Preparation and characterization of Bombyx mori silk fibroin and wool keratin

Silk and wool are well‐known protein‐based fibers. Their environmental stability, biocompatibility, and unique mechanical properties provide an important basis for using these natural proteins in biomedical applications. To use them as biomaterials in the form of fibers, films, or membranes, it is necessary to characterize these proteins in their solution and solid states because structural characteristics and morphological features have a great influence on the physical and mechanical properties of these new regenerated protein forms. Therefore, in the present study, silk fibroin and wool keratin were dissolved and their solution behaviors and secondary structures are analyzed and compared, using particle size distribution, molecular weight distribution (SDS‐PAGE), Fourier transform infrared, and X‐ray diffraction techniques. It was shown that keratin is more stable in solution and more amorphous in the solid state. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100:4260–4264, 2006     

Characterization of a Human Platelet Lysate-Loaded Keratin Hydrogel for Wound Healing Applications In Vitro

One of the promising approaches to facilitate healing and regenerative capacity includes the application of growth-factor-loaded biomaterials. Human platelet lysate (hPL) derived from platelet-rich plasma through a freeze-thaw process has been used as a growth factor rich therapeutic in many regenerative applications. To provide sustained local delivery of the hPL-derived growth factors such as epidermal growth factor (EGF), the hPL can be loaded into biomaterials that do not degrade rapidly in vivo. Keratin (KSO), a strong filamentous protein found in human hair, when formulated as a hydrogel, is shown to sustain the release of drugs and promote wound healing. In the current study, we created a KSO biomaterial that spontaneously forms a hydrogel when rehydrated with hPL that is capable of controlled and sustained release of pro-regenerative molecules. Our study demonstrates that the release of hPL is controlled by changing the KSO hydrogel and hPL-loading concentrations, with hPL loading concentrations having a greater effect in changing release profiles. In addition, the 15% KSO concentration proved to form a stable hydrogel, and supported cell proliferation over 3 days without cytotoxic effects in vitro. The hPL-loaded keratin hydrogels show promise in potential applications for wound healing with the sustained release of pro-regenerative growth factors with easy tailoring of hydrogel properties.     

Tissue Adhesives in Reconstructive and Aesthetic Surgery—Application of Silk Fibroin-Based Biomaterials

Tissue adhesives have been successfully used in various kind of surgeries such as oral and maxillofacial surgery for some time. They serve as a substitute for suturing of tissues and shorten treatment time. Besides synthetic-based adhesives, a number of biological-based formulations are finding their way into research and clinical application. In natural adhesives, proteins play a crucial role, mediating adhesion and cohesion at the same time. Silk fibroin, as a natural biomaterial, represents an interesting alternative to conventional medical adhesives. Here, the most commonly used bioadhesives as well as the potential of silk fibroin as natural adhesives will be discussed.     

Preparation of Silk Fibroin/Carboxymethyl Chitosan Hydrogel under Low Voltage as a Wound Dressing

At present, silk fibroin (SF) hydrogel can be prepared by means of electrodeposition at 25 V in direct current (DC) mode. Reducing the applied voltage would provide benefits, including lower fabrication costs, less risk of high voltage shocks, and better stability of devices. Here, a simple but uncommon strategy for SF-based hydrogel preparation using 4 V in DC mode is discussed. SF was mixed and cross-linked with carboxymethyl chitosan (CMCS) through hydrogen bonding, then co-deposited on the graphite electrode. The thickness, mass, and shape of the SF/CMCS hydrogel were easily controlled by adjusting the electrodeposition parameters. Morphological characterization of the prepared hydrogel via SEM revealed a porous network within the fabricated hydrogel. This structure was due to intermolecular hydrogen bonding between SF and CMCS, according to the results of thermogravimetric analysis and rheological measurements. As a potential wound dressing, SF/CMCS hydrogel maintained a suitable moisture environment for wound healing and demonstrated distinct properties in terms of promoting the proliferation of HEK-293 cells and antibacterial activity against Escherichia coli and Staphylococcus aureus. Furthermore, histological studies were conducted on a full-thickness skin wound in rats covered with the SF/CMCS hydrogel, with results indicating that this hydrogel can promote wound re-epithelization and enhance granulation tissue formation. These results illustrate the feasibility of using the developed strategy for SF-based hydrogel fabrication in practice for wound dressing.     

蚕丝蛋白开发利用的研究进展

较为全面地综述了最近几十年国内外关于蚕丝蛋白的组成、性能,介绍了蚕丝新用途和利用原理,蚕丝脱胶精炼方法,以及蚕丝在化妆品、医药、固定化酶载体材料、食品、再生丝和涂膜化纤表面等方面的应用和研究进展。     

An Injectable Enzymatically Crosslinked and Mechanically Tunable Silk Fibroin/Chondroitin Sulfate Chondro-Inductive Hydrogel

An injectable hybrid hydrogel is synthesized, comprising silk fibroin (SF) and chondroitin sulfate (CS) through di-tyrosine formation bond of SF chains. CS and SF are reported with excellent biocompatibility as tissue engineering scaffolds. Nonetheless, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate articular cartilage. As CS is one of the cartilage extracellular matrix (ECM) components, it has the potential to enhance the biological activity of SF-based hydrogel in terms of cartilage repair. Therefore, altering the CS concentrations (i.e., 0 wt%, 0.25 wt%, 0.5 wt%, 1 wt%, and 2 wt%), which are interpenetrated between SF β-sheets and chains, can potentially adjust the physical, chemical, and mechanical features of these hybrid hydrogels. The formation of β-sheets by 30 days of immersion in de-ionized (DI) water can improve the compression strength of the SF/CS hybrid hydrogels in comparison with the same SF/CS hybrid hydrogels in the dried state. Biological investigation and observation depicts proper cell attachment, proliferation and cell viability for C28/I2 cells. Gene expression of sex-determining region YBox 9 (SOX9), Collagen II α1, and Aggrecan (AGG) exhibits positive C3H10T1/2 growth and expression of cartilage-specific genes in the 0.25 wt% and 0.5 wt% SF/CS hydrogels.     

羊毛角蛋白粉末的制备及其结构与性能表征

以废旧毛涤混纺面料为原料,采用溶解-研磨法制备羊毛角蛋白粉末。通过对羊毛角蛋白粉末的外观形态、粒度、内部结构及热性能进行测试与分析,得出制备的羊毛角蛋白粉末表面粗糙且形态不规则,颗粒大小不匀,团聚现象比较明显;其平均粒径为203.026μm,粒径的一致性为0.873;角蛋白粉末含有大量氢键及酰胺键,二硫键已断裂,具有多肽的特征,属于蛋白质;当温度低于210℃时,角蛋白粉末具有热稳定性。     

Synthesis of pH and Glucose Responsive Silk Fibroin Hydrogels

Silk fibroin (SF) has attracted much attention due to its high, tunable mechanical strength and excellent biocompatibility. Imparting the ability to respond to external stimuli can further enhance its scope of application. In order to imbue stimuli-responsive behavior in silk fibroin, we propose a new conjugated material, namely cationic SF (CSF) obtained by chemical modification of silk fibroin with ε-Poly-(L-lysine) (ε-PLL). This pH-responsive CSF hydrogel was prepared by enzymatic crosslinking using horseradish peroxidase and H₂O₂. Zeta potential measurements and SDS-PAGE gel electrophoresis show successful synthesis, with an increase in isoelectric point from 4.1 to 8.6. Fourier transform infrared (FTIR) and X-ray diffraction (XRD) results show that the modification does not affect the crystalline structure of SF. Most importantly, the synthesized CSF hydrogel has an excellent pH response. At 10 wt.% ε-PLL, a significant change in swelling with pH is observed. We further demonstrate that the hydrogel can be glucose-responsive by the addition of glucose oxidase (GOx). At high glucose concentration (400 mg/dL), the swelling of CSF/GOx hydrogel is as high as 345 ± 16%, while swelling in 200 mg/dL, 100 mg/dL and 0 mg/dL glucose solutions is 237 ± 12%, 163 ± 12% and 98 ± 15%, respectively. This shows the responsive swelling of CSF/GOx hydrogels to glucose, thus providing sufficient conditions for rapid drug release. Together with the versatility and biological properties of fibroin, such stimuli-responsive silk hydrogels have great potential in intelligent drug delivery, as soft matter substrates for enzymatic reactions and in other biomedical applications.     

丝素蛋白微膜对花瓣生理影响分析研究

丝素蛋白的智能性水凝胶对采摘后玫瑰花瓣中过氧化物酶（ POD）,过氧化氢酶（ CAT）和超氧化物歧化酶（ SOD）的活性有着重要的影响。实验表明,涂抹了丝素的玫瑰花中POD、 CAT和SOD酶活性明显优于正常情况下玫瑰花中的酶活性,且其活性降低的趋势低于对照组,可见涂抹丝素对采摘后的玫瑰花具有延长保存时间的作用。本研究主要探究丝素对于玫瑰花的保鲜作用并初步确定对玫瑰花保鲜的最佳丝素浓度。     

Chemical,Thermal, Time,and Enzymatic Stability of Silk Materials with Silk I Structure

The crystalline structure of silk fibroin Silk I is generally considered to be a metastable structure; however, there is no definite conclusion under what circumstances this crystalline structure is stable or the crystal form will change. In this study, silk fibroin solution was prepared from B. Mori silkworm cocoons, and a combined method of freeze-crystallization and freeze-drying at different temperatures was used to obtain stable Silk I crystalline material and uncrystallized silk material, respectively. Different concentrations of methanol and ethanol were used to soak the two materials with different time periods to investigate the effect of immersion treatments on the crystalline structure of silk fibroin materials. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman scattering spectroscopy (Raman), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA) were used to characterize the structure of silk fibroin before and after the treatments. The results showed that, after immersion treatments, uncrystallized silk fibroin material with random coil structure was transformed into Silk II crystal structure, while the silk material with dominated Silk I crystal structure showed good long-term stability without obvious transition to Silk II crystal structure. α-chymotrypsin biodegradation study showed that the crystalline structure of silk fibroin Silk I materials is enzymatically degradable with a much lower rate compared to uncrystallized silk materials. The crystalline structure of Silk I materials demonstrate a good long-term stability, endurance to alcohol sterilization without structural changes, and can be applied to many emerging fields, such as biomedical materials, sustainable materials, and biosensors.     

丝素/羧甲基壳聚糖共混膜的结构与性能

利用丝素(SF)与羧甲基壳聚糖(CMCS)共混制取不同比例的SF/CMCS共混膜。研究了CMCS诱导的丝素构象转变行为,测试了共混膜的吸湿性、透湿性和保水性。当CMCS的质量分数为5%时,共混膜中丝素的构象以β-折叠为主;当CMCS的质量分数为10%时,共混膜中丝素的构象由β-折叠向α-螺旋发生转变;当CMCS的质量分数达到15%时,共混膜中丝素的构象向无规卷曲发生转变。当CMCS质量分数小于15%时,共混膜中SF与CMCS具有良好的相容性,溶胀度较小,吸湿性随CMCS含量的增加而迅速降低。     

静电纺再生丝素纳米纤维的结构与性能

静电纺丝获得的丝素纳米级纤维可作为细胞培养支架,用于纺丝工艺及后处理能改变丝素微细结构,影响其水溶性和力学性能。本文采用XRD、FTIR、固态13CNMR和DSC研究了不同工艺下丝素纳米纤维及经甲醇处理后的微细结构,比较了不同微细结构下的水溶性和力学性能。结果表明,电纺丝的微细结构受纺丝工艺影响,高电压、纺丝液中丝素质量分数大时纺得的电纺丝结晶度高,经甲醇处理后,β化程度提高;w(丝素)=11%、15%时制备的电纺丝断裂强度分别为8.5、11.9 cN/mm;w(丝素)=11%、19%,水溶性由51.2%下降到43.3%;w(丝素)=19%、电压32 kV制得的电纺丝甲醇处理前后水溶性从43.3%下降到6.6%,说明丝素纳米纤维结晶度提高,强度增加、水溶性下降,满足了细胞支架的要求。     

烯丙基丝素蛋白温敏水凝胶的合成及性能研究

为解决以柞蚕丝素蛋白(ASF)为基材的生物材料力学性能差、水溶失率高等问题,首先,以ASF为原料,烯丙基缩水甘油醚(AGE)为改性剂,在碱性条件下ASF与AGE发生亲核取代反应,形成具有反应性的烯丙基丝素蛋白(ASF-AGE);然后,以N-异丙基丙烯酰胺(NIPAAm)为单体,在不使用任何交联剂的情况下,将ASF-AGE与NIPAAm进行聚合,最终形成烯丙基丝素蛋白温敏p(ASF-AGE-NIPAAm)水凝胶。采用茚三酮比色法对ASF-AGE的氨基转化率进行测定,采用¹H NMR对ASF-AGE分子结构进行表征;采用XRD、DSC、压缩测试等方法研究ASF-AGE含量对水凝胶结晶结构、温敏特性、溶失稳定性和力学性能的影响。结果表明:烯丙基双键成功引入ASF大分子链上,ASF与AGE的质量比为1∶8,温度20℃,pH=10.5,反应24 h时,得到的ASF-AGE的氨基转化率为55.21%;ASF-AGE与NIPAAm聚合形成稳定形态的水凝胶,水凝胶的LCST约为32℃,具有明显的温敏特性;ASF-AGE与NIPAAm配比为4/6时,水凝胶具有良好的溶失稳定性和综合...     

Synthesis and properties of thermosensitive hydrogel of allyl silk fibroin

For the sake of solving the problems of poor mechanical strength and dissolve-loss ratio of Antheraea pernyi silk fibroin (ASF), allyl silk fibroin (ASF-AGE) was synthesized through nucleophilic substitution using ASF as substrate and allyl glycidyl ether (AGE) as modifier under basic conditions. And a series of gel were manufactured though in situ polymerization using ASF-AGE and N-isopropylacrylamide (NIPAAm) monomer without any crosslinking agent. The amino conversion rate of ASF was confirmed by ninhydrin colorimetry, the molecular structure of ASF-AGE was characterized by ¹H NMR and the influence of ASF-AGE content on crystal structure, temperature sensitivity, dissolution stability and mechanical properties of hydrogels were also investigated by XRD, DSC, compression test and so on. The results indicated that allyl was successfully introduced into ASF, the amino conversion rate of ASF was 55.21% when the reaction condition was 1∶8 mass ratio (ASF/AGE), T=20℃ and pH=10.5. The stable hydrogels can be obtained by ASF-AGE and NIPAAm polymerization. The hydrogels showed lower critical solution temperatures (LCST) at about 32℃, which revealed obvious thermosensitive characteristic. When the ratio of ASF-AGE to NIPAAm is 4/6, the hydrogel has good dissolution stability and comprehensive mechanical properties.     

凝固条件对纤维素/丝素蛋白纤维相形态及性能的影响

蛋白质纤维具有光滑柔顺、透气吸湿等优点,然而天然蛋白纤维产量有限。再生蛋白纤维的制备通常采用与其它成纤高分子接枝或共混的方法,有利于提高再生蛋白纤维的断裂强度。选用同为天然高分子的纤维素为基体,以共溶剂溶解纤维素与蛋白质,进而纺丝成形制得力学性能满足要求的纤维素/丝素蛋白共混纤维。为了探究凝固剂组成对纤维素/丝素蛋白共混纤维相形态及性能的影响,选用水、乙醇、乙醇/1-丁基-3-甲基咪唑氯盐([BMIM]Cl)等作为凝固剂。研究发现:乙醇作为凝固剂时,纤维素与丝素蛋白能很好地同时凝固;而当在乙醇凝固浴中加入适量的[BMIM]Cl径向均匀分散。通过对凝固剂组成的调控能有效提升纤维的力学强度。     

静电纺丝工艺参数对丝素/壳聚糖纳米纤维的形貌及直径的影响

以98%的甲酸为溶剂,不同质量分数的再生丝素溶液和3.5%的壳聚糖溶液以质量比70:30共混静电纺丝。用扫描电子显微镜(SEM)观察了丝素质量分数、电压和极距(喷丝口到收集装置的距离)对丝素/壳聚糖纳米纤维的形貌及直径的影响。正交试验结果表明:在丝素/壳聚糖溶液静电纺丝的工艺参数中,对纤维平均直径的影响因素由大到小依次为丝素质量分数、电压、极距。单因素试验表明:丝素/壳聚糖纳米纤维的平均直径及其分布范围随丝素质量分数的增加而增大;在15￣30kV范围内纤维的平均直径随电压增大而减小;当极距大于12cm时,对纤维直径影响不大。最佳工艺条件为:丝素质量分数13%,电压30kV,极距为12cm,制得的纳米纤维平均直径104nm。     

Comparative delivery of Diltiazem hydrochloride through synthesized polymer: Hydrogel and hydrogel microspheres

In this study, interpenetrating polymer network (IPN) hydrogel based on polyvinyl alcohol (PVA) networking with polyacrylic acid (PAA) were prepared by a non‐conventional emulsion method without any added crosslinker, using benzoyl peroxide as initiator and sodium chloride (NaCl) as additive. The IPN hydrogel was characterized by using Fourier transformed infrared (FTIR) spectrophotometry, Thermo gravimetric analysis (TGA), and Scanning electron microscopy (SEM). (PVA‐co‐PAA)/NaCl normal IPN hydrogel (H) were fabricated into hydrogel microspheres (HM) by modified emulsion crosslinking method using glutaraldehyde‐saturated toluene as crosslinker and were loaded with Diltiazem hydrochloride (DL). The IPN hydrogel showed more swelling in simulated intestinal fluid (SIF). (PVA‐co‐PAA)/NaCl HM formulation A1 showed comparatively higher DL entrapment (79%) and better control over DL release up to 24 h. By comparing antihypertensive activity of DL loaded two formulations in normotensive rats, HM formulation A1 found more effective in reducing blood pressure to 40.1%. The experimental results demonstrated that (PVA‐co‐PAA)/NaCl HM had the greater potential than normal hydrogel to be used as a drug carrier. A single use of the prepared hydrogel microsphere system of DL can effectively control hypertension in rats. The system holds promise for clinical studies. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

丝素-羟丙基壳聚糖微球的结构和释药性能

以胰岛素为目标药物,以丝素(SF)和羟丙基壳聚糖(HPCS)为包药材料,复凝聚法制备SF-HPCS载药微球。采用红外光谱(FTIR)、扫描电镜(SEM)、X射线衍射(XRD)、热重分析(TGA)等对载药微球的结构、外部形貌及热性能等进行了表征。结果表明,所制备的载药微球表面密实,平均粒径22.4μm,呈正态分布;载药微球对胰岛素的包埋率达73.6%,大于HPCS载药微球(64.3%)及壳聚糖(CS)载药微球(57.1%);SF-HPCS载药微球在人工胃液中4h内累计释药率为21.3%,在人工肠液中24h内累计释药率达81.2%,48h累计释药率为92.2%,释放过程平稳、缓慢。     

再生丝素/壳聚糖共混纳米纤维的结构与性能

以98%的甲酸为溶剂,不同质量分数的再生丝素溶液和3.5%的壳聚糖溶液以质量比70∶30共混静电纺丝。测定了壳聚糖的含量对共混膜的结构及力学、溶解等性能的影响。结果表明:随着壳聚糖相对含量的增加,丝素β化程度提高,纤维结晶度增大,丝素与壳聚糖以70∶30共混时的溶失率最小;甲醇处理后,溶失率明显降低;共混纳米纤维的断裂强度随着壳聚糖相对含量的增大而增加,柔软性也逐渐提高;共混纤维膜具有优异的抗菌性。     

玫瑰红色素对羊毛和蚕丝的媒染性能

以FeSO4和ZnSO4·7H2O为媒染剂,采用玫瑰红提取液对羊毛和蚕丝进行染色,探讨了媒染温度、时间、媒染剂用量、玫瑰红提取液用量对媒染性能的影响,以及染色织物的牢度。结果表明,媒染剂FeSO4和ZnSO4·7H2O用量为2g／L、提取液用量为10mL、羊毛和蚕丝织物均为2g时,染色织物具有较深的色泽和良好的色牢度。