell culture was also presented.
Acrylic Resins ; Cell Adhesion ; Cell Culture Techniques ; Temperature

Objective To evaluate the risk factors for surgical site infection(SSI)following ankle joint and Pilon fracture surgery,and provide theoretical basis for the prevention of postoperative SSI.Methods Clinical data of pa-tients who underwent ankle joint and Pilon fracture surgery in a hospital between June 2005 to May 2013 were sur-veyed retrospectively,risk factors for SSI were analyzed.Results Among 356 patients with ankle joint and Pilon fracture surgery,22 developed 25 times of SSI,SSI rate was 6.18%,case infection rate was 7.02%.Univariate analysis showed that elderly patients (> 60 years old),history of diabetes,pre-operative calcaneal traction, perioperative irrational antimicrobial use,incision type,and long duration of operation (>3 h)were risk factors for SSI following ankle joint and Pilon fracture surgery (all P <0.05).Multivariate logistic regression analysis showed that the independent risk factors for SSI were incision type(OR,3.58[95%CI ,3.24-12.07]),history of diabetes (OR,2.75[95%CI ,1 .54-4.75]),duration of operation(OR,2.15[95%CI ,1 .32-3.64]),and patients age(OR, 1 .68[95%CI ,1 .25-2.37]).Conclusion Occurrence of SSI following ankle joint and Pilon fracture surgery is related to multiple factors,corresponding prevention and control measures should be taken to reduce the occurrence of SSI.

Objective To evaluate retrospectively the causative factors and corresponding surgical treatments of cervical spinal cord injuries without fracture or dislocation. Methods 29 cases of cervical spinal cord injury without fracture or dislocation were studied to analyze their imaging manifestation, features and causative factors of the injury, and pathological elements. Different surgical operations were performed accordingly, and their short-term outcomes were evaluated. Results The analysis indicated that there were chiefly 3 groups of causative factors. About 24% of the patients were typical cervical disc protrusion or prolapse, about 52% of them suffered from obvious decrease or even disappearance of the storing space of spinal canal, and about 24% suffered from decrease of the storing space of spinal canal combined with segmental instability among the vertebras, or with cervical disc prolapse. The postoperative short follow-ups (a mean period of 8.5 months) for the 29 surgical treatments showed that their spinal cord functions had been improved significantly, with the recovery rate being 52.8% by Japanese Orthopaedic Association (JOA) scoring. Conclusions Despite some common features, the causative factors for the cervical spinal cord injury without fracture or dislocation are quite different. Consequently, in order to achieve satisfactory outcomes, corresponding surgical operations with different approaches should be performed, and proper internal fixation should be applied according to the specific indications.

Objective To explore the effects of walking in water on surface electromyography (sEMG) of lower limbs and neural func-tion in spinal cord injury (SCI) patients. Methods From June, 2014 to May, 2016, 42 patients with incomplete thoracolumbar SCI were di-vided into control group (n=21) and experimental group (n=21) randomly. All the patients accepted routine rehabilitation, while the experi-mental group accepted extra walking in water. Average electromyography (AEMG) of quadriceps femoris and tibialis anterior muscles was measured, and the motor and sense function was assessed with American Spinal Injury Association (ASIA) Classification before and six weeks after treatment. Results The AEMG (t>5.763, P<0.01) and the scores of motor and sense (t>10.871, P<0.001) increased in both groups after treatment, and increased more in the experimental group than in the control group (t>4.036, P<0.01). Conclusion Walking in water could further improve lower limbs muscle strength and neural function in patients with incomplete thoracolumbar SCI.

Objective To study the effects of polysaccharide from Ecklonia kurome on pulmonary interstitial fibrosis induced by bleomycin in rats.Methods The pulmonary interstitial fibrosis model was established by endotracheal injection of bleomycin in rats.25,50,100mg?mL-1 doses of the polysaccharide from Ecklonia kurome were ig administered to the rats respectively,and the contants of hydroxyproline in rat lung were determined by kits at 0,3,7,14,28 d after the model was made.Results At 7,28d after the model was made,the treatment groups of middle and high doses compared with the model group showed signi-ficantly decrease of hydroxyproline level(P

Background Heparanase degrade heparan sulfate side chains of heparan sulfate proteoglycans in the extracellular matrix.Heparanase induces angiogenesis and likely promotes the vascularization of tumor.ObjectiveThe present study is to investigate the expression of heparanase and perlecan in retinas with oxygen-induced retinopathy.Methods Sixty-five clean neonatal C57BL/6J mice were raised in a hyperbaric oxygen box with a volume percentage of 75%±2% for 5 days and then returned to the normal air room.Another 65 matched mice were raised in the normal environment as controls.Evans blue was infused by the superior vena cava in all the mice on postnatal days 12,13,17,21 and 30,afterwards fluorescein angiography was performed and then the mice were sacrificed.The retinas of mice were isolated and prepared and the retinal vessels were examined under a fluorescent microscope and optical microscope.Heranase and perlecan mRNA was detected using reverse transcription PCR (RT-PCR).Heranase and perlecan proteins were detected by Western blot.The analysis of variance was used to compare the mRNA and the protein levels of heranase and perlecan between the experimental and control groups.Results The expression of heparanase mRNA in the retinas of different ages of mice and the different groups showed significant differences (F_(group)=16.303,P=0.000;F_(time)=18.614,P=0.000;F_(interaction)=11.299,P=0.000),and the expression of heparanase mRNA was significantly enhanced in mice from postnatal days 12,13,17 and 21 compared with normal control mice (P=0.001,0.000,0.000,0.001,respectively).The expression of heparanase protein in the retinas of different ages of mice and the different groups followed the same tendency(F_(group)=458.134,P=0.000;F_(time)=78.466,P=0.000;F_(interaction)=71.398,P=0.000).The expression of perlecan mRNA in the retinas of different ages of mice and the different groups showed significant differences (F_(group)=7.351,P=0.013;F_(time)=9.098,P=0.000;F_(interaction)=3.349,P=0.000),and increase in differences also were clearly seen in mice from postnatal days 13,17 and 21 compared with normal control mice (P=0.048,0.000,0.003,respectively).Conclusion The expression of heparanase and perlecan is associated with the development and progression of retinal neovascularization,and perlecan and heparanase together produce a synergistic effect.Heparanase and perlecan may participate in the angiogenesis of oxygen-induced retinopathy.

Objective:To investigate the regulation of proliferation and differentiation of bone marrow mesenchymal stem cells(BM-SCs)by CKIP-1 in vitro.Methods:BMSCs from CKIP-1 nock out(KO)and wild type(WT)C57 mice were isolated and cultured u-sing adherence method in vitro.BMSCs of the 3rd passage were induced to osteogenic and adipgenic differentiation.Cell proliferation was examined by MTT assay.Cell surface markers were tested by FCM.The osteogenic and adipogenic differentiation was studied by alkaline phosphatase (ALP)staining,alizarin red staining and oil red O staining.Results:The proliferation and cell marke expression of the 2 groups were similar.ALP staining of KO group was strong than that of WT group after osteogenic induction.Alizarin red stai-ning showed that there were more mineralized nodules in BMSCs of KO group than in those of WT group.Oil red O staining of KO mice BMSCs was stronger than that of WT.Conclusion:CKIP-1 deficiency can enhance the osteogenic and adipogenic differentiation without influence on the proliferation of BMSCs.

Objective To investigate the association between endothelial dysfunction and arterial stiffness in continuous ambulatory peritoneal dialysis (CAPD) patients.Methods Ninetyfour stable CAPD patients from a single center were enrolled in this cross-sectional study.Ultrasound evaluation was conducted on brachial artery to estimate endothelial-dependent flow-mediated dilation (FMD).Automatice pulse wave velocity (PWV) measuring system was applied to examine the carotidfemoral PWV.Blood pressure and biochemical parameters were detected.Pearson's correlation and Stepwise multiple regression analysis were performed to explore the relationship between FMD and PWV.Results PWV was significantly higher in patients with diabetes as compared to those without diabetes[(13.25± 1.66) m/s vs (11.24±1.92) m/s,P ＜ 0.01].Furthermore,PWV was positively correlated with age(r=0.319,P=0.002),SBP (r=0.289,P=0.005) and C-reactive protein (r=0.211,P=0.041),was negatively correlated with albumin (r =-0.429,P =0.001) and FMD (r=-0.466,P=0.001).In multivariate regression analysis,diabetes mellitus,albumin,FMD,age and SBP were independently associated with PWV after adjustment.Conclusion Endothelial dysfunction is associated with greater arterial stiffness in CAPD patients.

Objective To study the way in showing collateral ligaments of knee by MRI and MRI findings of the normal collateral ligaments.Methods MR imaging examinations were performed in 55 healthy volunteers . MR imaging included T1WI and PDWI sequence on the coronal, sagittal and coronal posterior oblique plane, in order to observe the collateral ligaments, and to measure the medial collateral ligament and the lateral collateral ligament dimension, which were compared with the results of the cadaver published in the related anatomic literatures.Results (1)On sagittal MRI, the angle between the line of medial collateral ligament and the tibia long axis was 0.55°±0.25°,the angle between the line of lateral collateral ligament and the long axis of the fibular neck was 11.47°±1.61)°.(2)On coronal MRI, 96% of the medial collateral ligaments was completely showed on only one slice, at the same time, 82% of the lateral collateral ligaments was completely showed on only one slice .On 11°coronal posterior oblique plane,90% of the lateral collateral ligaments was completely showed on only one slice .(3)The normal medial and lateral collateral ligaments were hypointense string on both T1WI and PDWI coronal MR images , with an average length of 11.53 cm for the medial collateral ligament and 5.31 cm for the lateral collateral ligament.Conclusion Using sagittal plane completely shown the tibia and fibular neck as the standard plane and thickness of 3mm in examining the collateral ligaments of knee by MR imaging can display the medial and the lateral collateral ligament clearly .