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The study aimed to evaluate the safety and function of poly(lactic-acid-co-ε-caprolactone) (PLCL)/fibrinogen nanofibers (P/F-Ns), and provide theoretical basis for the clinical application. The surface morphology, mechanical properties, the hydrophilicity and the fibrinogen content of P/F-Ns were tested by scanning electron microscope, the material testing machine, the contact angle meter and the microplate reader, respectively. The cell adhesion, proliferation and ligament remodeling genes expression of Hig-82 cells on P/F-Ns were conducted through cell counting kit-8 (CCK-8) and real-time quantitative PCR analyses, respectively. The results showed that with the increase of the fibrinogen content, the pore sizes and hydrophilicity of three P/F-Ns increased, but the mechanical properties decreased. Cell adhesion and proliferation tests showed that P/F-N-2 held the best ability to promote cell adhesion and proliferation. The ligament remodeling genes expressions of Hig-82 cells on P/F-N-1, P/F-N-2 and P/F-N-3 were all up-regulated compared to P/F-N-0 on days 3 and 7. All the three P/F-Ns containing fibrinogen (P/F-N-1, P/F-N-2 and P/F-N-3) had better biocompatibility compared to P/F-N-0, and could be efficiently applied to the reconstruction of anterior cruciate ligament.
Subject(s)
Anterior Cruciate Ligament Reconstruction , Cell Adhesion , Fibrinogen , Materials Testing , NanofibersABSTRACT
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.
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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.
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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.
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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.
Subject(s)
Animals , Humans , Acellular Dermis , Cell Death , Cell Separation , Extracellular Matrix , Chemistry , Tissue Engineering , Methods , Tissue ScaffoldsABSTRACT
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.
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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.