Surface‐directed assembly of cell‐laden microgels

Cell‐laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well‐defined microarchitecture. In this article, we present a bottom‐up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well‐defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels (～100–200 µm in width) could self‐assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom‐up approach enables rapid fabrication of cell‐laden microgel assemblies with pre‐defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications. Biotechnol. Bioeng. 2010; 105: 655–662. © 2009 Wiley Periodicals, Inc.     

Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers

Elastin and silk spidroins are fibrous, structural proteins with elastomeric properties of extension and recoil. While elastin is highly extensible and has excellent recovery of elastic energy, silks are particularly strong and tough. This study describes the biophysical characterization of recombinant polypeptides designed by combining spider wrapping silk and elastin‐like sequences as a strategy to rationally increase the strength of elastin‐based materials while maintaining extensibility. We demonstrate a thermo‐responsive phase separation and spontaneous colloid‐like droplet formation from silk‐elastin block copolymers, and from a 34 residue disordered region of Argiope trifasciata wrapping silk alone, and measure a comprehensive suite of tensile mechanical properties from cross‐linked materials. Silk‐elastin materials exhibited significantly increased strength, toughness, and stiffness compared to an elastin‐only material, while retaining high failure strains and low energy loss upon recoil. These data demonstrate the mechanical tunability of protein polymer biomaterials through modular, chimeric recombination, and provide structural insights into mechanical design. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 693–703, 2016.     

Spider silk as a resource for future biotechnologies

Insect silks have been used by mankind for millennia to produce textiles and in particular, the cocoon silk of Bombyx mori was the base of one of the most important industries in history. In fact, B. mori is probably the only domesticated insect if not invertebrate in its true and strict sense, comparable to cattle and other livestock that humans have known and bred since the Neolithic period. In contrast, reports regarding the use of spider silk throughout history have the character of travellers’ tales or anecdotes, and serious attempts to exploit these biomaterials on a large scale have not been undertaken until recently. Indeed, the cannibalism of these carnivores makes their farming difficult and the production of significant yields of spider silk virtually impossible. Only today, with recombinant technologies available, does this problem seem to have been overcome. But why use spider silk at all – if we have the infrastructure to produce significant yields of silk from Bombyx? In contrast to most insects, spiders do not spin from labial glands, and many spiders possess different types of gland, most of them active throughout the whole lifespan. Typical orb‐weavers (Araneoidea) for instance possess up to seven different types of silk gland to produce different silk fibers and glues. Each of these products has evolved for a particular use and the respective material properties are highly adapted to that use. As the group of Araneae is about 400 million years old, the oldest fossil orb‐weaver is dated about 150 million years, and the use of silk is crucial to a spider's survival, we can expect that evolution will have “squeezed out every iota” to achieve optimum performance at minimum cost. Indeed, some dragline silks such as the major ampullate silks of some Nephila species show amazing mechanical properties that, in terms of toughness, are far superior to Bombyx silk. Labels like “stronger than steel” or “even better than Kevlar” were attached to them, and the Canadian‐based biotech company Nexia created the trademark “bio‐steel” for their prospective product. The discovery of these exceptional mechanical properties of those protein fibers triggered intense research on spider silk, with the goal of their commercial exploitation. But there is more to Arachne's weave and science is beginning to pick up those threads.     

Processing silk hydrogel and its applications in biomedical materials

This review mainly introduces the types of silk hydrogels, their processing methods, and applications. There are various methods for hydrogel preparation, and many new processes are being developed for various applications. Silk hydrogels can be used in cartilage tissue engineering, drug release materials, 3D scaffolds for cells, and artificial skin, among other applications because of their porous structure and high porosity and the large surface area for growth, migration, adhesion and proliferation of cells that the hydrogels provide. All of these advantages have made silk hydrogels increasingly attractive. In addition, silk hydrogels have wide prospects for application in the field of biomedical materials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:630–640, 2015     

Characterization and assembly of a GFP‐tagged cylindriform silk into hexameric complexes

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP‐silk fusion protein is predominantly α‐helical, and that pH can trigger a α‐ to β‐transition resulting in aggregation. Structural analysis by small angle X‐ray scattering suggests that the GFP‐Silk exists in the form of a hexamer in solution. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 378–390, 2014.     

Enzymatic hydrolysis of waste cellulose

Interstitial flow (IF) modulates both the biochemical and biophysical cues surrounding cells. It represents a very important regulating mechanism for cell/tissue function and has been commonly utilized in tissue engineering (TE). This article discusses the possible regulating mechanisms of IF on fibroblasts, the various fibroblast responses to IF, the current challenges in understanding the IF–fibroblast relationship and the application of IF for fibroblast involved TE. In particular, IF can affect fibroblast growth at both intracellular (e.g., calcium signaling, protein/proteinase secretion) and cellular (e.g., autocrine/paracrine signaling, proliferation, differentiation, alignment, adhesion, migration) levels. One major challenge for understanding IF–fibroblast interaction has been the determination of the flow and cell growth condition at microlevel especially in a three‐dimensional environment. To utilize IF and optimize the fluidic environment for TE, several influencing factors in the system including perfusate composition, flow profile, nutrient supply, signaling molecule effect, scaffold property, and fibroblast type should be considered. Biotechnol. Bioeng. 2010;107: 1–10. © 2010 Wiley Periodicals, Inc.     

Fabrication and evaluation of poly(epsilon-caprolactone)/silk fibroin blend nanofibrous scaffold

In this study we investigated the blend electrospinning of poly(?‐caprolactone) (PCL) and silk fibroin (SF) to improve the biodegradability and biocompatibility of PCL‐based nanofibrous scaffolds. Optimal conditions to fabricate PCL/SF (50/50) blend nanofiber were established for electrospinning using formic acid as a cosolvent and three‐dimensional (3D) PCL/SF blend nanofibrous scaffolds were prepared by a modified electrospinning process using methanol coagulation bath. The physical properties of 2D PCL/SF blend nanofiber mats and 3D highly porous blend nanofibrous scaffolds were measured and compared. To evaluate cytocompatibility of the 3D blend scaffolds as compared to 3D PCL nanofibrous scaffold, normal human dermal fibroblasts were cultured. It is concluded that biodegradability and cytocompatibility could be improved for the 3D highly porous PCL/SF (50/50) blend nanofibrous scaffold prepared by blending PCL with SF in electrospinning. In addition to the blending of PCL and SF, the 3D structure and high porosity of electrospun nanofiber assemblies may also be important factors for enhancing the performance of scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 265–275, 2012.     

A proposed model for dragline spider silk self‐assembly: Insights from the effect of the repetitive domain size on fiber properties

Dragline spider silk has been intensively studied for its superior qualities as a biomaterial. In previous studies, we made use of the baculovirus mediated expression system for the production of a recombinant Araneus diadematus spider silk dragline ADF4 protein and its self‐assembly into intricate fibers in host insect cells. In this study, our aim was to explore the function of the major repetitive domain of the dragline spider silk. Thus, we generated an array of synthetic proteins, each containing a different number of identical repeats up to the largest recombinantly expressed spider silk to date. Study of the self‐assembly properties of these proteins showed that depending on the increasing number of repeats they give rise to different assembly phenotypes, from a fully soluble protein to bona fide fibers with superior qualities. The different assembly forms, the corresponding chemical resistance properties obtained as well as ultrastructural studies, revealed novel insights concerning the structure and intermolecular interactions of the repetitive and nonrepetitive domains. Based on these observations and current knowledge in the field, we hereby present a comprehensive hypothetical model for the mechanism of dragline silk self‐assembly and fiber formation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 458–468, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk‐based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 292–333, 2013.     

昆虫的丝和丝腺

文中介绍了吐丝昆虫的种类、昆虫丝的结构与成分、丝与丝腺的类型、吐丝器及泌丝行为等。并简述了丝对泌丝昆虫生活的重要性及近期虫丝的研究动态。     

Self‐assembly of regenerated silk fibroin from random coil nanostructures to antiparallel β‐sheet nanostructures

In this work, we studied the effects of incubation concentration and time on the self‐assembly behaviors of regenerated silk fibroin (RSF). Our results showed the assembly ways of RSF were concentration‐dependent and there were four self‐assembly ways of RSF: (i) At relatively low concentration (≤0.015%), RSF molecules assembled into protofilaments (random coil), and then the thickness decreased and the secondary conformation changed to antiparallel β‐sheet; (ii) at the concentration of 0.015%, RSF molecules assembled into protofilaments (random coil), and then assembled into protofibrils (antiparallel β‐sheet). The protofibrils experienced the appearance and disappearance of phase periodic intervals in turn; (iii) at the concentration of 0.03%, RSF molecules assembled into bead‐like oligomers (random coil), and then assembled into protofibrils (antiparallel β‐sheet), and finally the height and phase periodic intervals of RSF protofibrils disappeared in turn; and (iv) at the relatively high concentration (≥0.15%), RSF molecules assembled into protofilaments (random coil), then aggregated into blurry cuboid‐like micelles (random coil), and finally self‐arranged to form smooth and clear cuboid‐like micelles (antiparallel β‐sheet). These results provide useful insights into the process by which the RSF molecules self‐assemble into protofilaments, protofibrils and micelles. Furthermore, our work will be beneficial to basic understanding of the nanoscale structure formations in different silk‐based biomaterials. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1181–1192, 2014.     

Modelling self assembly of natural silk solutions

We present a tentative interpretation of the origin of nematic liquid crystalline order exhibited by various natural silk fibroin solutions, notably those of orb-weaving spiders and the domestic silkworm Bombyx mori. It is thought that liquid crystalline rheology is exploited during the spinning process. We discuss in this approach the response of the liquid crystalline phase diagram to equilibrium physiological conditions and to parameters characterising the amino acid sequence of the fibroin molecules. The phase diagram is sensitive in this latter respect to sequence mutations, such that it may constitute a source of evolutionary selection pressure.     

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers

Spider dragline silk is a remarkable fiber made of unique proteins—spidroins—secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L^–1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m⁻³. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.     

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.     

蛋白质内含子介导蛛丝功能化平台的建立

为建立高效快捷的蛛丝功能化修饰平台,蛋白质内含子的反式剪接技术被首次应用于重组蛛丝的功能化修饰。在体外通过Ssp Dna B的反式剪接作用,在蛋白质水平上将12 k Da泛素相关修饰蛋白(SUMO)与蛛丝蛋白(W2CT)连接形成功能化蛛丝蛋白SUMOW2CT。修饰后SUMOW2CT与W2CT均能形成纳米至微米级的丝纤维,但SUMOW2CT自动成丝速度明显下降且产量约为W2CT的一半。与W2CT丝纤维(W)相似,SUMOW2CT丝纤维(UW)不具有超收缩能力和对2%SDS不耐受,但机械性能低于W2CT丝纤维。功能化蛋白SUMOW2CT形成的丝纤维中SUMO蛋白仍保持着正确三维结构,可被SUMO蛋白酶酶切。外源功能化蛋白质虽在一定程度上降低了丝的形成速度和机械性能,但修饰上的功能化蛋白仍保持着生物活性,表明断裂蛋白质内含子介导的蛛丝修饰平台成功建立,也为蛛丝的功能化修饰和应用奠定了坚实的技术基础。     

Engineered disulfides improve mechanical properties of recombinant spider silk

Nature's high‐performance polymer, spider silk, is composed of specific proteins, spidroins, which form solid fibers. So far, fibers made from recombinant spidroins have failed in replicating the extraordinary mechanical properties of the native material. A recombinant miniature spidroin consisting of four poly‐Ala/Gly‐rich tandem repeats and a nonrepetitive C‐terminal domain (4RepCT) can be isolated in physiological buffers and undergoes self assembly into macrofibers. Herein, we have made a first attempt to improve the mechanical properties of 4RepCT fibers by selective introduction of AA → CC mutations and by letting the fibers form under physiologically relevant redox conditions. Introduction of AA → CC mutations in the first poly‐Ala block in the miniature spidroin increases the stiffness and tensile strength without changes in ability to form fibers, or in fiber morphology. These improved mechanical properties correlate with degree of disulfide formation. AA → CC mutations in the forth poly‐Ala block, however, lead to premature aggregation of the protein, possibly due to disulfide bonding with a conserved Cys in the C‐terminal domain. Replacement of this Cys with a Ser, lowers thermal stability but does not interfere with dimerization, fiber morphology or tensile strength. These results show that mutagenesis of 4RepCT can reveal spidroin structure‐activity relationships and generate recombinant fibers with improved mechanical properties.     

Vibrational infrared conformational studies of model peptides representing the semicrystalline domains of Bombyx mori silk fibroin

The structural organization of Bombyx mori silk fibroin was investigated by infrared (IR) spectroscopy. To this aim, (AG)15 and other model peptides of varying chain length, containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid phase method and their spectroscopic properties were determined. Both the position and the relative content of Y, V, and S residues in the (AG)n model system appeared critical in determining the preferred conformation, i.e., silk I, silk II, and unordered structures. Curve fitting analysis in the amide I range showed that the model peptides with prevailing silk II structure displayed different beta-sheet content, which was dependent on the degree of interruption of the (AG)n sequence. In this regard, the bands at about 1000 and 980 cm(-1), specifically assigned to the AG sequence of the B. mori silk fibroin chain, were identified as marker of the degree of interruption of the (AG)n sequence.A stable silk I structure was observed only when the Y residue was located near the chain terminus, while a silk I --> silk II conformational transition occurred when it was positioned in the central region of the peptide.Analysis of the second-derivative spectra in the amide I range allowed us to identify a band at 1639 cm(-1) (4 --> 1 hydrogen-bonded type II beta-turns), which is characteristic of the silk I conformation.     

Facts and myths of antibacterial properties of silk

Silk cocoons provide protection to silkworm from biotic and abiotic hazards during the immobile pupal phase of the lifecycle of silkworms. Protection is particularly important for the wild silk cocoons reared in an open and harsh environment. To understand whether some of the cocoon components resist growth of microorganisms, in vitro studies were performed using gram negative bacteria Escherichia coli (E. coli) to investigate antibacterial properties of silk fiber, silk gum, and calcium oxalate crystals embedded inside some cocoons. The results show that the previously reported antibacterial properties of silk cocoons are actually due to residues of chemicals used to isolate/purify cocoon elements, and properly isolated silk fiber, gum, and embedded crystals free from such residues do not have inherent resistance to E. coli. This study removes the uncertainty created by previous studies over the presence of antibacterial properties of silk cocoons, particularly the silk gum and sericin. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 237–245, 2014.     

Evolution of aerial spider webs coincided with repeated structural optimization of silk anchorages

Physical structures built by animals challenge our understanding of biological processes and inspire the development of smart materials and green architecture. It is thus indispensable to understand the drivers, constraints, and dynamics that lead to the emergence and modification of building behavior. Here, we demonstrate that spider web diversification repeatedly followed strikingly similar evolutionary trajectories, guided by physical constraints. We found that the evolution of suspended webs that intercept flying prey coincided with small changes in silk anchoring behavior with considerable effects on the robustness of web attachment. The use of nanofiber based capture threads (cribellate silk) conflicts with the behavioral enhancement of web attachment, and the repeated loss of this trait was frequently followed by physical improvements of web anchor structure. These findings suggest that the evolution of building behavior may be constrained by major physical traits limiting its role in rapid adaptation to a changing environment.     

银纳米线对再生蚕丝性能的影响

近年来,再生蚕丝作为一种重要的生物资源已被广泛应用于人造组织、生物支架等领域,对其力学性能提出了较高的要求。为提高再生蚕丝的力学性能,采用化学还原法合成直径为80~100 nm、长度为8~11 μm的银纳米线（AgNWs）,并将其作为共混材料,通过湿法纺丝工艺制备再生蚕丝,探讨AgNWs浓度对再生蚕丝力学性能和结构的影响。结果表明：不同浓度AgNWs均有利于提高再生蚕丝的力学性能,AgNWs浓度为4‰时,再生蚕丝的力学性能最佳,断裂强度可达233.41 MPa,断裂伸长率可达88.37%,显著高于普通的再生蚕丝（158.13 MPa和15.02%）。扫描电镜和红外光谱发现,添加AgNWs对再生蚕丝的形貌和直径无明显改变,但可促进丝素蛋白β-折叠构象形成。