Silk fibroin/chitosan/heparin scaffold: preparation,antithrombogenicity and culture with hepatocytes

Antithrombogenicity is very important for tissue engineering scaffolds used in situations involving contact with blood. A silk fibroin/chitosan (SFCS) scaffold has been developed for liver tissue engineering with porous structure, suitable mechanical properties and biocompatibility. Because the interaction between silk fibroin and blood coagulation factors can lead to blood coagulation, the anticoagulant property of the SFCS scaffold should be improved. Heparin was added into SFCS scaffold under mild conditions. The effects of heparin on the morphology, swelling properties, structure, porosity, mechanical properties, antithrombogenicity and cytocompatibility of the SFCS scaffold were studied. SFCS scaffold containing heparin maintains the porous structure and good mechanical properties of the fibroin‐based scaffold; moreover, it is not cytotoxic. Addition of heparin leads to the SFCS scaffold being blood‐compatible and an effective heparin‐delivering system. The anticoagulant property of the SFCS scaffold can be improved by the addition of heparin, which may be helpful for scaffolds used in situations involving contact with blood. Copyright © 2009 Society of Chemical Industry     

丝素膜上接枝褐藻多糖硫酸酯及其体外抗凝血性能的研究

以戊二醛为交联剂,将褐藻多糖硫酸酯接枝在NH3等离子体处理的丝素膜的表面上。主要探讨了pH、反应温度、反应时间对丝素膜接枝效果的影响。用XPS分析了丝素膜表面化学元素。采用部分凝血活酶时间(APTT)、凝血活酶时间(PT)、凝血酶时间(TT)评价了接枝膜的体外抗凝血活性。得出最佳接枝工艺为:pH=2,反应温度40℃,反应时间6h。在该工艺条件下,接枝丝素膜APTT的最佳值为72s,APTT、TT凝血时间比空白对照分别延长37s和6s以上,具有抗凝血性能。经稳定性测试,固定的褐藻多糖硫酸酯基本上不脱落,从而获得了一种具有抗凝血性的新颖生物材料。     

Surface modification of polyethylene by plasma pretreatment and UV‐induced graft polymerization for improvement of antithrombogenicity

The purpose of this study was to enhance blood compatibility of polyethylene (PE) films. Glycidyl methacrylate (GMA) was grafted onto the surface of PE by Ar plasma pretreatment and UV‐induced graft polymerization without photo‐initiator, then heparin was immobilized onto the poly (glycidyl methacrylate) segments. The surface compositions and microstructure of GMA graft polymerized PE films were studied by X‐ray photoelectron spectroscopy (XPS) and Attenuated Total Reflectance Fourier Transfer Infrared (ATR‐FTIR) spectroscopy. It was confirmed that heparin was successfully immobilized onto the surface of PE films by XPS analysis. The antithrombogenicity of the samples was determined by the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and plasma recalcification time (PRT) tests and platelet adhesion experiment. Results indicated that the antithrombogenicity of modified PE was improved remarkably. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2014–2018, 2004     

Surface modification of polypropylene film by radiation‐induced grafting and its blood compatibility

To endow blood‐compatible properties onto polypropylene (PP) film, we grafted 2,3‐epoxypropyl methacrylate (EPMA) to PP film with a preirradiation grafting technique and then introduced various functional groups onto the grafted PP film. The EPMA grafting extent was dependent on the absorbed dose, reaction time, and temperature. The reactions of hydroxylation, iminodiacetation, sulfonation, phosphonation, and amination were performed under various conditions to introduce functional groups into the epoxy group of EPMA‐grafted PP films, respectively. We also immobilized heparin on aminated PP film to compare blood compatibility with various functionalized samples. The grafting, functionalization, and heparinization reaction were confirmed by Fourier transform infrared spectroscopy in the attenuated total reflectance mode and electron spectroscopy for chemical analysis. The blood compatibility of various functional groups and heparin‐introduced samples as well as control samples was examined by the determination of platelet adsorption and thrombus formation. For the examination of the blood compatibility of functionalized PP samples, acid citrate dextrose human whole blood and platelet‐rich plasma were used. The amount of the formed thrombus and the adherent platelets on functionalized PP sample surfaces were evaluated by an in vitro method following Imai and Nose's technique and by scanning electron microscopy, respectively. The blood compatibility of various functional‐group‐introduced PP films after grafting was better than that of the PP control. Phosphoric‐acid‐group‐ and heparin‐introduced PP films had especially good blood compatibility. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1726–1736, 2003     

Composition optimization of polyvinyl alcohol/rice starch/silk fibroin‐blended films for improving its eco‐friendly packaging properties

Biopolymers obtained from renewable sources have been exploited in developing biomaterials with eco‐friendly properties. Most biopolymers have some limitations because of their poor mechanical properties, high water solubility, or low transparencies. In this study, some biopolymers, that is, silk fibroin (SF) and rice starch (RS) were used as starting materials together with polyvinyl alcohol (PVA) as a film‐former for preparing novel eco‐friendly films. The film preparations were done by solution casting with two different sequences of blending and the film compositions were optimized. Results from UV, SEM, and film properties testing on mechanical properties, degree of swelling, water solubility, and also oxygen permeability indicated that all film properties depended on their compositions and order of blending. The PVA/RS film obtained is transparent with good mechanical properties and low water solubility. The addition of SF increases the permeability of oxygen and also the degradability of the films. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Physical properties of silk fibroin/chitosan blend films

Silk fibroin/chitosan blend films were examined through IR spectroscopy to determine the conformational changes of silk fibroin. The effects of the fibroin/chitosan blend ratios (chitosan content) on the physical and mechanical properties were investigated to discover the feasibility of using these films as biomedical materials such as artificial skin and wound dressing. The mechanical properties of the blend films containing 10–40% chitosan were found to be excellent. The tensile strength, breaking elongation, and Young's modulus were affected by the chitosan contents of the blend films, which were also related to the density and degree of swelling. The coefficient of water vapor permeability of the blend films increased linearly with the chitosan content, and the values of 1000–2000 g m^?2 day^?1 were comparable to those of commercial wound dressings. Silk fibroin/chitosan blend films had good oxygen and water vapor permeabilities, making them useful as biomaterials. In particular, the blend film containing 40–50% chitosan showed very high oxygen permeability. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 928–934, 2001     

Preparation of insoluble fibroin/collagen films without methanol treatment and the increase of its flexibility and cytocompatibility

It is still a critical challenge to increase the flexibility of regenerated fibroin materials in dry and near dry states. In this study, a novel biocompatible and water‐stable film composed of fibroin and collagen was successfully prepared from aqueous fibroin solution without methanol treatment. The result of contact angle measurement indicates that hydrophilicity is evidently increased when collagen was added to fibroin film. The elongation at break in wet state is also increased because of the blending of collagen, which implied the improvement of flexibility. More importantly, the blend films containing 20% collagen become flexible when placed at above 65% humidity in atmosphere. It means that the blend films could be fabricated to different conformations easily through adjusting humidity in atmosphere. HepG2 cells were cultured on fibroin and fibroin/collagen films to investigate the cytocompatibility of these films. Scanning electron microscopy and MTT analysis demonstrated that the adding of collagen evidently improved HepG2 proliferation in over 10 days culture. The excellent cytocompatibility, the flexibility in the near dry state as well as the green preparation process of fibroin/collagen blend films make them become the promising biomaterials for different medical applications. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Improved hemocompatibility of silk fibroin fabric using layer‐by‐layer polyelectrolyte deposition and heparin immobilization

Silk fibroin has long been used as implantable surgical sutures and it has acceptable mechanical properties and patency rates in animal models and in clinical end‐uses. However, fibroin has been shown to be hemolytic and can cause damage to red blood cells. So to be used as an implantable vascular prosthesis its hemocompatibility needs to be improved. This study has taken two sequential steps to address this problem. First, to create a positively charged layer on the fibroin fibers' surface, a 1.5 and 2.5 bilayers polyelectrolyte surface deposition layer‐by‐layer technique was used with the positive counterion poly(allylamine hydrochloride) and the negative counterion poly(acrylic acid). Second, negatively charged low molecular weight heparin was then immobilized on these positively charged self‐assembled surfaces. The presence of the heparin was confirmed with Alcian Blue staining and a toluidine blue assay, and the increased roughness and hydrophilicity of the modified surfaces were characterized by scanning electron microscopy, contact angle measurements, and atomic force microscopy. In addition, a negligible hemolytic effect, reduced protein adsorption, and a higher concentration of free hemoglobin measured by a kinetic clotting time test were found to be enhanced with the use of 2.5 bilayers compared to the 1.5 bilayers self‐assembly technique. Given the success of these preliminary results, it is anticipated that this novel approach of surface modification and heparin immobilization will demonstrate long‐term patency during future animal trials of small caliber silk fibroin vascular grafts. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40772.     

Preparation of insoluble fibroin films without methanol treatment

Insoluble fibroin films were prepared without methanol treatment. A 15 wt % fibroin solution was obtained through concentration, and then, the insoluble film was achieved by the adjustment of the drying temperature and rate. These films were examined through Fourier transform infrared–attenuated total reflection, X‐ray diffraction, and scanning electron microscopy to determine the structure of the silk fibroin. The physical and mechanical properties were investigated to discover the feasibility of using these films as biomedical materials. When fibroin films were dried above 60°C, abundant β‐sheet crystals existed in the fibroin films, and many globule‐containing micelles aggregated and interacted with each other, which resulted in excellent mechanical properties of the regenerated fibroin films in the wet state. Interestingly, the amide III band of the random coil structure in the fibroin films dried at 70 and 80°C was shifted to a lower frequency, 1228 cm^?1, which meant that a partly orientated structure formed. This may have also affected the mechanical properties of the fibroin films. The tensile strength and breaking elongation of the films dried at 70°C were 29.8 MPa and 59.6%, which is distinctly superior to fibroin films treated with methanol. If the drying temperature was raised to 60°C, the stability of the films in water was also excellent. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 2168–2173, 2005     

Effects of addition of condensed tannin on the structure and properties of silk fibroin film

Condensed tannin is a polymeric polyphenol compound, which has considerable protein‐binding ability and a variety of bioactivities, such as anti‐inflammatory, antithrombotic and antimicrobial activities. In this study, silk fibroin film was modified by adding condensed tannin. It was found that strong intermolecular interactions occurred between silk fibroin and tannin, and incorporation of 1–10% (w/w) tannin in the blend films resulted in more compact, stiffer but less elastic films. Swelling and dissolution trials showed that the addition of tannin significantly reduced the swelling and solubility of the films. The mechanical properties, swelling and solubility of the blend films varied as a function of tannin concentration, showing optimum values when containing 10% (w/w) tannin. The silk fibroin–tannin films all showed significant antioxidant activity and antibacterial activity against Gram‐positive bacteria, and the activities were positively related to the concentration of tannin. The results emphasized that the addition of condensed tannin could simultaneously improve the structural stability of silk fibroin film and impart functional properties. © 2016 Society of Chemical Industry     

Glycerin and ethanol as additives on silk fibroin films: Insoluble and malleable films

Silk fibroin (SF) films have been largely studied as biomaterials due to their biocompatibility and biodegradability. Casting a SF aqueous solution at room temperature is a common technique to produce SF films at relative low cost and processing time; however, their brittleness and solubility in water make them unsuitable for certain biomedical applications. In this study, the incorporation of additives, ethanol and glycerin, are presented as an alternative to both improve mechanical properties of SF films and decrease their solubility in water. SF films with additives were further characterized using scanning electron microscopy, X‐ray diffraction, Fourier transformed infrared spectroscopy with attenuated total reflection, analysis of water solubility, mechanical test of traction, and in vitro cytotoxicity experiments. The results show that SF films containing additives are stable in water due to the effect of glycerin and ethanol, and do not require post‐treatments. Furthermore, great improvements on elongation of the films were achieved, mainly in the presence of both additives. In addition, all films were not toxic to cells, which is a first indication of their biocompatibility. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

肝素化聚乙烯醇性能研究

用聚乙烯醇(PVA)缩醛化方法,共价键结合肝素。用Schiff试剂染色法、红外光谱分析、X射线光电子能谱法(ESCA)、元素分析等测试方法证明醛基和肝素的存在。力学性能测定表明,肝素化聚乙烯醇的拉伸强度达到12 25MPa,断裂伸长率为400%。生物学指标说明,在全血凝固时间实验(CT)中,肝素化聚乙烯醇的抗凝时间达3h,在活性部分凝血时间实验(APTT)中,缩醛化聚乙烯醇共价键结合肝素非常牢固,没有肝素脱落到血液中,证明肝素化聚乙烯醇具有显著的抗凝血性。     

褐藻多糖硫酸酯/甲基丙烯酸甲酯接枝聚合及抗凝血性能研究

研究了Cu(Ⅱ)离子存在时,褐藻多糖硫酸酯与甲基丙烯酸甲酯(MMA)的接枝聚合反应。主要探讨了V(MMA)/V(H2O)、Cu(Ⅱ)浓度、反应时间、温度对接枝率的影响。采用FT-IR对接枝共聚物进行了表征。接枝共聚物的体外抗凝血活性采用部分凝血活酶时间(APTT)、凝血活酶时间(PT)、凝血酶时间(TT)来评价。结果表明,褐藻多糖硫酸酯的水溶液在Cu(Ⅱ)离子存在时,可以引发MMA的自由基聚合反应。当褐藻多糖硫酸酯的质量浓度为5 6g/L,V(MMA)/V(H2O)=0 20,Cu(Ⅱ)浓度为3 26mmol/L,反应温度85℃,反应时间为3h时,接枝率为197.0%。APTT、TT、PT实验证明,接枝产物的APTT、TT比空白对照物分别延长20s和5s以上,具有抗凝血性能。     

Production of chitosan PVA PCL hydrogels to bind heparin and induce angiogenesis

New blood vessel formation is an essential part of wound healing to provide cells with the nutrients and oxygen for their survival. Many nonhealing ulcers fail to heal because of poor blood supply and skin grafts will also fail to take on poorly vascularized wound beds. There is a real need for proangiogenic biomaterials to assist wound healing. In vivo heparin binds proangiogenic growth factors and helps regulate new blood vessel formation, hence heparin containing biomaterials are attractive. To achieve a hydrogel with high heparin binding capacity a composite of chitosan, poly(vinyl alcohol) (PVA) and polycaprolactone (PCL) was produced. Chitosan is a biodegradable natural polymer with great potential for biomedical applications due to its biocompatibility, high charge density and nontoxicity. PVA is biocompatible and nontoxic with good chemical stability, film-forming ability, and high hydrophilicity. PCL has physicochemical and mechanical properties comparable to those of the biological tissues and due its hydrophilic nature helps in the sustained release of drugs. Accordingly in this study we explored a range of PCL concentrations from 4% to 16% added to hydrogels composed of chitosan and PVA. Heparin was blended into the polymer mixture and the nanoporous structure was created by freeze-drying the PCL hydrogel. The physical properties of the hydrogels were evaluated by Fourier transform infrared spectroscopy (FTIR) and XPS confirmed the presence of sulfur on the surface of the hydrogels. Their porous morphology was investigated by scanning electron microscope (SEM). The Chick Chorionic Allantoic Membrane (CAM) assay was used to study the angiogenic potential of these materials and histology (H&E and Goldner trochome) was used to confirm the presence of new blood vessels inside the hydrogels. We report that the addition of 8% PCL to the hydrogels gave porous structures containing heparin, which significantly increased new blood vessel formation into the hydrogels. These hydrogels offer a new approach to biomaterials, which could be added to wounds to improve vascularization.     

Surface Modification and Blood Compatibility of Polyacrylonitrile Membrane with Immobilized Chitosan–Heparin Conjugate

Polyacrylonitrile (PAN) membranes were hydrolyzed with sodium hydroxide to introduce carboxyl groups, and then grafted with chitooligosaccharides (COS), followed by the immobilization of heparin (HEP). The properties of heparin and chitosan-immobilized PAN membranes were characterized with infrared spectroscopy. The surface densities of carboxyl groups, COS, and HEP on the PAN membranes were determined by the uptake of dye. Blood compatibility was evaluated by the activated partial thromboplastin time (APTT). The protein affinity was determined by human plasma total protein. The results showed that the amount of immobilization of COS and HEP increased with the density of carboxyl groups. The heparinized PAN membrane showed longer APTT and decreasing adsorption of plasma proteins. Less than 10% of heparin was released within 24 h, thus demonstrating the stability of the heparinization. These results demonstrated that the blood compatibility of PAN membranes could be improved by the immobilization of chitosan–heparin conjugate. The results obtained in this study will be helpful for the development of modified PAN for clinical applications.     

Chitosan‐finished Antheraea mylitta silk fibroin nonwoven composite films for wound dressing

In this study, we aimed to produce nonwoven wound‐dressing films made of Antheraea mylitta (tasar) silk fibroin by a solution‐casting method. These nonwoven films were finished with chitosan solutions of different concentrations ranging from 0.75 to 2% w/v with a pad–dry method to fabricate nonwoven composite films. Chitosan‐finished tasar fibroin nonwoven composite films (CMTFFs) showed higher mechanical and dynamic mechanical properties as compared to nonwoven tasar fibroin. The physical, structural, and thermal properties of the films were investigated. The hemocompatibility, cytocompatibility, and biodegradation tests showed that the CMTFF was a promising material for use as a wound dressing. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 44341.     

Rapid formation of flexible silk fibroin gel‐like films

Flexible silk fibroin gel‐like films with microporous morphology were prepared from B. mori silk fibroin fibers directly solubilized in formic acid/CaCl₂ solvent. These films were characterized by several analysis techniques to determine the structure and properties of films. The pore size of gel‐like films can be adjusted through SF concentration and Ca ions concentration. The controllable pore size in gel‐like films was grew from 3–5 μm to 100 μm under the increase of fibroin concentration from 1.0 wt % to 8.0 wt %. At the same time, the water content of silk fibroin gel‐like film decreased from 83.5 ± 3.4% to 68.2 ± 2.6%. With increasing Ca ions contents from 2.0 wt % to 10.0 wt % in dissolution process, the pore size and water content of silk fibroin gel‐like films grew larger, especially its water content values reached 86.2 ± 4.0% at 10.0 wt % Ca ions concentration. At wet condition, the gel‐like film with β‐sheet structure showed higher breaking stress (4.26 ± 0.31 MPa) and elongation (45.45 ± 15.79%) at 8.0 wt % concentration. With the preparation method, the membrane is hydrophilic and the pore size is adjustable, which contributes to high toughness and favorable cell growth environment, suggesting that these silk fibroin gel‐like films can be a potential candidate scaffold for biomedical applications, such as wound dressing, facial mask, contact lenses, etc. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41842.     

Chemical grafting of sulfobetaine onto poly(ether urethane) surface for improving blood compatibility

Poly(ether urethane)s (PEUs) are widely used as blood‐contact biomaterials because of their good biocompatibility and mechanical properties. Nevertheless, their blood compatibility is still not adequate for more demanding applications. Surface modification is an effective way to improve blood compatibility and retain bulk properties of biomaterials. The purpose of the present study was to design and synthesis a novel non‐thrombogenic biomaterial by modifying the surface of PEU with zwitterionic monomer. In this study, sulfobetaine was grafted onto PEU surface through the following reaction steps: (1) Poly(propylene oxide) (PPO) was reacted at both chain ends with hexamethylene diisocyanate (HDI), and OCN–PPO–NCO was obtained; (2) OCN–PPO–NCO was reacted at one chain end with N,N‐dimethylethanolamine (DMEA) and OCN–PPO–N(CH₃)₂ was formed; (3) the sulfobetaine was prepared by a ring‐opening reaction between OCN–PPO–N(CH₃)₂ and 1,3‐propanesultone (PSu); (4) the sulfobetaine was grafted onto PEU surface by the reaction between NCO and the N–H bonds of PEU. The surface composition of films and the hydrophilicity on the PEU surface were investigated by X‐ray photoelectron spectroscopy analysis and contact angle measurements, respectively. The blood compatibility of PEU was evaluated by platelet‐rich plasma contact experiments and the results were observed by scanning electron microscopy. The state of platelet adhesion and shape variation for the attached platelets was described. The modified surface showed excellent blood compatibility, featured by low platelet adhesion. Copyright © 2003 Society of Chemical Industry     

纤维素与透明质酸、肝素、丝素、甲壳素湿纺复合生物纤维的制备与性能研究

室温下,将再生纤维素与透明质酸(HA)、肝素(Hep)、丝素(SF)、甲壳素(N-ACS)等的复合纤维的纺丝液溶于NaOH溶液中,通过喷丝头(孔径0.1mm)喷入含有40%~43%硫酸铵的10%硫酸溶液中,得到产率为75%~98%的新型白色、柔韧性较好的复合生物纤维。FT-IR结果显示,复合纤维组分间存在物理吸附和氢键相互作用;少量的HA、Hep、SF和N-ACS等纤维的添加可增加复合纤维的韧能,其中纤维素-SF复合纤维在SF含量为10%时纤度为9.9旦,韧度值为1.08g/旦,伸长率为35.0%,机械性能相对最好。SEM图像中可以看出复合纤维表面呈现条纹、鳞状、填充和均匀包覆的结构,复合纤维间存在一定的相容性;复合纤维直径为19~55μm,密度为0.1~0.36旦/μm。     

玉米芯木聚糖硫酸酯的合成、表征及其抗凝血活性

以碱法提取的玉米芯木聚糖为原料,LiCl为催化剂,在非质子溶剂中,采用三氧化硫-吡啶法合成了玉米芯木聚糖硫酸酯（XS）,通过正交试验对合成工艺进行了优化,并采用凝胶渗透色谱（GPC）、红外光谱（FT-IR）、元素分析和¹³C NMR对其结构与性能进行了表征,进一步考察了其体外抗凝血活性。结果表明：在DMF溶剂中,当三氧化硫-吡啶络合物与木糖羟基单元的物质的量之比为1.5：1时,于55℃反应3 h,木聚糖的酯化效果最好,所制备的XS的取代度（D_S）为1.53,得率为78.2%,重均相对分子质量为36 754,分散系数为1.191。结构表征发现,硫酸酯基已经成功引入木聚糖中。体外抗凝血活性结果表明：XS可以延长APTT和TT,具有一定的抗凝血活性;当取代度为1.53、质量浓度为20 mg/L时,XS的APTT、PT和TT分别为36.37、14.22和14.70 s,与阳性对照肝素钠基本相当。