A universal self‐eroding sacrificial bioink that enables bioprinting at room temperature

Natural polymer‐based hydrogel bioinks are widely used in bioprinting due to their suitability for recapitulation of in vivo cellular activities. However, preservation of the target geometry in a cell‐laden hydrogel is difficult to achieve. The aim of this study was to develop a universal sacrificial bioink that allows high cell viability and a better shape fidelity in the cell‐laden construct. A polysaccharide‐based universal sacrificial bioink was developed for microextrusion‐based bioprinting and was optimized to erode in 48 hours in the cell culture medium without formation of any undesired by‐products. The sacrificial hydrogel was prepared from alginate and agarose via a microwave oven assisted method and bioprinted at room temperature to generate microchannels in the cell‐laden hydrogel or to support a tubular structure and its biocompatibility determined by live/dead assay. Bioprinting time was significantly reduced, down to a few minutes for a large‐scale tissue model (1 minute 52 seconds for a 2 cm tubular structure), by means of a high bioprinting speed up to 25 mm/s. After 48 hours in the cell culture, the sacrificial bioink completely detached from the cell‐laden construct without causing any changes in its printed shape. Cell viability in the cell‐laden construct was observed to be more than 95% at the end of 3‐day culture. This novel sacrificial bioink enables bioprinting at room temperature without affecting oxygen and nutrient penetration into the cell‐laden hydrogel and allows retention of high cell viability and shape fidelity.     

Degree of Biomimicry of Artificial Spider Silk Spinning Assessed by NMR Spectroscopy

Biomimetic spinning of artificial spider silk requires that the terminal domains of designed minispidroins undergo specific structural changes in concert with the β‐sheet conversion of the repetitive region. Herein, we combine solution and solid‐state NMR methods to probe domain‐specific structural changes in the NT2RepCT minispidroin, which allows us to assess the degree of biomimicry of artificial silk spinning. In addition, we show that the structural effects of post‐spinning procedures can be examined. By studying the impact of NT2RepCT fiber drying, we observed a reversible beta‐to‐alpha conversion. We think that this approach will be useful for guiding the optimization of artificial spider silk fibers.     

Production and processing of spider silk proteins

Natural spider silk fibers have impressive mechanical properties (outperforming many man‐made fibers) and are, moreover, biocompatible, biodegradable, and produced under benign conditions (using water as a solvent at ambient temperature). The problems associated with harvesting natural spider silks inspired us to devise a method to produce spider silk‐like proteins biotechnologically (the first subject tackled in this highlight); we subsequently discuss their processing into various materials morphologies, and some potential technical and biomedical applications. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3957–3963, 2009     

Correlation between processing conditions,microstructure and mechanical behavior in regenerated silkworm silk fibers

Regenerated silkworm fibers spun through a wet‐spinning process followed by an immersion postspinning drawing step show a work to fracture comparable with that of natural silkworm silk fibers in a wide range of spinning conditions. The mechanical behavior and microstructure of these high performance fibers have been characterized, and compared with those fibers produced through conventional spinning conditions. The comparison reveals that both sets of fibers share a common semicrystalline microstructure, but significant differences are apparent in the amorphous region. Besides, high performance fibers show a ground state and the possibility of tuning their tensile behavior. These properties are characteristic of spider silk and not of natural silkworm silk, despite both regenerated and natural silkworm silk share a common composition different from that of spider silk. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012     

一种天然纳米纤维捕鸟蛛丝的物理化学结构表征

蜘蛛丝作为功能性结构材料, 其独特的纤维成型方法与优良的结构和性能引起许多人的关注. 从20世纪80年代开始有关蜘蛛丝的研究报道日益增加[1]. 与高温高压下或由溶剂纺丝成型的合成纤维相比, 蜘蛛丝在空气中凝固成型, 丝纤维成型安全、无害, 从腹部若干不同吐丝器产生不同种类的丝具有不同的用途[2]. 蜘蛛拖曳丝(dragline silk)的比强度大于钢丝, 且具有较大的断裂伸长率(9%～30%)[3,4], 抗张强度1.1～1.4 GPa. 在相对湿度50%和应变速率100%/min的条件下, 模量值可达10～50 GPa. 在所有已知纤维品种中, 蜘蛛丝的断裂能是最高的. 此外, 蜘蛛丝在许多方面的综合性能优于最优良的人造纤维. 另外, 蜘蛛丝的细度为已知纤度最小的天然有机纤维, 这种高性能丝具有捕捉昆虫甚至鸟类的功能, 因此蜘蛛丝是具有特异功能的天然纤维材料. 目前, 蜘蛛丝结构和性能的研究主要包括其化学组成[5]、结晶结构[6,7]、结构模型[8,9]以及其NMR表征[10]等, 这些研究揭示了蜘蛛丝的氨基酸组成、分子量及其分布、结晶度、晶胞尺寸、链构象以及结构模型等. 这些研究主要集中在少数几种蜘蛛品种上, 如金色圆网织网蛛(Nephila clavipes)、十字圆蛛(A.diadematus)和大腹圆蛛(A.ventrocosus)等. 目前, 已知的蜘蛛种类大于30 000种[11], 以蜘蛛丝为例的生物大分子材料研究是一个挑战性的课题. 国内蜘蛛丝的研究仅有大腹圆蛛拖曳丝蛋白一级结构的研究报道[12,13]. 本文报道了广西捕鸟蛛丝的红外光谱、形貌结构和原子力显微镜的初步研究结果.     

A General Strategy for Extrusion Bioprinting of Bio‐Macromolecular Bioinks through Alginate‐Templated Dual‐Stage Crosslinking

The recently developed 3D bioprinting technology has greatly improved the ability to generate biomimetic tissues that are structurally and functionally relevant to their human counterparts. The selection of proper biomaterials as the bioinks is a key step toward successful bioprinting. For example, viscosity of a bioink is an important rheological parameter to determine the flexibility in deposition of free‐standing structures and the maintenance of architectural integrity following bioprinting. This requirement, however, has greatly limited the selection of bioinks, especially for those naturally derived due to their commonly low mechanical properties. Here the generalization of a mechanism for extrusion bioprinting of bio‐macromolecular components, mainly focusing on collagen and its derivatives including gelatin and gelatin methacryloyl, is reported. Specifically, a templating strategy is adopted using a composite bioink containing both the desired bio‐macromolecular component and a polysaccharide alginate. The physically crosslinkable alginate component serves as the temporal structural support to stabilize the shape of the construct during bioprinting; upon subsequent chemical or physical crosslinking of the bio‐macromolecular component, alginate can be selectively removed to leave only the desired bio‐macromolecule. It is anticipated that this strategy is general, and can be readily expanded for use of a wide variety of other bio‐macromolecular bioinks.     

Spinnenseidenproteine: Grundlage für neue Materialien

Spider silk is a biomaterial with extraordinary properties. It is extremely tough and at the same time highly elastic – a combination not found in other polymers. Due to its outstanding potential, spider silk has long been desired as a material for technical applications. This review highlights recent developments in the field of spider silk technology, insights into silk structure, and the natural silk spinning process. Due to the recent progress, spider silk products might be available in the near future, reflecting a new generation of environmentally friendly polymer products.     

Spinnenseide in der plastischen Chirurgie. Wunderwerkstoff der Natur

The experimental data show that the production of artificial nerve grafts with spider silk is a potential alternative therapy. The biologically favourable properties of the fibres from the spider Nephila clavipes should be used in human nerve reconstruction. The biological fibres promote the proliferation of cells. They are immunologically tolerated and not rejected. The muscle attraction line is kept intact which prevents muscle degeneration to a large extent. The spider fibres accelerate the migration of peripheral schwann cells into the nerve construct and promote the alignment of the nerve cells. For surgical interventions the biomechanical stability of spider silk and the composition from essential amino acids make the spider silk fibres interesting as a matrix for the cellular regeneration and in particular as a guiding structure for nerve regeneration.     

Basic Principles in the Design of Spider Silk Fibers

The prominence of spider silk as a hallmark in biomimetics relies not only on its unrivalled mechanical properties, but also on how these properties are the result of a set of original design principles. In this sense, the study of spider silk summarizes most of the main topics relevant to the field and, consequently, offers a nice example on how these topics could be considered in other biomimetic systems. This review is intended to present a selection of some of the essential design principles that underlie the singular microstructure of major ampullate gland silk, as well as to show how the interplay between them leads to the outstanding tensile behavior of spider silk. Following this rationale, the mechanical behavior of the material is analyzed in detail and connected with its main microstructural features, specifically with those derived from the semicrystalline organization of the fibers. Establishing the relationship between mechanical properties and microstructure in spider silk not only offers a vivid image of the paths explored by nature in the search for high performance materials, but is also a valuable guide for the development of new artificial fibers inspired in their natural counterparts.     

从天然动物丝到丝蛋白基材料的研究

作为具有优异综合力学性能的天然蛋白质纤维,丰产的动物丝特别是蚕丝长期伴随着人们的日常生活,近十余年来,各种具有特色的功能性丝蛋白基材料更是层出不穷.但在探索动物丝和丝蛋白基材料的过程中,动物丝纤维是经由蚕或蜘蛛等动物的纺器而纺制得到的简单事实往往被忽视;换言之,动物丝实际上是动物对丝蛋白进行体内“加工”后的产物,也是丝蛋白基材料中的一种.因此,天然动物丝中独特的各等级间构效关系与丝蛋白基材料的构效关系之间并不存在着必然的传承效应.本文着重介绍了我们在对动物丝和丝蛋白基材料探索中的经验和体会,即在强调以丝蛋白分子链结构与性能及其之间的关系为研究重点的基础上,从比较和发掘各种天然动物丝的特性入手,进而了解丝蛋白分子链在本体和溶液中的行为,并通过对动物丝蛋白分子链聚集态结构的调控,以达到设计制备一系列多形貌和多功能的动物丝蛋白基材料的目的.     

蜘蛛与蚕的人工强制纺丝研究进展

为制备高性能人造蜘蛛丝和蚕丝,人们在蜘蛛丝蛋白的基因重组、再生蜘蛛丝蛋白或蚕丝蛋白的仿生纺丝,以及蜘蛛与蚕的人工强制纺丝方面开展了大量工作.研究了人工强制纺丝,比较了蜘蛛牵引丝和蚕丝在不同纺丝条件下的力学性能.结果表明:在一定范围内增加纺丝速率有利于提高蜘蛛丝和蚕丝的力学性能,此外,环境温度、纺丝状态等条件对丝的性能也...     

光交联自支撑丝素蛋白水凝胶的三维快速成型

将丝素蛋白(SF)光诱导自交联原理与挤出式三维(3D)打印相结合, 开发了光交联自支撑SF水凝胶的原位成型加工技术. 采用旋转流变仪、 光流变测试系统和改装的挤出式3D打印设备等对SF溶液的流变性能、 光交联性能和成型加工性能等进行研究. 结果表明, SF溶液主要表现为黏性特征, 结构强度和稳定性均较差. 利用SF的光诱导自交联特性, 以三联吡啶氯化钌[Ru(Ⅱ)]和过硫酸钾(KPS)为蓝光引发体系, 可实现SF水凝胶的快速光交联成型. SF光交联行为符合指数函数增长模型, 因“滤镜效应”, 当Ru(Ⅱ)的浓度为0.05 mmol/L时, SF具有最佳的光交联性能. 通过调节气压、 针头孔径、 移动速度及固化速率等参数, 采用3D打印设备可实现从单层几何结构到多层三维网络构型SF凝胶材料的高效、 精准构建, 为SF的生物3D打印提供了新思路.     

Characterization of Hydrogels Made of a Novel Spider Silk Protein eMaSp1s and Evaluation for 3D Printing

Recombinantly produced spider silk proteins have high potential for bioengineering and various biomedical applications because of their biocompatibility, biodegradability, and low immunogenicity. Here, the recently described small spider silk protein eMaSp1s is assembled into hydrogels, which can be 3D printed into scaffolds. Further, blending with a recombinantly produced MaSp2 derivative eADF4(C16) alters the mechanical properties of the resulting hydrogels. Different spider silk hydrogels also show a distinct recovery after a high shear stress deformation, exhibiting the tunability of their features for selected applications.     

WISE NMR characterization of nanoscale heterogeneity and mobility in supercontracted Nephila clavipes spider dragline silk

The addition of water to spider dragline silk results in fiber contraction to 50% its initial length and significant changes to the mechanical properties of the silk. This event has been termed supercontraction. A decrease in strength and increase in elasticity have been reported when the silk is in contact with water. Two-dimensional wide-line separation (WISE) nuclear magnetic resonance (NMR) is implemented to correlate (13)C chemical shifts with mobility by observing the corresponding (1)H line widths and line shapes in water-saturated spider dragline silk. The WISE NMR spectrum of the native silk exhibits (1)H line widths that are approximately 40 kHz for all carbon environments characteristic of a rigid organic system. In contrast, the water-saturated case displays a component of the (1)H line that is narrowed to approximately 5 kHz for the glycine C(alpha) and a newly resolved alanine helical environment while the alanine C(beta) corresponding to the beta-sheet conformation remains broad. These results indicate that water permeates the amorphous, glycine-rich matrix and not the crystalline, polyalanine beta-sheets. A delay time is added to the WISE NMR pulse sequence to monitor spin diffusion between the amorphous, mobile region and the crystalline domains. The time required for spin diffusion to reach spatial equilibrium is related to the length scale of the polyalanine crystallites. This technique is employed to measure crystalline domain sizes on the nanometer length scale in water-solvated spider dragline silk. These results provide further insight into the structure of spider silk and mechanism of supercontraction.     

Reconfigurable Bioinspired Framework Nucleic Acid Nanoplatform Dynamically Manipulated in Living Cells for Subcellular Imaging

In nature, the formation of spider silk fibers begins with dimerizing the pH‐sensitive N‐terminal domains of silk proteins (spidroins) upon lowering pH, and provides a natural masterpiece for programmable assembly. Inspired by the similarity of pH‐dependent dimerization behaviors, introduced here is an i‐motif‐guided model to mimic the initial step of spidroin assembly at the subcellular level. A framework nucleic acid (FNA) nanoplatform is designed using two tetrahedral DNA nanostructures (TDNs) with different branched vertexes carrying a bimolecular i‐motif and a split ATP aptamer. Once TDNs enter acidic lysosomes within living cells, they assemble into a heterodimeric architecture, thereby enabling the formation of a larger‐size framework and meanwhile subcellular imaging in response to endogenous ATP, which can be dynamically manipulated by adjusting intracellular pH and ATP levels with external drug stimuli.     

Hydrophobic and Hofmeister effects on the adhesion of spider silk proteins onto solid substrates: an AFM-based single-molecule study

AFM-based single-molecule force spectroscopy has been used to study the effect of Hofmeister salts and protein hydrophobicity on the adhesion of recombinant spider silk proteins onto solid substrates. Therefore, a molecular probe consisting of a spider silk protein and an AFM tip has been developed, which (i) is a well-defined, small system that can be simulated by molecular dynamics simulations, (ii) allows access to the whole soluble concentration range for ions, and (iii) provides the distribution of desorption forces rather than just ensemble-averaged mean values. The measured desorption forces follow the Hofmeister series for anions (H2PO4-, Cl-, I-) with a stabilizing energy of more than 15 kBT for 5 M NaH2PO4. Moreover, this effect is influenced by the hydrophobicity of the spider silk protein, indicating that hydrophobic and Hofmeister effects are closely related.     

Sonochemical Degradation of Gelatin Methacryloyl to Control Viscoelasticity for Inkjet Bioprinting

Inkjet printing enables the mimicry of the microenvironment of natural complex tissues by patterning cells and hydrogels at a high resolution. However, the polymer content of an inkjet-printable bioink is limited as it leads to strong viscoelasticity in the inkjet nozzle. Here it is demonstrated that sonochemical treatment controls the viscoelasticity of a gelatin methacryloyl (GelMA) based bioink by shortening the length of polymer chains without causing chemical destruction of the methacryloyl groups. The rheological properties of treated GelMA inks are evaluated by a piezo-axial vibrator over a wide range of frequencies between 10 and 10 000 Hz. This approach enables to effectively increase the maximum printable polymer concentration from 3% to 10%. Then it is studied how the sonochemical treatment effectively controls the microstructure and mechanical properties of GelMA hydrogel constructs after crosslinking while maintaining its fluid properties within the printable range. The control of mechanical properties of GelMA hydrogels can lead fibroblasts more spreading on the hydrogels. A 3D cell-laden multilayered hydrogel constructs containing layers with different physical properties is fabrictated by using high-resolution inkjet printing. The sonochemical treatment delivers a new path to inkjet bioprinting to build microarchitectures with various physical properties by expanding the range of applicable bioinks.     

Nephila clavipes spider dragline silk microstructure studied by scanning transmission X-ray microscopy

Nephila clavipes dragline silk microstructure has been investigated by scanning transmission X-ray microscopy (STXM), a technique that allows quantitative mapping of the level of orientation of the peptide groups at high spatial resolution (<50 nm). Maps of the orientation parameter P2 have been derived for spider silk for the first time. Dragline silk presents a very fine microstructure in which small, highly oriented domains (average area of 1800 nm2, thus clearly bigger than individual beta-sheet crystallites) are dispersed in a dominant, moderately oriented matrix with several small unoriented domains. Our results also highlight the orientation of the noncrystalline fraction in silk, which has been underestimated in numerous structural models. No evidence of either a regular lamellar structure or any periodicity along the fiber was observed at this spatial resolution. The surface of fresh spider silk sections consists of a approximately 30-120 nm thick layer of highly oriented protein chains, which was found to vary with the reeling speed, where web building (0.5 cm/s) and lifeline (10 cm/s) spinning speeds were investigated. While the average level of orientation of the protein chains is unaffected by the spinning speed, STXM measurements clearly highlight microstructure differences. The slowpull fiber contains a larger fraction of highly oriented domains, while the protein chains are more homogeneously oriented in the fastpull fiber. In comparison, cocoon silk from the silkworm Bombyx mori presents a narrower orientation distribution. The strength-extensibility combination found in spider dragline silk is associated with its broad orientation distribution of highly interdigitated and unoriented domains.     

Spider silk and amyloid fibrils: a structural comparison

Although spider silks have been studied for decades, the assembly properties of the underlying silk proteins have still not been unravelled. Previously, the detection of amyloid-like nanofibrils in the spider's silk gland suggested their involvement in the assembly process.Recombinantly produced spider silk also self-assembles into nanofibrils. In order to investigate the structural properties of such silk nanofibrils in more detail, they have been compared to amyloid-like fibrils to highlight structural similarities.     

蜘蛛吐丝过程中钾的作用

用电感耦合等离子体质谱(ICP-MS)对蜘蛛Nephila丝腺体和丝进行测定,结果表明,钾在丝中的含量明显高于在丝腺体中的含量.同时,在蜘蛛丝蛋白溶液中加入氯化钾,溶液出现乳白色浑浊,表明有呈β-折叠构象的微纤产生.浊度测试发现,丝蛋白微纤会逐渐聚集成较大颗粒而在溶液中形成沉淀.另外,红外光谱和拉曼光谱亦证明钾能够使蜘蛛丝蛋白膜发生从无规线团/螺旋到β-折叠的构象转变.有理由认为钾在蜘蛛吐丝过程中起重要作用,它的存在有利于丝蛋白形成β-折叠结构.