BACKGROUND:Due to the much higher requirement of biocompatibility and anticoagulant of smal-diameter vascular grafts than those of large-diameter ones, in situ blood vessel regeneration occurs as a new research direction. OBJECTIVE:To summarize the recent research development of electrospun smal-diameter scaffolds and to explore the application of in situ blood vessel regeneration and the development tendency. METHODS:The first author retrieved China National Knowledge Infrastructure database, Wanfang data and ISI Web of Knowledge foreign database to retrieve literatures addressing the fabrication of electrospun smal-diameter nanofibrous vascular grafts, surface modification and mimicking extracel ular matrix, as wel as the evaluation of biocompatibility and security after grafting. RESULTS AND CONCLUSION:Electrospun smal-diameter nanofibrous vascular grafts have emerged as promising candidates in vascular tissue engineering. By using both natural and synthetic polymers, the scaffolds can achieve a good balance between mechanical property and biocompatibility. Meanwhile, the fabrication of multi-layered vascular scaffolds, functional surface modification and mimicking extracel ular matrix structural y and functional y are now becoming attractive research directions. However, at current stage, electrospun vascular scaffolds used clinical y are basical y formed by synthetic materials, which have limited biocompatibility and anticoagulant activity. In this case, more efforts should be paid to find an optimal ratio between natural and synthetic materials for the improvement of biocompatibility and anticoagulant ability of smal-diameter vascular grafts.

BACKGROUND:Thrombogenesis is the most common cause of failure in the implantation of tissue engineered small-caliber vesselgrafts.And immobilizing heparin onto the surfaces of vascular scaffoidgrafts is often applied to improve their blood compatibility and patency.OBJECTIVES:To investigate the small intestinal submucosa(SIS)surface after heparinizadon with hypothermic plasma technique,to ireprove the blood compatibility of SIS and to explore the possibility for the construction of small-caliber vascular grafts with modified SIS seaffolds in vivo.DESIGN:Single exponent study.SETTING:Department of Orthopaedics,the Six People's Hospital Affiliated to Shanghai Jiao Tong University;ShanghaiInstitute for Microsurgery of Extremities.MATERIALS:This study was performed in the Shahghai Institute for Microsurgery of Extremities from January to October 2006.The jejuna were taken from farm pigs.METHODS:The SIS surface of pigswere processed by argon plasma(20mL/min)technique at different time periods(0,2,4,6,8,10,12,and 14 s),which were then immediately immerged in heparin sodium solution for 24 hours.Dogs were divided into two groups.The SIS films were sewn into a 3-mm diameter tube and implanted into the defect of a canine femoral by anastomosis as a vascular graft.The observation lasted for 6 weeks.MAIN OUTCOME MEASURES:The surface morphologies of SIS were observed under scanning electron microscope(SEM).The antithrombogenicity of SIS films was tested by water contact angle,blood coagulation time and platelet adherence observation by SEM.The efficiency of the SIS graft was evaluated by the patency in the circulation of blood with colour doppler detection and histology.RESULTS:Heparinized SIS showed great different surface morphology comparing with untreated SIS.Untreated SIS surface looked like wrinkled film,but on heparinized SIS surface spread with uniform micro-dots,which looked like a layer of heparin adhesion.Water contact angle decreased with the increase of plasma irradiation time.Prothrombin time (PT),partial thromboplastin time(APTT),and thrombin time(TT)of the SIS films modified with heparin were prolonged.Platelets adhered much more on untreated SIS film than on heparinized SIS film.Vascular graft from SIS embolized in the lumina completely at day 3 after anastomosis.Heparinized SIS graft kept patency for six weeks,and the inner surface of graft was covered with full endothelial cells.CONCLUSION:Hydrophilicity and antithrombogenicity of heparinized SIS are increased obviously after hypothermia plasma treatment.

The poly ester-amide (PEA) nanofiber membrane was prepared by electrospinning and the surface topography of nanofiber membrane was observed by scanning electron microscope (SEM). It was found that with the increasing of spinning fluid concentration from 10% to 20%, the diameter of prepared fibers was increased from 180 nm to 305 nm. The chemical structure of PEA was detected by infrared spectrum, which showed that electrospinning had no significant effect on PEA. X-ray diffraction spectrogram and differential scanning calorimetry revealed that the crystallinity of PEA was decreased after electrospinning. Mechanical property test demonstrated that the mean breaking strength and elongation at break of PEA nanofiber membrane with (0.50±0.05) mm thickness were (1.00±0.18) MPa, and (18.20±2.86)%, respectively. MTT results showed that the endothelial cells proliferated actively on the PEA nanofiber membrane, and presented good adhesive and growth status.

ObjectiveTo investigate the effect of artesunate on the expression of vascular endothlial growth factors(VEGF)and VEGFR in SHI-1 cell line.MethodsEnzyme-linked immunosorbent assay analysis was performed to detect the amount of VEGF in culture supernatants of SHI-1 cell in the condition of artesunate or not. The expression of VEGFR1 and VEGFR2 in SHI-1 cell in the condition of artesunate or not were detected by flow cytometry.ResultsWithout artesunate,the concentration of VEGF in the culture supernatant of SHI-1 cell were (980.3±2.2) pg/ml in 24 h and (982.4±2.3) pg/ml in 48 h. The expression of VEGFRI in SHI-1 cell were (6.40±3.11) ％ in 24 h and (6.45±2.85) ％ in 48 h. The expression of VEGFR2 in SHI-1 cell were (13.90±2.26) ％ in 24 h and (13.95±1.96) ％ in 48 h. With artesunate at 5, 10, 20 ng/ml, the concentration of VEGF in culture supematant of SHI-1 cell were (234.6±1.8)pg/ml, (114.9±1.6)pg/ml, (108.8±1.5) pg/ml in 24 h and (62.3±1.7) pg/ml, (60.9±1.6) pg/ml, (32.7±1.7) pg/ml in 48 h, respectively. The levels of VEGF in SHI-1 cells treated with artesunate at different concentrations decreased significantly (P ＜0.05).There was significant difference between 24 hours group and 48 hours group(P ＜0.05).The expression of VEGFR1 in SHI-1 cell were (4.30±2.21) ％, (4.20±1.37) ％, (3.90±1.86) ％ in 24 h and (3.80±2.87) ％, (3.60±1.73) ％, (3.00±1.82) ％ in 48 h, respectively. The expression of VEGFR1 in SHI-1 cell treated with artesunate at different concentrations were not significantly different (P ＞0.05). No significant difference between 24 hours group and 48 hours group was observed (P ＞0.05). VEGFR2 expression of SHI-1 cell were(4.40±1.15) ％, (3.10±0.68) ％, (1.10±0.72) ％ in 24 h and (3.00±1.68) ％, (2.20±0.93) ％, (0.60±0.92) ％ in 48 h, respectively. The results indicated that the expression of VEGFR2 in SHI-1 cells treated with artesunate at different concentrations reduced significantly (P ＜0.05),but there was no significant difference between 24 h group and 48 h group (P ＞0.05). ConclusionThe concentration of VEGF in SHI-1 cell was high, and artesunate can down-regulate the expression of VEGF and VEGFR2,but the effect of artesunate on the VEGFR1 was not significant.

BACKGROUND:The electrospinning technique has been used to prepare biological scaffolds to simulate nano-fiber structure of extracelular matrix; therefore, widespread attention has been paid to the electrospinning technique in the field of regenerative medicine and tissue engineering. OBJECTIVE: To review the articles about increasing electrospun nanofiber scaffold porosity, enlarging pore diameter, promoting cel infiltration with related technologies, in order to discover the most practical and economical technology. METHODS:The first author retrieved CNKI database, Wanfang database and PubMed with the keywords of “cel infiltration, 3D scaffold, electrospinning” in Chinese and English, respectively. Literature retrieval period was from January 2004 to October 2014. RESULTS AND CONCLUSION:Electrospinning technology is the most effective method for preparation of nanofiber scaffolds. Electrospinning scaffolds as tissue engineering scaffolds have become an issue of concern in the basic research year by year. However, the internal nano-scale pore of nanofiber scaffolds limits the cels to grow on the surface, so recent research has been focused on highly porous three-dimensional structure which can promote the permeable growth of cels instead of two-dimensional scaffolds. Several techniques have been used, which go from the adjustment of materials and speed of electrospinning to the applications of various kinds of complicated machines. However, the existing researches are stil not mature and stable, the majority of which are applied onlyin vitro as cel implantation or subcutaneous implantation in smal animals. The above-mentioned methods stil need long-term comparative studies to confirm the feasibility in the tissue-engineered repair of organs.

Objective:To investigate the sensitivity and the specificity of scorpions amplification refractory mutation system ( ARMS) in comparing with that of direct DNA sequencing in the detection of BRAF gene mutations in patients with papillary thyroid microcarcinoma.Methods:Direct sequencing and ARMS were used simultaneously to detect BRAF mutation status in 56 patients with PTMC.Results:BRAF mutations were identified in 46 cases with a mutation rate of 82.9%by ARMS,while in 18 cases with a mutation rate of 32.1%by direct sequencing.Besides,the sensitivity of ARMS was 100%and that of direct sequencing was 39.1%.There were significant differences of both mutation rate and sensitivity between two methods ( P<0.01 ).Conclusion: Compared to direct sequencing,ARMS gains a higher sensitivity in the detection of BRAF mutations in samples with tiny lesions.

Biologic scaffold materials composed of extracellular matrix (ECM) are typically obtained in processes that involve decellularization of tissues or organs. Decellularized tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the structure and potential loss of composition. The efficiency of cell removal from a tissue is dependent on the origin of the tissue and the physical, chemical, and enzymatic methods that are used. Each of these treatments affects the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining ECM scaffold, and all of the treatment methods affect the host response to the material as well. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making correct decisions regarding the agents and techniques utilized during processing. In this paper, the most commonly used decellularization methods are described, and consideration given to the effects of these methods upon the biologic scaffold material and recently described antigen removal strategy are presented.
Acellular Dermis ; Animals ; Cell Death ; drug effects ; Cell Separation ; Extracellular Matrix ; chemistry ; Humans ; Tissue Engineering ; methods ; Tissue Scaffolds

Objective To establish a mouse model of spleen deficiency and dampness stagnation syndrome combining atopic dermatitis(AD)and explore the feasibility of modeling by comparing 2,4-dinitrochlorobenzene(DNCB)-induced atopic dermatitis model of mouse,"external dampness+improper diet+irrigation of senna"-induced spleen deficiency and dampness stagnation syndrome model of mouse,as well as both in combination of model mouse.Methods The construction of a mouse(Balb/c)with spleen deficiency and dampness stagnation syndrome was explored by using the method of"external dampness+improper diet+irrigation of senna",and then DNCB was applied to induce the AD-like lesions in Balb/c mice to establish a mouse model of spleen deficiency and dampness stagnation syndrome combining atopic dermatitis.The general condition and body weight of mice in each group were observed,and the symptoms of spleen deficiency and dampness were scored.The severity of AD was evaluated by comparing the skin lesion degree,EASI score,transcutaneous water loss value(TEWL),spleen index and thymus index.The levels of creatinine,glucose,total cholesterol,triglyceride,gastrin,and amylase were measured.Results(1)During the modeling period of spleen deficiency and dampness stagnation syndrome,compared with the normal group,spleen deficiency and dampness stagnation syndrome group,spleen deficiency and dampness stagnation syndrome combined with atopic dermatitis group showed obesity,listlessness,filthy and greasy hair,diarrhea,and poor cleanliness around the anal.After combining with the application of the atopic dermatitis model,the body weight of the mice in atopic dermatitis group(P＜0.001),spleen deficiency and dampness stagnation syndrome group(P＜0.05)and spleen deficiency and dampness stagnation syndrome combined with atopic dermatitis group(P＜0.001)decreased sharply compared with the normal group.(2)Compared with the atopic dermatitis group,the degree of skin lesions,EASI score(P＜0.05)and TEWL(P＞0.05)were higher in the spleen deficiency and dampness stagnation syndrome combined with atopic dermatitis group.(3)Compared with the normal group,the spleen index of the atopic dermatitis group increased(P＜0.001)and the thymus index decreased(P＜0.001).Compared with the atopic dermatitis group,the spleen index(P＞0.05)and thymus index(P＜0.05)of the spleen deficiency and dampness stagnation syndrome combined with atopic dermatitis group decreased.(4)The results of serum biochemical indexes showed that compared with the normal group,the levels of creatinine(P＜0.01),glucose(P＜0.001),total cholesterol(P＞0.05),triglyceride(P＞0.05)and gastrin(P＜0.001)in the spleen deficiency and dampness stagnation syndrome group were increased,and the level of amylase was decreased(P＜0.01).Compared with the atopic dermatitis group,the levels of creatinine(P＞0.05),glucose(P＜0.05),total cholesterol(P＞0.05),triglyceride(P＞0.05),gastrin(P＜0.001)increased and the level of amylase decreased(P＞0.05).Conclusion A mouse model of spleen deficiency and dampness stagnation syndrome combining atopic dermatitis,which was induced by the combination of DNCB and"external dampness+improper diet+irrigation of senna",can not only show obvious TCM indications of spleen deficiency and dampness syndrome,but also show the characteristics of AD.This model can be used as a reliable animal model of combination of disease and syndrome.It provides reference for further study on pathological mechanism,pharmacodynamic evaluation and pharmacological mechanism of spleen deficiency and dampness stagnation syndrome combining atopic dermatitis.

Objective:To evaluate the effect of danshensu on proliferation and apoptosis of M5-stimulated HaCaT cells (a psoriasis-like cell model) , and to explore its relationship with Yes-associated protein (YAP) expression.Methods:HaCaT cells were stimulated with M5, a mixture containing 10 μg/L interleukin (IL) -1α, IL-17, IL-22, tumor necrosis factor (TNF) -α and oncostatin M, for 48 hours to establish a psoriasis-like cell model. Then, the cell model was divided into several groups to be treated with 0 (control group) , 0.125, 0.25 and 0.5 mmol/L danshensu respectively, and HaCaT cells receiving no treatment served as the blank control group. Real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of YAP respectively in these groups; methyl thiazolyl tetrazolium (MTT) assay was conducted to estimate the cellular proliferative activity after 24-, 48- and 72-hour treatment with danshensu, flow cytometry to evaluate the effect of danshensu on cell cycle and apoptosis, and Western blot analysis to determine expression of cell cycle-related proteins (cyclin A, cyclin B1, cyclin D, cyclin E) and apoptosis-related proteins (cleaved caspase-3, Bcl-2, BAX, p53 and p21) . One-way analysis of variance was used for comparing means in several groups, and least significant difference (LSD) - t test for multiple comparisons. Results:The mRNA and protein expression of YAP significantly differed among the blank control group, control group, 0.125-, 0.25- and 0.5-mmol/L danshensu groups (both P < 0.001) , so did the cellular proliferative activity at 24, 48 and 72 hours (all P < 0.001) . The 0.125-, 0.25- and 0.5-mmol/L danshensu groups all showed significantly decreased mRNA and protein expression of YAP (mRNA: 1.76 ± 0.04, 1.54 ± 0.05, 1.33 ± 0.05 respectively; protein: 1.78 ± 0.06, 1.49 ± 0.32, 1.27 ± 0.04 respectively) , and cellular proliferative activity at 48 hours (1.66 ± 0.04, 1.52 ± 0.02, 1.34 ± 0.04 respectively) compared with the control group (mRNA: 2.04 ± 0.04; protein: 2.10 ± 0.06; cellular proliferative activity: 1.82 ± 0.03; all P < 0.05) . Flow cytometry showed significant differences in the proportions of cells at G0/G1, S and G2/M phases as well as in the apoptosis rates among the above 5 groups (all P < 0.001) . Compared with the control group, the 0.125-, 0.25- and 0.5-mmol/L danshensu groups showed significantly higher proportions of cells at G0/G1 and G2/M phases, but lower proportions of cells at S phase (all P < 0.05) . Additionally, the apoptosis rates were significantly higher in the 0.25- and 0.5-mmol/L danshensu groups than in the control group (both P < 0.05) . Western blot analysis revealed significant differences in the expression of cell cycle-related proteins and apoptosis-related proteins among the above 5 groups (all P < 0.001) . Conclusion:Danshensu can inhibit the proliferation of the psoriasis-like cell model and promote its apoptosis, likely by suppressing YAP expression.

BACKGROUND:Currently,electrospun nanofibers,which are biomimetic materials of natural extracellular matrix and contain a three-dimensional network of interconnected pores,have been successfully used as scaffolds for various tissue regeneration,but are still faced with the challenge of extending the biomaterials into three-dimensional structures to reproduce the physiological,chemical as well as mechanical properties of the tissue microenvironment. OBJECTIVE:To summarize the process and principles of electrostatic spinning and to explore the applications of the resulting electrospun nanofibers in tissue regeneration of skin,blood vessels,nerves,bone,cartilage and tendons/ligaments. METHODS:With"electrospinning,electrospun nanofibers,electrospun nanofiber scaffolds,tissue regeneration"as the Chinese and English search terms,Google Academic Database,PubMed,and CNKI were searched,and finally 88 articles were included for review. RESULTS AND CONCLUSION:(1)The electrospun nanofibers are a natural fibrous extracellular matrix mimetic material and contain a three-dimensional network of interconnected pores that have been successfully used as scaffolds for a variety of tissue regeneration applications.(2)Several papers have described the great potential of electrospun nanofiber scaffolds applied to the regeneration of skin,blood vessels,nerves,bones,cartilage and tendons/ligaments,providing a solid theoretical basis for its final application in clinical disease treatment,or for its transformation into practical products to enter the market.(3)However,the current research results are mostly based on cell experimental research results in vitro,and whether it can be finally applied to human body still needs clinical verification.(4)At present,many kinds of electrospun products for various clinical needs have been commercialized in and outside China,indicating that the research field of electrospun nanofiber scaffolds for soft and hard tissue regeneration has great research value and application potential.