Review Article

Engineering Silk for Versatile Applications

N Gokarneshan¹*, SH Ritti², B Sainath², D Anita Rachel³, N Saravanan⁴, K Velumani⁴, V Tamilanban⁴, R Hari Priya⁵ and B Padma⁵

¹Department of Textile Chemistry, SSM College of Engineering, India

²Department of Textile Technology, Rural Engineering College, India

³Department of Fashion Designing, Footwear Design and Development Institute, India

⁴Department of Textile Technology, SSM College of Engineering, Komarapalayam, India

⁵Department of Costume Design and Fashion, Dr. SNS Rajalakshmi College of Arts and Science, India

Submitted: July 27, 2024; Published: August 23, 2024

*Corresponding author: N Gokarneshan, Department of Textile Chemistry, SSM College of Engineering, Komarapalayam, Tamil Nadu, India

How to cite this article: N Gokarneshan*, SH Ritti, B Sainath, D Anita Rachel, N Saravanan, K Velumani, et al. Engineering Silk for Versatile Applications. JOJ Material Sci. 2023; 8(5): 555750. DOI:10.19080/JOJMS.2024.08.555750

Abstract

Silks spun by the arthropods are “ancient” materials historically utilized for fabricating high-quality textiles. Silks are natural protein-based biomaterials with unique physical and biological properties, including particularly outstanding mechanical properties and biocompatibility. Current goals to produce artificially engineered silks to enable additional applications in biomedical engineering, consumer products, and device fields have prompted considerable effort toward new silk processing methods using bio-inspired spinning and advanced biopolymer processing. These advances have redefined silk as a promising biomaterial past traditional textile applications and into tissue engineering, drug delivery, and biodegradable medical devices. In this review, we highlight recent progress in understanding natural silk spinning systems, as well as advanced technologies used for processing and engineering silk into a broad range of new functional materials.

Keywords: Natural materials; Arthropod; Drug delivery; Nuclear magnetic resonance; Proteins; Biomaterials; Biomedical engineering; Biomedical equipment; Biopolymers; Tissue engineering

Introduction

Silks are protein-based biopolymers spun by thousands of arthropod species, particularly silkworms and spiders, for various applications throughout their lifetime, including orb web construction for prey capture and housing for protection and reproduction [1-5]. In general, silks possess extraordinary physical and biological properties including superior mechanical properties to biocompatibility, making them attractive materials. Over thousands of years, silk fibers unraveled from cocoons spun by the silkworm Bombyx mori (B. mori) have been used on the commercial scale for fabricating soft and durable textiles for apparel and clinical sutures.

In recent decades, with an improved understanding of the physical properties of different silks from spiders to insects, spider silks have drawn considerable attention due to their superior mechanical properties compared to silks spun by other insects, for example, silkworms and the caddisfly [6,7]. Some of the silks provide an excellent combination of high tensile strength and extensibility and are able to absorb more energy per weight compared to synthesized industrial materials with high strength like Kevlar. The dragline silk spun from spiders typically possesses a tensile strength >800MPa and an astonishing toughness >110MJ/m3. These outstanding mechanical properties make spider silks one type of promising high-performance structural biomaterial for consideration in future applications [8-10]. However, unlike silkworm silks, spiders cannot produce sufficient natural silk at a commercial scale that is required for most applications, although they were used by the ancient Greeks for wound treatments and by natives in New Guinea to make fishing nets and bags. To overcome the challenge of producing spider silk at a large scale, technologies are being developed to generate artificial spider silks with physical properties similar to those of natural silk by biomimicking the natural spinning process with silk-like recombinant proteins [11- 14].

Aside from using silks for textiles, since the late 1990s, considerable efforts have been made to extend the applications of silks into other fields such as biomedical engineering, tissue engineering, and biomedicine by taking advantage of the biocompatibility and enzymatic degradability of silk-based materials [15]. Toward these needs, silk-based materials, including natural and recombinant silks, have been fabricated in a variety of material forms, including films, sponges, scaffolds, fibers, tubes, and nano-/micro-particles for specific applications. In this review, mechanisms of natural silk spinning and techniques developed to artificially transform silk into next-generation materials will be reviewed [16,17]. Further, challenges and opportunities for future engineering of silk materials will be discussed.

Structure of Natural Silks

In nature, a large variety of silks with structures and properties are produced by different species of spiders and insects. For example, silkworms primarily produce silk to construct cocoons for self-protection during metamorphosis. Female orb-weaving spiders can spin up to seven different types of silks that are specifically used for making webs, prey capture, and reproduction. Embioptera (Webspinners) produce nano-sized silk fibers for protection and breeding. Trichoptera (Caddisfly) use silk to hunt and protect themselves in their aquatic environment. Surprisingly, in most structural silks across species, the molecular structure is conserved in high molecular weight amphiphilic sequences composed of highly repetitive amino acid motifs (Figure 1(a)). These repetitive motifs typically form into nanocrystalline β-sheet domains via strong and stable hydrogen bonding, while the nonrepetitive sequences form less structured regions, including helices, coils, and turns. In this review, we primarily focus on cocoon silk from the silk moth B. mori and spider dragline silk from Nephila clavipes (N. clavipes) since the majority of silkbased studies are on these two types of silk [18-22]. In general, cocoon silk and spider dragline silk are composed of hierarchical structures where semi-crystalline nanofibrils organize into fiber with typical diameters of 1–20μm (Figure 1(b)). The molecular structure of cocoon silk protein (fibroin) consists of a heavy (H) chain of ∼390 kDa and a light (L) chain of ∼26kDa, connected by a disulfide bond at the C-terminus (CT) of H-chain, forming a H–L complex. The highly repetitive GAGXGA (X = S, Y, V) sequence in the heavy chain makes up the bulk of the crystalline/semi-crystalline domains in the fibers [23-28]. Spider dragline silk typically refers to major ampullate silk that most studies are carried out with toward fundamental understanding of the structure-property relationships as a guide to artificial production of synthetic spider silks using recombinant DNA technology. The core constituents of spider major ampullate silk are two fibrous proteins called major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), each with a molecular weight of 250–350 kDa.24 Both proteins contain large central, repetitive motifs of poly(Ala), poly(Gly–Ala), Gly–Gly–X or Gly–X–Gly (X = Gln, Leu, Tyr) where ploy(Ala) and ploy(Gly–Ala) form the β-sheet (dominated by antiparallel) structures organized into nano crystallites with approximate sizes of 2 × 5 × 7nm3.

A variety of techniques have been utilized to gain insight into molecular structures of silks, including x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR)/Raman spectroscopy, transmission electron microscopy (TEM), and nuclear magnetic resonance (NMR) spectroscopy [29-31]. However, only a limited molecular-level picture has been obtained along with some remaining challenges such as the molecular structure of the amorphous regions in silk fibers and protein– protein interactions in the producing glands and the spun fibers. More effort is required to achieve complete understanding of silk structure-function relationships for providing an improved guide in the design and assembly of synthetic silks to recapitulate native properties where needed. Nevertheless, inspiration has driven a great deal of progress into silk-based biomaterials in recent biomedical research.

Natural Spinning of Silks

Over millions of years, silkworms and spiders have developed sophisticated biological fiber spinning systems where silks are generally spun via a protein self-assembly process from highly concentrated protein solutions (known as “dope”) or gels into solid fibers. Specifically, the silk proteins are synthesized and stored in silk glands at high concentrations (∼25-50wt. % for silkworms and spiders) [32-36]. Upon spinning, the dope is forced to flow through a specially shaped spinning duct and is subjected to a series of physiological changes such as shear, pH, metal gradient, and salt gradients. During the process, the silk proteins undergo phase transitions along with self-assembly to form solid, semi-crystalline, insoluble fibers. Among the factors involved, pH gradients and shear forces are key in influencing the molecular structures and dynamics of the silk proteins. The flanking, nonrepetitive domains (N- and C-termini) of the silk proteins are sensitive to pH. Thus, for B. mori silk fibroin, the N-terminal domain undergoes a pH-sensitive conformational transition from random coil to well-ordered β-sheets at around pH 6.0, forming micelle-like oligomers. For spider silk protein, the N- and C-termini generally possess well-defined structures with five helices. In the gland, the C-terminal domains form into parallel-oriented dimers via a stable disulfide bond and electrostatic interactions [37- 41]. Such dimers further form into micelle-like structures where hydrophobic repetitive domains are hidden within the cores. These micelle-like structures are stable under physiological conditions in the gland. In contrast, the N-terminal domain is monomeric at physiological conditions in the gland and forms an antiparallel dimer when the pH level decreases in the duct that connects the gland to the spinneret. Site-specific mutations revealed that the N-terminus prevents premature aggregation of the protein and enables rapid fiber formation.

Shear force also plays a crucial role in the self-assembly of silk proteins during fiber formation. Rheological studies of the flow characteristics of native silk dopes showed that a higher shear rate (>5 s−1) led to instability of the proteins in silk dopes for both the silkworm and spider, leading to the formation of insoluble solid silk [42]. Further analysis indicated that both native silk dopes responded to shear force like a typical molten polymer, which inspires the development of artificial spinning systems as mimics of the natural process.

Artificial Processing of Silk

Bio-inspired spinning

Inspired by the natural biological spinning process, different approaches have been developed to artificially spin silk fibers with the goal to generate the molecular and hierarchical structures found in natural spider and silkworm silks. In general, artificial spinning systems can be classified into two categories: wet spinning and dry spinning systems. In wet spinning, the silk protein solution is extruded through a spinneret directly into a coagulation bath that initiates solidification into fibers via precipitation [43-47]. In contrast, dry spinning solidification of the fiber occurs due to the evaporation of a volatile solvent. Three common types of spinning dope have been used in bio-inspired spinning including native liquid silk isolated from silkworm or spider glands, regenerated silk solution, and recombinant silk solution. Regenerated silk is generally referring to silk protein extracted from natural silk fibers [48-50]. For instance, regenerated B. mori silk is typically obtained following a three-step process: fiber degumming by boiling the cocoons in basic solution; fiber dissolution by using denaturing agents [LiBr, HFIP, CaCl2, Ca(NO3)2]; solution purification by dialysis and centrifugation. Recombinant silk refers to the silkinspired protein synthesized using recombinant DNA techniques. Several types of silk protein motifs or silk-inspired proteins have been expressed successfully by genetically modified organisms such as bacteria, yeast, insect cells, plant cells, and mammalian cells. However, compared to natural silk proteins, the recombinant silk proteins are generally lower in molecular weight (<300kDa) due to the challenges in replicating the full sequence of natural silk genes in a heterologous host system [51,52].

Various artificial spinning methods have been invented with an emphasis on mimicking the natural spinning system. Some studies have shown that the artificially generated silks can exceed natural silk in terms of some of the mechanical properties summarizes the properties of some artificial fibers produced with various spinning dopes and methods [53-59]. The as-spun fibers normally show weak mechanical properties and require postspinning treatments, including stretching and alcohol treatment to enhance the mechanical properties. Recently, a sophisticated artificial spinning system was developed by combining shear force and pH gradients. Escherichia coli was used to express a water-soluble silk-like protein containing domains from the silks of two species of spider, Euprosthenops australis and Araneus ventricosus. The concentrated recombinant silk protein solutions were pumped through a glass capillary into an acidic bath, mimicking the conditions experienced by natural silk as it passes through a spider silk gland and ducts. The recombinant silk protein was solidified into fibers via self-assembly with a diameter of 10-20μm and showed some physical properties comparable to those of natural silk fibers, but with lower toughness [60-64].

A dry spinning method was developed to directly produce high performance regenerated silk fibers and involved spinning nematic silk microfibril solutions into fibers with direct extrusion into air. The nematic microfibril solution was prepared by partially dissolving native silkworm silk fibers into microfibrils, where the hierarchical structures in natural silk fibers were maintained in solution. The as spun regenerated silk fibers showed a maximum modulus of 11 ± 4GPa, higher than that of some natural spider silk. In addition, a microfluidic-based system was applied to mimic the natural spinning system. The regenerated silk fibers were dryspun from regenerated silk fibroin aqueous solution and showed higher toughness than degummed natural silk fibers, with a tensile strength of 614MPa, a strain of 27%, and a toughness of 101kJ/kg.

Electrospinning

Electrospinning has also been applied to produce artificial silk fibers with a wide range of diameters, from the nanoscale to microscale. The electrospun silk fibers h can be used to construct biomimic scaffolds based on the diameters and overall morphology. To fabricate the electrospun silk fibers, organic solvent-based and aqueous-based systems have been reported, including an allaqueous solution at low silk concentration (<10%, w/v%). The asspun fibers showed good biocompatibility [65]. While electrospun silk fibers possess structures and morphologies to mimic natural silk fibers, their mechanical properties are generally weak, which limits applications for higher performance structural materials.

Multidimensional assembly

In nature, silk is primarily produced in fiber form, followed by further construction into multidimensional form such as orb webs and cocoons. Artificial methods for spinning silk fibers were reviewed; here, the focus is on techniques developed for generating multidimensional (2D, 3D, and 4D) silk-based materials. Most multidimensional silk-based materials are generated via selfassembly [66-69]. For example, silk films are typically prepared by casting silk solution into a premade mold/surface with the subsequent slow evaporation of solvent (typically water). During the drying process, silk fibroin undergoes self-assembly where the final physical properties and molecular structures can be controlled by solvent, humidity, and temperature, combined with the rate of drying. Silk films prepared from aqueous silk solutions and cured at lower humidity (<75% RH) and ambient temperatures exhibit good water solubility, useful for fabricating silk-based bioresorbable devices The silk film holds and transfers prefabricated electrical circuits to the biological system. Furthermore, by addition of the salt and plasticizer, along with calcium chloride and glycerol, highly flexible silk films can be obtained. The plasticization of silk into skin-like softness was achieved by the addition of Ca2+ and the subsequent hydration via ambient humidity. The gold-based thin conductive layers on silk films form wrinkled structures during plasticization by ambient humidity, resulting in highly conductive and stretchable electrodes. Additionally, to tune the mechanical properties and molecular structures, a variety of post-treatment methods were utilized, including water-annealing, methanol, and stretching. In general, these treatments result in the formation of β-sheet structures (with variations in sizes and distribution depending on the treatment), which serve as the building blocks to form crystalline domains in the film.

Hydrogels are another important form of biomaterials with appealing applications in tissue engineering. Given the biocompatibility, biodegradability, and excellent mechanical properties of silk fibroin, processing silk into hydrogels has been explored. Typical methods to obtain silk hydrogels are via sol–gel transition, where a molecular network forms by controlling the formation of β-sheet structures as crosslinks for silk. A series of environmental factors including pH, salts, temperature, and solvents have been utilized toward this goal. Generally, a lower pH, higher salt concentration, and a higher incubation temperature facilitate increased kinetics toward the sol–gel transition. Recently, a robust silk-based hydrogel was generated via a combination of sol–gel transition and solvent-exchange. In this process, the silk fibroin transitioned from random coils and/or helical structures to β-sheets, which served as the physical crosslinks. The hydrogels possessed combined high strength and toughness for machining, such as by laser cutting and mechanical turning. Furthermore, silk hydrogels have been explored for various applications such as biocompatible scaffolds in cartilage regeneration and as biodegradable microfluidic devices.

Different types of silk-based micro-/nano-materials have also been developed with silk with promising applications from drug delivery to filtration to tissue engineering. Microfluidic-based strategies were developed to spin liquid native silk, obtained directly from the silk gland of B. mori silkworms, into micrometerscale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. Such microparticles enabled the encapsulation, storage, and release of active biomolecules such as functional antibodies. Silk microfibers were fabricated by alkali hydrolysis of natural silk fibers, where alkali (sodium hydroxide) initiated hydrolysis of amide bonds and resulted in the chopping of the natural silk fibers into microfibers. The silk microfibers obtained in this process were used as reinforcements in fabricating 3D silk-based scaffolds with a combination of tunable mechanical properties, surface roughness, and porosity. Biological studies showed that the scaffolds supported human bone marrow-derived mesenchymal stem cell (hMSCs) differentiation toward bone-like tissues in vitro and possessed minimal immunomodulatory responses in vivo, indicating potential utility as biomaterials for tissue engineering bone.

Silk nanoparticles are an interesting form with potential in drug delivery. These particles can be fabricated using a variety of methods, exploiting silk self-assembly. The most direct way to process silk into nanomaterials is physical milling, based on chopping and grinding natural silk fibers into nanoscale formats [70]. However, this approach normally requires a long-time frame and generally results in random nanoparticle aggregates with a broad size distribution. An alternative process to generate silk nanoparticles is via wet chemistry using regenerated silk solution. The self-assembly of silk in this process is controlled by monitoring environmental factors including pH, salt concentration, and solvent composition. For example, in the presence of solvents such as methanol, ethanol, and dimethyl sulfoxide (DMSO), silk fibroin undergoes structural changes and phase separation, forming nanoparticles. In addition, salting out represents another approach for the production of silk nanoparticles, where the nanoparticles are formed due to hydrophobic interactions between silk protein chains and the decrease in water molecules in the high concentration salt bath. An intriguing strategy for preparing silk nanoparticles directly during the dissolution step utilizes a formic acid (FA)/lithium bromide (LiBr) dissolving system with an optimized ratio to control the degree of dissolution degree of silk degummed fibers. After the usual dialysis and centrifugation processes that are used to prepare silk solutions, silk nanoparticles were formed directly and showed spherical shapes, no aggregation, and dimensions of about 100–200nm. In addition, silk-based ultrathin filtration membranes were formed with silk nanofibers (SNF) generated directly from exfoliated natural silk fibers. The membranes possessed a narrow distribution of pore sizes, ranging from 8 to 12nm, a very high pure water flux, and high separation efficiency for dyes, proteins, and colloids of nanoparticles.

By controlled self-assembly, silk fibroin can form 3D bulk materials (silk monoliths), which can be further machined into predesigned shapes to fit various biomedical applications, such as orthopedic fixation devices. illustrates the processing of silk fibroin into 3D bulk materials via different routes; controlled evaporation of water from aqueous silk solution results in a sol–gel–solid transition and solidification. An alternative route is to dissolve silk in organic solvents, such as HFIP, followed by long-term methanol treatment to induce β-sheet structures. After removing the solvent by thorough drying, silk bulk materials were obtained. Recently, a heat compression-based method was developed to transform amorphous silk directly into compact bulk materials without added solvent [71,72]. This method is advantageous over traditional solution self-assembly methods based on the shortened time and improved efficiency, along with the flexibility of fabricating composite materials. In addition, the exploration of processing solid silk materials provides an important path to engineer silks with tools developed for commercial polymer processing; thus, a significant step toward industrial production of silk-based bulk materials.

To achieve high-performance biomaterials with desirable functions, artificial approaches inspired from natural material engineering processes to construct hierarchical structures are an intriguing strategy. Recently, a bio-inspired process was developed to generate hierarchically defined structures with multiscale morphology by using regenerated silk fibroin. The combination of protein self-assembly and microscale mechanical constraints was used to form oriented, porous nanofibrillar networks within predesigned macroscopic structures. This approach provided a path to fabricate macroscale material geometries including anchors, cables, lattices, and webs with predefined mechanical and physical properties.

Light-assisted processing

Femtosecond lasers have attracted attention for the precise, non-thermal processing of materials with control over structural damage. Recently, this optical technique has been applied to processing silk hydrogels under ambient conditions with an emphasis on non-invasive shaping and hetero structuring of silk. Based on the nonlinear multiphoton interactions of silk with a few-cycle femtosecond pulses, two approaches were proposed for optically processing silk: plasma-assisted ablation with higher laser intensity and photon-induced bulging with lower laser intensity [73-76]. Plasma-assisted ablation facilitates noninvasive shaping of silk including localized nano cutting and micro patterning. Photon-induced bulging allows micro welding of silk with materials such as metal, glass, and Kevlar, and with strength comparable to pristine silk [77]. Molecular structural analysis revealed that the polypeptide backbone remained intact while the weak hydrogen bonds were disrupted. Using this approach, silk-based functional topological microstructures, such as Mobiüs strips, chiral helices, and silk-based sensors, were fabricated.

Biomicrofabrication of silk

Inspired by advanced micro/nano-manufacturing technologies in the semiconductor industry, several multiscale manufacturing methods have been used to engineer silk-based materials, including photolithography, soft lithography, nanoimprinting lithography, and scanning probe lithography covering 2D to 3D and nanoscale to macroscale systems. A combined electronbeam lithography (EBL) and ion-beam lithography (IBL) system was used to generate complex and arbitrary 3D silk-based microstructures. Briefly, the IBL constructs the designed 3D structures from top to bottom, since the ions cross-link the silk protein while the EBL generates the structures from bottom to top since the electrons can penetrate and cross-link the silk protein. In addition, soft lithography techniques were applied to generate silkbased micro/nano-patterned structures, including 3D silk-based photonic crystals or silk inverse opals (SIOs) as biocompatible optical devices. Generally, the SIOs were fabricated by infiltrating silk solution into a pre-templated 3D poly (methyl methacrylate) (PMMA)/polystyrene (PS) sphere array [78]. Furthermore, the structure of SIOs could be tailored by water vapor (WV) annealing or exposure to ultraviolet (UV) radiation exposure to obtain structural color-coded patterns.

3D bioprinting

Integrating advanced 3D printing technology with silk processing offers an enabling approach to generate different material platforms, such as tissue engineering scaffolds and smart devices. Silk-based “bioinks” can be either pure silk solutions or functional silk solutions tailored by chemical modifications, by doping, or by mixing with functional components. Directwriting of concentrated regenerated silk fibroin (28-30wt.%) into a methanol-rich bath results in 3D, micro-periodic scaffolds. The printed single filament is at diameter as small as 5μm. The printed scaffold supports the adhesion and growth of human bone marrow-derived mesenchymal stem cells (hMSCs). In addition, the scaffold increases the production of glycosaminoglycan and the chondrogenic differentiation. This method has been further extended by integrating hydroxyapatite (HAP) into the silk for bone regeneration. By incorporating functional materials/ dopants such as polyol, synthetic nanoclay, gelatin, polyethylene glycol (PEG), glycerol, and Konjac gum into silk solution we can further create a variety of silk-based bioinks which allows freestanding bioprinting, and structural enhancement. Dopants can also include cells for the direct construction of 3D structures. Silk/ polyethylene glycol (PEG) bioink containing human bone marrow mesenchymal stem cells (hMSCs) was used to print a variety of tissue constructs with high resolution and homogeneity. The cellloaded constructs maintained their shape over at least 12 weeks in culture [79-84]. Furthermore, a higher concentration silk solution (10 wt. %) facilitated cell growth, suggesting that these silk/PEG gels may provide suitable scaffold environments for cell printing.

Besides inject printing, light-based 3D printing technology has also been applied to construct silk-based 3D structures. Recently, a silk-based bioink was developed by functionalizing silk using glycidyl methacrylate (GMA), which was used to build complex organ structures, including the heart, vessel, brain, trachea, and ear with the assistance of digital light processing (DLP) (Figure 2). The printed structures showed excellent structural stability and cytocompatibility.

Conclusion

Silk, a widely applied material for consumer use in textile applications for centuries, has been reinvented and redefined in recent years as a versatile biomaterial for many biomedical applications and other utilities. With advanced spectroscopy and biotechnology, a lot of intriguing physical and biological properties of silks have been revealed, particularly, the outstanding mechanical properties and biocompatibility of the protein in material formats. These appealing properties have attracted interest from many fields including chemistry, physics, and bioengineering. In general, two primary focuses have been established with the same goal of exploring broad applications of silk from high performance structural biomaterials to biomedical applications. One is to develop technologies to generate artificial fibers superior to the natural silk fibers. Based on the fundamental understanding of composition, molecular structure, and selfassembly in natural silks, silk-like proteins (recombinant silk proteins) have been generated, particularly spider silk-mimetic proteins, and these have been used as spinning dopes to form artificial fibers. However, due to the complex physiology involved in the natural silk spinning process, full-mimic artificial spinning is difficult to achieve. More effort is needed to better understand the details of the natural process and then to optimize the artificial spinning system. The other focus is to develop technologies to process silk into functional materials that can be used in biomedical applications, such as tissue engineering and implantable medical devices. In recent years, silk has been demonstrated as a versatile biomaterial that can be engineered using a variety of technologies, such as film casting, gelation, lithography, and 3D printing. However, most of these processing techniques rely on silk fibroin solution to generate the materials, which can be time and solvent intensive. The recent discovery of silk processing with classical polymer processing techniques such as thermal molding shows promise to boost future industrialization of silk processing. Recent studies showed that regenerated amorphous silk fibroin can be directly molded into pre-designed shapes or parts with heat compression, which provides a new path for engineering silk materials as is performed with synthetic polymers.

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