Fibre-reinforced hydrogels with high optical transparency

Cellulose fibre

Cellulose, the most abundant natural polymer, ^8,9 is composed of linear polymer chains of β-1,4-linked glucose residues. The structural repeat unit of cellulose is shown in Fig. 1. The six hydroxyl groups per mer in cellulose make it amenable for chemical functionalization to change its properties. For example, acetylation has been shown to change its optical properties. ¹⁰ Most of the cellulose on our planet is made by vascular plants, such as trees. Wood derived celluloses also have lignins and hemicelluloses intimately mixed with the cellulose. Other than vascular plants, cellulose is also made by natural organisms such as algae, slime mould, several bacteria and tunicates. ¹¹ Of all the sources of cellulose, bacterial (sometimes referred to as microbial) cellulose and plant based (e.g. wood and cotton) cellulose have been widely studied in recent years because of its interesting wound care, tissue engineering, structural and optical properties. ^9,10,12–26 Cellulose fibres also have advantageous gas barrier properties in addition to their excellent transparency. ²⁷

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1.

Structural repeat unit of cellulose

Cellulose is a polymorphic polymer. Depending on the source and processing method of the cellulose, the molecular weight, crystal structure and percent crystallinity can vary greatly, which in turn substantially impact the properties of the material. Cellulose I is most commonly produced in nature by plants and is a crystalline form of cellulose in which the cellulose chains align parallel to each other. ¹¹ Cellulose I is also produced by bacteria such as Acetobacter xylinum. In those bacteria, nanofibres are ejected from a series of pores on the cell wall and aggregate to form ∼15–40-nm ribbon shaped long fibres. A wet and supercritical CO₂-dried mat of bacterial cellulose is shown in Fig. 2. Cellulose I contains two crystalline sub-allomorphs in varying amounts that are termed I_α and I_β. The crystal packing, hydrogen bonding and molecular conformation of I_α and I_β differ, which could influence the structural and optical properties. ¹¹ Bacterial and algal cellulose are I_α rich, with bacterial cellulose consisting of 100% pure I_α form. In contrast, cellulose found in wood, ramie, cotton and tunicates is rich in the I_β form. Note that cellulose I_α exists in a metastable state and when annealed undergoes a phase transformation to the I_β form. Another crystalline state of cellulose that has an anti-parallel chain arrangement is termed cellulose II. Cellulose II is produced by some organisms and mutants of A. xylinum, which normally produces cellulose I. Cellulose can also exist in an amorphous state. The cellulose produced naturally can contain varying amounts of Cellulose I, Cellulose II and amorphous cellulose and have vastly different properties depending on the cellulose phase and morphology of the material.

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2.

a Plant based cellulose powder, b cotton based cellulose powder and c supercritical CO₂-dried mat of bacterial cellulose as viewed in the SEM; d mat of bacterial nanocellulose fibres over a person’s hand (for size reference) ²⁹

One interesting method to produce cellulose nanofibres is a top-down method, which involves high power sonication. Zhao et al. ²⁸ showed that sonication can be used to generate ultrafine cellulose nanofibres from wood and other natural sources. Sonication produces uniformly sized nanofibres compared to other methods with the resultant fibres in the 25–120 nm diameter range. The process is environmentally friendly, scalable, and facilitates retention of the hierarchical structure of natural materials.

Of particular interest for this paper are celluloses that are naturally made into nanofibres or can be processed into nanofibres, because nanofibres are expected to provide a good route to high strength and high transmittance hydrogels. An excellent review of cellulose nanofibres and nanocomposites is given by Eichhorn et al. ¹⁸ Cellulose nanofibres that are particularly relevant to incorporation in a hydrogel as a reinforcing element are briefly described below. Nanofibre mats (also known as pellicles), such as those shown in Fig. 2, could be utilized as reinforcing agents in hydrogels. The density of the mat can be controlled by optimizing the growth time. Shorter growth times lead to thinner, less dense pellicles. One could also employ top-down processing methods to produce cellulose nanofibre mats. For example, high power sonication was shown to be very effective in extracting nanofibres from natural materials including cotton, bamboo, wood, ramie and hemp. ³⁰ Furthermore, nanofibrillated cellulose (NFC) produced by dissolving wood pulp fibres and grinding them iteratively can also be used to generate mats of cellulose nanofibres. ⁹ Finally, directed assembly has also recently been demonstrated to be a viable technique for producing patterned structures made of bacterial cellulose. Microfluidic channels with flowing media containing cellulose producing bacteria were utilized to create novel web and linear patterns of bacterial cellulose fibres (Fig. 3). ³¹

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3.

Optical and SEM images of patterned bacterial cellulose fibres in both a grid and a linear array pattern using microfluidic directed assembly techniques ³¹

Chitin and chitosan

Chitin, or poly(1→4)-2-acetamido-2-deoxy-β-d-glucose (Fig. 4a ), is the second most abundant natural polymer. ³² Polysaccharides chitin and chitosan can be regarded as chemical derivatives of cellulose in which the cellulose C-2 hydroxyl group is replaced by an acetamide group or amine group, respectively, in either chitin or chitosan. Chitin acts as a reinforcement material for cell walls in lower animals and plants whose foods are rich in nitrogen. Sources of chitin include the cuticular and exoskeletons of invertebrates including crabs, shrimp and insects, as well as cell walls of fungi, moulds and yeasts. Commercial chitin is typically purified from shell wastes of crabs, shrimp and krill generated from the seafood industry. Chitosan is the de-acetylated form of chitin, poly(1→4)-2-amino-2-deoxy-β-d-glucose (Fig. 4b ) and exists naturally in a few species of fungi. ³² Chitosan is commercially produced via deacetylation with concentrated alkali solutions. Chitin and chitosan most often exist as copolymers of glucosamines and acetylated glucosamines. Therefore, the degree of acetylation is one of the most important structural parameters for describing these polysaccharides. Similarly to cellulose, the properties of chitin and chitosan can be modified using various reactions involving the –OH and –NH₂ groups. Typical reactions and products include chitosan salts, N-acylation, Schiff bases and their reduced products, reaction with halogen-substituted compounds, and chelation of metal ions. ³²

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4.

Chemical structure of a chitin and b chitosan ³²

Chitin and chitosan fibres are typically made by wet spinning, ³² in which fibres are produced by dissolving the polymer in a solvent and then extruding the solution via fine holes into a non-solvent. The precipitate is in the form of a filament, which can be washed, drawn and dried to form fibres. Dissolution of chitin has proven difficult, but can be achieved with dimethylacetamide (DMAc) or N-methyl-2-pyrrolidone and lithium chloride. ³² Dissolution is greatly improved by treatment of chitin with p-toluene sulfonic acid in i-propanol. ³³ The viscosity of the polymer solution can be controlled via the chitin pretreatment (up to 2 h showed added effects), LiCl concentration (optimum 5–9%), chitin concentration (optimum spinnable solution with 5–9% chitin) and temperature. Chitin purified from red crabs was made into fibres by Nakajima et al. ³⁴ via dissolution in an amide–LiCl system. Filaments of 5 μm diameter were spun from a spinneret of 0·05 mm diameter, using a butyl alcohol coagulant. The resulting fibres had demonstrated a tensile strength of 490 MPa. Chitin fibres of 20–80 μm diameter were made by Agboh ³³ via extrusion through 50–150 μm diameter holes directly into a coagulation bath, or via extrusion through spinnerets with 150–400 μm diameter into an air gap before introduction into the coagulation bath. Coagulants used included 75 : 25 DMAc/H₂O at 40–75°C or methanol at room temperature. Chitosan is a more easily soluble polymer than chitin. Fibres have been made by dissolving chitosan (2–4%) in aqueous acetic acid (0·5–1%) and extruding into 5% NaOH, ³⁵ Na₂SO₄ with NaOH, ³⁶ 2% Na lauryl sulphate, ³⁷ or CuSO₄–NH₄OH or CuSO₄–H₂SO_4. ³⁸ In the latter case, the fibres were a copper–chitosan complex, but the copper was removable afterward.

Natural chitin exists in three crystal structures: α, β and γ. ^32,39 The structural differences between these polymorphs lead to differences in mechanical behaviour, as outlined in Table 1. Chitin fibres can also be made in highly oriented structures when stretched via dry-jet wet-spinning, ³² or other stretching processes. The mechanical properties of chitin fibres are determined by the synthesis conditions and spinning techniques utilized. ³² The dry strength of chitin fibres has been shown to increase with acetylation. ³⁶ Fibre wet strength showed an initial decrease, followed by subsequent increase upon further acetylation. ³⁶ This can be explained because the acetamide groups form hydrogen bonds, causing the crystallinity and strength of inter-chain forces in chitin to increase. ³²

Alginate

Alginate, a natural polysaccharide, is extracted from brown seaweeds by treatment with aqueous alkali solutions, such as NaOH. During this process, the salt form of natural alginate is converted to water-soluble sodium alginate, ⁴⁰ which can be precipitated by addition of calcium chloride. Water-soluble sodium alginate powder can be produced following purification. ⁴¹ Alginate is a block copolymer composed of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) residues (Fig. 5). ⁴² Alginates extracted from different seaweed species have different M/G ratios, resulting in different physical properties. ⁴² Additionally, the polymer chains can be composed of three types of blocks, ⁴³ as described in Table 2, which also lead to differences in ⁴² physical properties.

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5.

Chemical structures of a β-d-mannuronic acid and b α-l-guluronic acid ⁴²

To make alginate fibres, sodium alginate is first dissolved in water. Solution viscosity can be controlled by varying concentration and molecular weight. To form filaments by extrusion through spinneret holes, viscosities in the range of 10 000–20 000 mPa are needed. ⁴² This can be achieved with high concentrations of low molecular weight or low concentrations of high molecular weight. To balance production efficiency with fibre strength, viscosities of 40–100 mPa·in 1% solution are ideal. ⁴⁴ Wet-spinning processes typically use spinning solutions with 5–6% sodium alginate. ⁴² Variations in temperature (depolymerization at T>50°C for several hours) and pH (unaffected in pH range from 5 to 11) can also affect the solution viscosity and must be controlled during synthesis. ⁴² Extrusion of sodium alginate solution through spinneret holes into a calcium chloride bath results in water insoluble calcium alginate fibres. Calcium ions coordinate and pack within interstices between buckled guluronic acid units, resulting in a firm gel structure. ⁴⁵ Gelation time varies linearly with alginate concentration, increases notably with fibre radius and is inversely proportional to calcium concentration. ⁴⁶ There is no relationship between gelation time and G/M ratios. ⁴⁶ Extrusion into a methanol, ethanol, isopropanol, or acetone coagulation bath will result in sodium alginate fibres, but these are of lower tenacity than calcium alginate fibres. ⁴⁴ For wound healing applications, it is desirable to produce calcium sodium alginate fibres, because the calcium ions provide integrity to the wet fibres, while the sodium ions provide absorbency. To produce these fibres, calcium alginate fibres are washed with HCl to replace some calcium ions with hydrogen ions, which are subsequently replaced with sodium ions by treatment with sodium carbonate or sodium hydroxide. ⁴⁷ The ratio of calcium to sodium ions can be tailored to specifically engineer the desired gel swelling and absorbencies.

Collagen

Collagen is a rather stiff and hard protein with several levels of hierarchy, as depicted in Fig. 6. Tropocollagen molecules, which have lengths of ∼280 nm and diameters of ∼1·5 nm, ⁴⁸ comprise three polypeptide strands (alpha chains), each with the conformation of a left-handed helix with approximately 0·87 nm per turn. ^39,49 The amino acid sequence in each alpha chain varies with collagen type. ³⁹ Three left-handed helices are twisted together into a right-handed triple helix, a cooperative quaternary structure stabilized by numerous hydrogen bonds. ⁵⁰ Each triple-helix associates into a right-handed super–super–coil referred to as the collagen microfibril. Microfibrils, in turn, arrange themselves into fibrils, which are organized into fibres, or bundles of fibrils with diameters between 0·2 and 12 μm. ³⁹ In some tissues, the fibres organize themselves into more complex patterns, leading to two- and three-dimensional networks. Aside from amino acid sequence, other distinctions between the collagen types are due to variations in length of non-helical fraction, helix length and the number and nature of carbohydrate attachments. ^39,50 Collagen in skin, tendon and bone is mostly type I, whereas type II makes up cartilage and type III is a major constituent of blood vessel walls. ⁵⁰

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6.

Schematic illustrating the structural features of collagen, which range from the amino acid sequence to collagen fibres with lengths of ∼10 μm ⁴⁸

Collagen can be extracted from tissue using two different methods – one resulting in molecular collagen and the other resulting in fibrillar collagen. ⁵¹ Isolation of molecular collagen requires cleavage of the non-triple helical telopeptides of collagen molecules via use of a proteolytic enzyme, such as pepsin. ⁵² This process renders the collagen molecules soluble in an aqueous solution, because it is the telopeptides that form intermolecular cross-links in vivo. The resulting molecular collagen is called soluble collagen or atelocollagen. These molecules can be reconstituted into various polymorphs of collagen, but the packing is not as efficient as with intact molecules because the telopeptides facilitate fibril formation. ⁵³ Repetitive precipitation of the pepsin-solubilized collagen with a neutral salt results in purification. ⁵¹

Isolation of fibrous collagen involves sequential removal of non-collagenous material from the tissue. ⁵¹ Salt extraction removes non-collagenous moieties that are soluble in aqueous conditions and also collagen molecules that have not yet been covalently integrated into the fibrils. Low molecular weight solvents are used to extract lipids, and acids are used to extract acidic proteins and glycosaminoglycans (GAGs) upon weakening of interactions between these species and collagen fibrils. Interactions between basic proteins and collagen fibrils are facilitated by alkaline extraction. Glycoproteins, proteoglycans (PGs) and elastins can be removed from tissue with enzymes such as collagenase. This sequential process results in an insoluble, intact collagen matrix, called insoluble collagen or fibrillar collagen.

The complex structure of natural fibres dictates their mechanical behaviour. For example, the structure of collagen fibres in vivo (e.g. in tendons) is characterized by waviness with a spatial period 2l ₀ and the angle θ₀. ³⁹ This wavy structure plays an important role in deformation; under tensile stress the bent fibres stretch out, and when the load is removed the waviness returns. When stretched beyond the straightening of the waviness, damage begins to occur. As can be seen in Fig. 7, the deformation of collagen occurs in three distinct stages. The first region, known as the Toe Region, is where the tendon operates under normal physiological conditions. The maximum permissible strain that a collagen molecule can experience without damage can be calculated according to: ³⁹ (1) where r is the ratio between wave amplitude and wavelength. A linear slope characterizes Region II. A study employing synchrotron small angle X-ray scattering (SAXS) to follow the molecular D spacing (typically ∼67 nm periodicity) of rat tail collagen as a function of imposed stretching showed negligible strains in the fibrils during the Toe Region, but linear deformation even at the molecular level once the transition to Region II occurs (Fig. 8). ⁵⁴ In Region III, the slope begins to decrease and additional strain leads to failure.

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7.

Stress–strain curve of tendon collagen exhibiting three characteristic stages of deformation: I, toe; II, linear; and III, failure ³⁹

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8.

Tensile load and corresponding increase in D-period of rat tail collagen during tensile loading combined with synchrotron X-ray scattering ⁵⁴

Spider silk

Silks, in general, exhibit interesting properties. In particular, spider silks are remarkable because of their mechanical properties, including a unique combination of tensile strength and extensibility. Its toughness exceeds that of steel and Kevlar. Many different types of spider silk exist, with structures and compositions that vary with their specific functionality related to catching prey. The two most important varieties of spider silks are the major ampullate silk, which composes the frame and radii of spider webs, and flagelliform silk, which composes the capture spiral of webs. ^55,56 Proteins produced in the major ampullate silk gland have a molecular weight of >300 kDa, diameters from 1 to 20 μm (depending on species) and a core–shell type structure, as illustrated in Fig. 9. ^56,57 Spidroins, the two structural proteins in the core, are composed mostly of glycine, alanine and proline, as depicted in Fig. 10. The spidroins resemble block copolymers containing blocks of polyalanine that form β-sheet stacks, and either (GGX) _n blocks (where X is tyrosine, leucine, or glutamine), which form 3₁₀-helices, or GPGXX blocks, which form β-turn spirals. ⁵⁶ The β-sheet stacks provide high tensile strength, whereas the turn spirals provide elasticity. The extremely high tensile strength (similar to Kevlar) and moderate elasticity make these silks ideal for use as the scaffold upon which other silks are attached during web construction and as a lifeline for escape in the event of a predator attack. Flagelliform silks, on the other hand, have a molecular weight of ∼500 kDa, are highly elastic, are composed of only one major protein containing greater amounts of proline and valine and less alanine. ⁵⁶ Flagelliform silks also are composed of (GGX) _n and GPGXX blocks as well as a polar hydrophilic spacer, which may be important for cross-linking and hydration. ⁵⁸

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9.

Schematic of a cross-section of major ampullate silk fibre ⁵⁶

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10.

The repetitive blocks of amino acids that make up the common secondary structural patterns in spider silk proteins ⁵⁶

Spider silk proteins can be synthesized in the laboratory using E. coli (HMS174(DE3) K-12 derivatives) as host cells via a process of high density fermentation and subsequent purification. ^56,59 Although naturally occurring spider silks are insoluble in water, recombinantly produced proteins with identical primary amino acid sequences exhibit varying solubilities, enabling versatility in solvent choice and therefore the ability to process these proteins into fibres, films, foams, spheres, hydrogels and capsules. ^56,60 The resulting structures and functions of the obtained materials are directly controlled by the content and distribution of crystalline β-sheets, a process that can be controlled by the mode of processing and environmental factors including solvent, pH, water and concentration of protein and salt. ⁶¹ Natural spider silk fibre production utilizes processes of ion exchange, water extraction, acidification and exposure to mechanical forces. To mimic these processes in the lab, potassium phosphate has been added to the synthesized proteins, and then hand-drawing was applied to subject the solution to shear forces. This resulted in production of smooth, β-sheet rich fibres. ⁶² Spider silk fibres have also been formed by electrospinning. ⁶³ Spin dopes prepared by dialyzing genetically engineered silk (MaSpI protein analogues) in urea containing Tris buffer and salts yielded fibres of 10–60 μm diameter with molecular orientation and mechanical properties similar to those of natural spider silk. ⁶⁴ Spin dopes derived from ADF-3 recombinant spider-silk protein yielded fibre diameters up to 40 μm with fibre toughness and modulus comparable to native dragline silks, but lower tenacity. ⁶⁵ Fibres with an average diameter of 300 nm were generated from Nephila clavipes MaSpI silk dope prepared in hexafluoroisopropanol, and control of conformation was achieved; results indicated that molecular alignment might be possible, potentially offering improved fibre properties. ⁶⁶

By adding methanol to recombinant forms of the dragline silk protein ADF-4, the silk has been shown to self-assemble into nanofibres with diameters of ∼3 nm and lengths less than 1 μm. ⁶³ Over the course of a few days, these nanofibres transformed into a hydrogel fibre network. These hydrogels exhibited a nonlinear viscoelastic material response with low stiffness and strength. Cross-linking was induced by visible light after applying ammonium peroxodisulfate and tris(2,20-bipyridyl) dichlororuthenium(II) to the surface of the hydrogel. In another approach, addition of sodium phosphate to aqueous solutions of engineered spidroins was shown to induce their self-assembly into β-sheet rich nanofibrils ⁶⁷ that hierarchically assemble into a sample spanning network that ultimately immobilizes the solvating water, yielding a hydrogel. ⁵⁶

Mechanical properties of natural and synthetic fibres are compared in Table 3. Natural fibres have a wide range of mechanical properties as would be expected after many years of being optimized by nature for their specific application. For example, the spider major ampullate silk exhibits significantly higher stiffness and strength than the viscid silk, but the two silks have nearly identical toughness due to the differences in extensibility required for their function (Fig. 11). The range in mechanical properties and deformation mechanisms available in natural fibres offer great potential for incorporation into hydrogels for various applications. Note that for applications requiring significant energy absorption, natural fibres exhibit significantly higher toughness (although lower strength) compared with traditional engineering materials of comparable density. Combination of natural and synthetic fibres into hydrogel composites could lead to materials with previously unattained mechanical properties.

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11.

Stress–strain curves for major ampullate gland silk (red line) and viscid silks (blue line) from the spider Araneus diadematus; E _init indicates initial stiffness ⁷¹

Polymer fibres

Polymer fibres are one type of synthetic fibre. The polymers are normally produced from petroleum based chemicals through chemical processing. There are many different types of polymer fibres having a wide range of mechanical and optical properties of interest for consideration as hydrogel reinforcement elements. In particular, polymer nanofibres are quite appealing for a transparent hydrogel reinforcement, because their size can be controlled to be less than the wavelength of visible light and their composition and surface chemistry tailored to achieve a suitable distribution within the hydrogel matrix. The most common classes of polymers include polyamides, polyesters, polyvinyls, polyolefins, acrylics and aramids. The following sections discuss the different classes of synthetic polymer nanofibres.

Polyamide fibres

Nylon 6 and Nylon 6,6 are two very common polyamide fibres commercially produced. Their chemical structures are shown in Fig. 12. One of the principal methods of producing Nylon nanofibres is via the electrospinning process, as shown in Fig. 13a . In this case, the Nylon is typically dissolved in a solvent such as a formic acid/acetic acid solution and then electrospun. By adjusting the electrospinning parameters (e.g. voltage, collector–syringe distance, solution concentration and constituents), it is possible to control the fibre morphology and the resultant mechanical properties. For example, Pant et al. ⁷² were able to systematically vary the modulus, ultimate tensile strength and strain to failure by using a formic acid solution to reduce the molecular weight of some of the dissolved Nylon chains by combining it with fresh solutions of Nylon to electrospin micro–nano Nylon fibrous mats. ⁷² Figure 13 shows the resultant morphology for four permutations. As the concentration of degraded Nylon increases, one notices an increase of smaller diameter fibres present in the micrographs. In Fig. 13d, a spider web-like network of Nylon nanofibres is present between the mat of ∼500 nm and 1 μm diameter fibres. The mechanical properties changed drastically with the appearance of the smaller nanofibres, as indicated by the stress–strain curves of Fig. 14. The tensile mechanical properties derived from Fig. 14 are shown in Table 4. The nanofibres have a much higher specific surface area compared with the microfibres and may also have relatively fewer defects within the fibre, which likely result in the improved mechanical properties reported in Table 4.

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12.

a Structure of Nylon 6 and b structure of Nylon 6,6

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13.

FE-SEM images of electrospun Nylon 6 nanofibrous mats containing a 0, b 5, c 10 and d 20 wt-% of solvent-degraded solution in freshly prepared solution ⁷²

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14.

a 0, b 5, c 10 and d 20 wt-% degraded Nylon 6 solutions in freshly prepared solution ⁷²

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Table 4.

Tensile strength, Young’s modulus and strain at break of Nylon 6 mats containing different fractions of solvent-degraded polymer solution ⁷²

Mats obtained from the different fractions of solvent (wt-%) degraded solution in freshly prepared solution	Tensile strength/MPa	Young’s modulus/MPa	Strain at break
0	7·2±2·3	49±8	0·51±0·08
5	9·3±2·1	69±7	0·60±0·07
10	10·1±2·4	56±5	0·72±0·05
20	13·3±1·8	64±9	1·13±0·12

Bazbouz and Stylios ⁷³ tested the mechanical properties of single Nylon 6 nanofibres as opposed to a whole mat as described for the Pant et al. research cited earlier. Figure 15 shows one of the experimental methods used by Bazbouz and Stylios to test the individual Nylon nanofibres, and Fig. 16 shows an example of the resultant stress–strain curve for a single 800 nm diameter Nylon 6 nanofibre. From the data in Fig. 16, the mechanical properties of a single Nylon 6 nanofibre are: Young’s modulus of 901·65 MPa, tensile strength of 304 MPa, yield stress of 136·9 MPa, yield strain of 15% and strain at break of 40%.

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15.

Individually aligned nanofibres are visible by light scattering. They are gripped between the spring and the fixed head through the cut cardboard frame. Non-required nanofibres were removed to prepare an electrospun single nanofibre for tensile testing ⁷³

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16.

Average stress–strain curve of a single electrospun Nylon 6 nanofibre ⁷³

Polyester fibres

A pure polyester, poly(ethylene terephthalate) (PET), nanofibre and a polyester block copolymer, poly(hexamethylene adipate)–PEO (PHA–b–PEO), nanofibre are good examples of polyester nanofibres that have practical application in the areas of filtration, medicine, pharmacy and agriculture. Their chemical structures are shown in Fig. 17.

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17.

PET mer (left) and PHA-b-PEO mer (right)

PET is a common polymer that has been widely used in textiles. Nanofibre mat morphologies of PET have been used as filtration membranes for juice purification. ⁷⁴ A hybrid PET–chitosan nanofibre mat showed improved anti-microbial activity compared with the PET nanofibre control and also showed better fibroblast compatibility. ⁷⁴ Duzyer et al. ⁷⁴ focused on the thermo-mechanical and structural properties of native PET nanofibres produced via electrospinning and the impact of various electrospinning parameters on the resultant properties. One parameter varied was the solution concentration (their electrospinning method was the solvent spinning method as described below in Synthesis of natural and synthetic fibres section). The diameter distributions and the measured tensile mechanical properties are shown in Table 5. One can see that the 13 and 16 wt-% solutions yielded nanofibres that were just submicrometre, on average. Wang et al. also adopted a similar method and studied the impact of PET molecular weight and polymer concentration on the resultant nanofibre morphology as shown in Fig. 18. The figure shows the enhanced fibre size homogeneity with increasing concentration of PET. ⁷⁵

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18.

The scale bar is 50 μm. ⁷⁵

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Table 5.

Diameter distributions and mechanical properties of the polyester nanofibres as a function of PET concentration ⁷⁴

PET/wt-%	Minimum diameter/μm	Maximum diameter/μm	Average diameter/μm	Young’s modulus/MPa	Ultimate tensile strength/MPa	Strain at Break
13	0·38	1·10	0·82	2·399	∼9·5	0·41
16	0·46	1·10	0·92	3·621	∼15·5	0·35
20	1·00	5·14	3·00	2·957	Not applicable	Did not fail

Sun et al. ⁷⁶ focused their efforts on developing a ‘green electrospinning’ process to yield nanofibres of a biodegradable aliphatic polyester compared to other biodegradable polymers such as poly(E-caprolactone) (PCL) and polylactic acid (PLA), which can be electrospun only using solutions containing harmful organic solvents. The synthesis process involved the following basic steps: polyester synthesis (melt polycondensation), suspension preparation (dissolution in acetone, addition of water and surfactant, evaporation of acetone), suspension concentration using dialysis and electrospinning. The resultant nanofibrous mat and fibre morphology are shown in Fig. 19. The figure shows a randomly oriented mat with fibres that are about 350–550 nm in diameter for the 4% PEO variety of PHA-b-PEO. The fibres are water stable after two days with only a slight increase in surface roughness, making them suitable for biological applications requiring uniformity and smoothness.

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19.

a SEM at 1000× of the 4% PEO PHA-b-PEO nanofibres, b zoom image at 20 000× of 4% PEO PHA-b-PEO nanofibres before water treatment and c the same as b but after water treatment for 2 days at room temperature ⁷⁶

Polyvinyl fibres

One polyvinyl family fibre that has been studied in great detail is polyvinyl alcohol (PVA). The structure of PVA is shown in Fig. 20. These fibres have many uses, such as reinforcement fibre in concrete.

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20.

Chemical structure of PVA

When comparing the surface area between a film of a material and a nanofibrous mat of the same material, the nanofibrous mat has a very large relative surface area. Therefore, potential medical applications for transdermal patches consisting of drug loaded nanofibre mats exist. Toward that end, Ngawhirunpat et al. ⁷⁷ studied the fabrication of capsaicin (CC)-loaded PVA nanofibre mats and compared their CC release properties to those of PVA films. During their electrospinning fabrication process, they examined the morphology of the as-spun PVA fibre mats that were loaded with CC as shown in Fig. 21. The average diameter of the fibres was in the 165 nm range (in the collected electrospun mat). Ngawhirunpat et al. also examined the tensile mechanical properties of the PVA fibre mats and films and found the average mat tensile strength to be 16·5 MPa and the as-cast films strength to be 20·7 MPa. ⁷⁷

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21.

Scanning electron micrographs of as-spun PVA fibre mats from 10% w/v PVA solutions loaded with CC at 0·05% by weight of PVA ⁷⁷

Most electrospun nanofibres have random orientations of fibres in the mat, similar to the mat shown in Fig. 21. However, some researchers have achieved uniaxial alignment by electrostatic repulsion induced self-assembly. Yan et al. ⁷⁸ have taken electrospinning self-assembly to the next level by developing the capability to self-assemble electrospun nanofibres into honeycomb-patterned nanofibrous structures (HNFSs). The authors have identified a range of experimental conditions over which PVA self-assembles into the structures shown in Fig. 22. For PVA, the pore size is ∼130–250 μm and the depth is over 150 μm. The porous structures could find end application as templates to synthesize other HNFSs such as oxides. The novel structures may also find use as tissue engineering scaffolds, catalyst supports, filters, sensors, micro-containers, battery electrodes and many other application areas. The authors attribute the unique microstructures to the delicate balance between surface tension and electrostatic repulsion forces.

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22.

SEM images of PVA HFNSs produced by electrospinning ⁷⁸

Polyolefin fibres

Polyethylene and, in particular, ultra-high molecular weight polyethylene (UHMWPE) fibres are of keen interest for many applications because of their unique combination of chemical and physical properties, which include high strength, abrasion resistance, impact resistance, good dielectric properties, as well as approval for use in food and biomedical applications. The fibres have traditionally been prepared by gel spinning technology relying on a series of draws that resulted in strengths of 1·5 GPa and an as-prepared modulus of 140 GPa. ⁷⁹ Rein et al. ⁷⁹ have tried to simplify the fabrication process by developing a simple one-step electrospinning fabrication method for UHMWPE nanofibrous mats. The morphology of the resultant nanofibres is shown in Fig. 23. In the studied parameter space, the fibres were a minimum of about 250 nm in diameter. The mechanical properties for a 65 μm diameter yarn of the UHMWPE nanofibres included a strength of about 129 MPa, an initial elastic modulus of 390 MPa and a strain to failure of about 33%. ⁷⁹

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23.

SEM image showing UHMWPE nanofibres electrospun from solutions in a 1 : 1 (w/w) mixture of p-xylene and cyclohexanone (CH) at a UHMWPE concentration of 0·025 wt-% ⁷⁹

Polypropylene (PP) is another important type of polyolefin for many applications such as filter media, biomedical replacement materials, reinforced composite materials and antistatic materials. Prior to about 2007 it had been extremely difficult or impossible to fabricate fine, uniform filaments (e.g. nanofibre filaments) of PP. All of that changed in 2007 when Wang et al. ⁸⁰ succeeded in developing a new method to form continuous yarns of nano-sized thermoplastic fibres. Their method is described by the flowchart in Fig. 24. The as-prepared isotactic PP nanofibres produced via the method shown in Fig. 24 are shown in Fig. 25 for hot drawn ratios of 2, 8 and 25. Based on the images in Fig. 25, it appears that there is at least a qualitative reduction in the fibre diameter as hot drawn ratio is increased. By analysing the images quantitatively, Wang et al. were able to produce a diameter histogram for each hot drawn ratio. The data are shown in Fig. 26 and reveal that the diameter distribution has a smaller range and also a smaller mean diameter (∼250–300 nm) for the highest hot drawn ratio PP nanofibre sample.

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24.

Flowchart describing the novel nano-PP fibre formation process developed by Wang et al. ⁸⁰

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25.

SEM images of iPP nanofibres from the CAB/iPP = 90/10 blends with hot drawn ratios of a 2, b 8 and c 25 ⁸⁰

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26.

Diameter distributions of iPP nanofibres prepared by removing the CAB matrix of CAB/iPP = 90 : 10 with three hot drawn ratios. a Hot drawn ratio = 2 (bar width = 200 nm); b Hot drawn ratio = 8 (Bar width = 100 nm); c Hot drawn ratio = 25 (Bar width = 50 nm)

Acrylic fibres

Poly(methyl methacrylate) (PMMA) is a very common acrylic polymer that is used for optical applications because it is clear in the visible spectrum and shatter resistant in comparison with glass. It is also used in various medical technologies and human implants. The chemical structure of PMMA is shown in Fig. 27. Because of its blend of optical properties, good mechanical properties and cost advantage compared to polycarbonate, Carrizales et al. ⁸¹ undertook a detailed study of the morphological and mechanical properties of electrospun PMMA nanofibres in mat form. The as-prepared PMMA nanofibres are shown in Fig. 28, and have an average diameter of 543±140 nm (as measured at the locations indicated in Fig. 28). ⁸¹ The tensile mechanical properties of the PMMA nanofibres are given in Table 6, and provide a suitable baseline for future research in this area.

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27.

Chemical repeat unit of PMMA

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28.

SEM of electrospun PMMA ⁸¹

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Table 6.

Tensile mechanical properties of PMMA nanofibre mats ⁸¹

	Modulus/MPa	Peak stress/MPa	Strain at break	Energy to break/N mm
Measured value for PMMA nanofibre mats	12·9±6·0	0·3±0·2	0·4±0·01	0·03±0·03

Functionalized PMMA nanofibres have also been developed with interesting properties/capabilities related to embedding fragrances and flavours into the fibres. For example, Uyar et al. ⁸² developed electrospun PMMA with cyclodextrin–menthol inclusion complexes. Cyclodextrins (CD) have been proven useful for stabilizing flavour/fragrance ingredients in the food, cosmetics and textile fields. The utility of CDs is derived from their toroidal structure, akin to crown ether molecules. Similar to crown ethers’ capability for binding cations or molecules with great size related specificity, the CDs can also stabilize molecules with particular size, conformation and charge. The chemical structures and general dimensions for γ, β and α CDs are shown in Fig. 29. Compared with electrospinning neat PMMA with menthol, the PMMA with CD-menthol produced nanofibre mats with reduced nanofibre diameters, as shown in Fig. 30.

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29.

a The chemical structure of cyclodextrin showing the functional groups; b, c and d dimensions of γ, β and α CDs, respectively ⁸²

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30.

SEM images of electrospun nanofibres of a PMMA/menthol, b PMMA/α-CD-menthol-IC, c PMMA/β-CD-menthol-IC and d PMMA/γ -CD-menthol-IC. Insets show higher magnification images ⁸²

Polyaramid fibres

While cellulosic media are used traditionally for filtration applications, polyaramid fibre mats provide a good non-woven high temperature option for filtration. One form of aramid polymer, termed meta-aramid due to its chemical structure (Fig. 31), has recently been successfully prepared in nanofibre mat form via electrospinning from a solvent system. Yao and Kim ⁸³ used a LiCl/DMAc solvent to electrospin meta-aramid nanofibres. The size and shape of the electrospun meta-aramid fibres are shown in Fig. 32. In all of the studied concentrations of aramid in the solvent system, the resultant fibres are sub-500 nm in diameter. The relationship between aramid concentration and resultant fibre diameter was also investigated (Fig. 33). The approximate tensile modulus for a meta-aramid nanofibre mat produced via electrospinning from an 11 wt-% aramid solvent was 370 MPa as determined through analysis of Yao and Kim’s reported stress–strain curve. The ultimate tensile strength was about 15 MPa and the strain to failure was approximately 13%.

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31.

Structural repeat unit of poly(meta-aramid)

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32.

a 8·5 wt-%; b 9 wt-%; c 11 wt-%; d 13 wt-% ⁸³

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33.

Dependence of fibre diameter on concentration of aramid in the electrospinning solvent system ⁸³

Glass fibres

Glass fibres have long been utilized to improve the mechanical properties of polymers through the design and fabrication of composites. As one can see from Table 7, the tensile strength and modulus of glass fibres are very high compared with those of natural fibres, and the low cost of glass compared with carbon fibres makes them compelling in many applications. The mechanical properties of E-glass and S-glass (two common compositional glass variants for fibre applications), as reported in Table 7, are for traditional diameter glass fibres (e.g. in the diameter range of ∼9–15 μm). While the tensile strength dramatically increases as the diameter of glass fibres decreases below about 12 μm (Fig. 34), the fibreglass manufacturers do not make smaller nanofibres due to human health concerns. However, with careful environmental controls and processing protocols, the health concerns could be mitigated; though, the extra costs ensued by implementing such controls may only permit niche, high value applications.

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34.

Tensile strength of glass fibres as a function of fibre diameter (Courtesy of K. K. Chawla)

Weichold et al. ⁸⁴ have developed an electrospinning process to generate silica nanofibre mats with filament diameters of 600–1000 nm. The process involves electrospinning a tetraethylorthosilicate precursor followed by subsequent heat treatment at ∼1000–1100°C. The best mechanical properties were observed when the heat treatment was performed at 1065°C. The morphology of the 1065°C treated nanofibre mat is shown in Fig. 35. Weichold et al.’s experiments have shown that non-woven randomly oriented silica nanofibres can be readily synthesized via electrospinning, and the mechanical testing indicated that some limited improvements in tensile performance over neat epoxy could be expected for epoxy reinforced with the silica nanofibre mats, but that the real improvements of up to 200% increase in strength (over the neat epoxy and much higher than the improvement afforded by unidirectional glass fibre reinforcements) would occur during bending and under complex loads. Bending and complex loads are especially important for aerospace applications.

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35.

SEM image of the 1065°C treated silica nanofibre mat ⁸⁴

Crystalline ceramic fibres

Crystalline ceramic fibres are also interesting reinforcements as a result of their improved mechanical properties compared with glass fibres. As with all of the aforementioned fibres described, it is beneficial from an optical transparency perspective to have very small diameter fibres so that an effective medium theory can be used and visible light does not scatter via standard geometrical means at the refractive index gradient locations within the composite material. As an example, transparent alumina (Al₂O₃) nanofibres have been formed using an electrospinning method at 7–9 kV. ⁸⁵ The alumina precursor used was aluminium 2,4-pentanedionate in acetone with polyvinylpyrrolidone (PVP) as a polymeric additive. Using a slow ramp rate of 0·5° min^–1, the authors were able to maintain the as-spun nano-fibrillar nature of the fibres, as shown in Fig. 36 for fibres heat-treated at 1300°C for 1 h.

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36.

Scanning electron micrographs of the Al₂O₃–PVP composite fibres calcined at 1300°C for 1 h ⁸⁵

The TEM image shown in Fig. 37 reveals the ∼150 nm diameter Al₂O₃ nanofibres after 1500°C heat treatment for 1 h. The authors state that the fibres are transparent, but do not differentiate between visual transparency and electron transparency. To determine visual transparency, one may use clear index matching fluids and a light microscope or other optical source. The authors found no evidence of carbon in the spectrum obtained via energy dispersive spectroscopy and no evidence of a D or G carbon peak in the Raman spectrum ⁸⁶ of the as-fired nanofibres. No mechanical testing or composite synthesis was reported by the authors. However, the alumina nanofibres have potential utility as reinforcements in hydrogel or other composite systems.

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37.

TEM images of the electrospun Al₂O₃ fibres fired at 1500°C/1 h (bar = 100 nm) ⁸⁵

Hydrogel materials

Many different materials have been applied to fabricate hydrogels. Synthetic polymeric materials have been widely investigated for biomedical applications, including poly(vinyl alcohol) (PVA), poly(2-hydroxyethyl methacrylate) (pHEMA), poly(ethylene glycol) (PEG) and silicone (or polysiloxanes). These polymers provide wide flexibility in structural and mechanical properties, optical properties and other important parameters.

Poly(vinyl alcohol) (PVA)

Poly(vinyl alcohol) (PVA) is a water-soluble polymer of great interest for pharmaceutical and biomedical applications owing to its biocompatibility (Fig. 41). ¹⁰⁰ This polymer is produced from hydrolysis of poly(vinyl acetate) (PVAc). Poly(vinyl alcohol) hydrogels are formed by cross-linking PVA chains in various ways. Chemical cross-linking of PVA hydrogels has been applied to form acetal bridges between the pendant hydroxyl groups of the PVA chains using aldehydes like glutaraldehyde, acetaldehyde and formaldehyde. Nevertheless, tedious procedures of extracting toxic reaction residues are required for biomedical applications. In order to avoid this, γ-irradiation was employed to cross-link PVA chemically. However, this technique showed bubble formation, rendering it unusable for many applications. On the other hand, PVA chains feature a unique capability to form crystallites by hydroxyl bonds and van der Waals forces. These crystallites can serve as physical cross-linkers between the PVA chains. Due to the three-dimensional structures of the crystallites, the physically cross-linked PVA hydrogels demonstrate enhanced mechanical strength compared with the chemically cross-linked ones. An efficient way to crystallize aqueous PVA solutions was demonstrated by Peppas and other researchers using freezing and thawing techniques. ^101,102 Increased PVA crystallinity was obtained by increasing freezing time. Yet, long-term stability of the structure and morphology of the PVA hydrogels was a potential issue because slow recrystallization or dissolution of the PVA crystallites could happen at different conditions. In order to avoid any harsh environment to prepare PVA hydrogels, Schmedlen et al. ¹⁰³ investigated photoactive PVA derivatives functionalized with aminobutyraldehyde diethyl acetal. The photoactive PVA not only showed rapid photopolymerization under ultraviolet (UV) exposure, but also exhibited compatibility with cell seeding.

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41.

Chemical structures of synthetic polymers used for biomedical applications

Hydrogel properties

Hydrogels are characterized as highly swollen cross-linked polymeric networks and can be classified based on the polymer source and cross-linking type. ^125,126 Hydrogels made from both naturally and synthetically derived polymers have been investigated. Natural hydrogels potentially produce favourable biodegradation products and allow for directing/controlling cell behaviour such as adhesion, growth, migration and differentiation. Natural scaffolds, such as collagen, HA, alginates and chitosan, have been widely tested and applied clinically. Nevertheless, naturally derived materials suffer a greater risk of contamination and immune reaction than synthetically derived materials. On the other hand, synthetic hydrogels can be processed from a plethora of options and provide higher flexibility to tune chemical, physical and biological properties. Combinatorial uses of both naturally and synthetically derived materials in hydrogels have also attracted great attention, with the goal of combining the advantages from each component. Hydrogels can be cross-linked via chemical and/or physical bonds. Chemical hydrogels are formed by covalently cross-linked networks, which are more stable and provide higher mechanical strength compared with physical hydrogels. In contrast, physical hydrogels allow cross-linker formation requiring no addition of toxic reactants such as chemical hydrogels, and so avoid potential side effects.

Several important factors play a role in determining the physical–chemical properties of hydrogels: comonomer composition, volume fraction, average molecular weight between cross-links and correlation length (Fig. 42), and polymerization conditions (pH, reaction time, temperature). ^97,127 Altering the composition of a hydrogel by increasing the relative amount of a physically stronger component will lead to increased strength of the hydrogel, e.g. by replacing acrylates with methacrylates, or it could alter hydrophilicty by replacing 2-hydroxyethyl methacrylate with methacrylic acid, for example. Volume fraction is defined as the ratio of the volume occupied by the polymer to that of the hydrogel. When the hydrogel is swollen in equilibrium state, volume fraction describes the degree of the swelling and sometimes is estimated from the weight fraction because it is more convenient to determine the weights of the polymer and hydrogel. Cross-link density of the hydrogel determines the average molecular weight between cross-links, which is correlated with the distance between cross-links, called correlation length or network pore size. All of these factors influence hydrogels in various ways, including permeability, molecular diffusivity, elastic deformation and modulus. For example, Table 8 reports mechanical properties of an HEMA–acrylic acid comonomer hydrogel, varying up to orders of magnitude as a result of pH and cross-linker concentration. ¹²⁸ In other studies, increasing the fraction of N-vinyl-2-pyrrolidone (NVP) relative to HEMA or methyl methacrylate (MMA) resulted in one to two orders of magnitude reduction in Young’s modulus, which is due to the highly hydrophilic nature of NVP relative to HEMA or MMA, causing an increase in swelling. ^127,129,130 In a hydrogel containing ionic monomers, varying the ratio of cationic and anionic monomers affected swelling, leading to maximum swelling (60 or 100%) when all of the monomer was either positively or negatively charged, but less than 20% swelling when the cationic and anionic monomers were present in equal amounts. ^127,131 As the volume degree of swelling of polyacrylamide gels cross-linked with N,N-methylene-bisacrylamide changed from 4 to 8, the elastic modulus decreased by nearly two orders of magnitude (from 10 ⁵ to 10 ³ Pa). ¹³² In the work by Greenberg and Kusy, ¹³³ increasing allyl acrylate cross-link agent concentration in acrylic acid polymers was shown to increase dynamic modulus significantly and glass transition temperature linearly. ¹²⁷ These examples illustrate the sensitivity, and therefore tailorability, of hydrogel properties to composition and synthesis conditions.

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42.

a Simplified structure of cross-linked hydrogel. Cross-linking point is indicated by black dots. The distance between cross-links is called correlation length, ξ, or network pore size. b The cross-linking density of hydrogels strongly influences gel properties, including modulus/stiffness, hydrophobicity, permeability/diffusivity, water content/gel swelling and hydrolytic degradation rate ⁹⁷

Mechanical properties

Natural hydrogels such as cartilage are strong and tough, whereas many synthetic hydrogels are weak. Cartilage contains micrometre scale fibres that toughen and strengthen the gel matrix, improving the mechanical properties of the overall composite. The fibres also play the role of arresting cracks that propagate through the gels, which otherwise have no barrier to propagation under tension. Understanding the roles of the hydrogel matrix and the reinforcement fibres in determining the overall mechanical properties can enable tailoring of these composites for specific applications.

As is the case for any material, the mechanical behaviour and properties of fibre-reinforced hydrogels are highly dependent on composition and processing. To achieve sufficient mechanical performance of a hydrogel in a specified application, requirements for mechanical properties, including elasticity, compressibility, viscoelastic behaviour, tensile strength and failure strain, must be specified, characterized and controlled. ¹³⁴ Both the hydrogel matrix and the fibre properties are important in improving mechanical properties of hydrogel composites. The tensile strength of a fibre-reinforced hydrogel is largely dictated by the matrix properties, whereas the modulus is more dependent on the amount and properties of the reinforcing fibres. A strong fibre–hydrogel interface, low stress concentration and fibre orientation are required to increase tensile strength, whereas fibre concentration, fibre wetting in the matrix phase and high fibre aspect ratio determine tensile modulus. ^7,135

In hydrogels, mechanical properties are affected by polymer and cross-linker characteristics, gelling conditions (e.g. temperature and pH), swelling and degradation. ¹²⁷ For example, high guluronic acid containing alginate polymers yielded stronger, more ductile hydrogels than high mannuronic acid containing alginates. ¹³⁶ Increases in polymer volume fraction have also resulted in an increase in both the compression modulus and the equilibrium shear modulus for alginate, ¹³⁷ PEG ¹³⁸ and PVA hydrogels, ¹³⁹ for example. The mechanical properties and swelling degrees of hydrogels can also be tightly tailored by employing different kinds of cross-linking molecules and controlling the cross-linking densities. ¹⁴⁰ Gel swelling usually results in a decrease in the mechanical strength of hydrogels, ^127,138 but these can be independently controlled in covalently cross-linked hydrogels by varying both the cross-linker type and its density. ¹⁴⁰ Hydrogel degradation and dissolution usually lead to a weakening of the gels ^136,137 unless tissue in-growth acts to strengthen them ¹³⁸ or these properties are decoupled. ¹⁴¹ Overall, by controlling the specific polymer and processing methods, a wide range of tensile properties is available from hydrogels. ¹³⁶

Hydrogels on their own have poor mechanical properties, in general, but incorporation of fibres can enable improvement and tailoring of mechanical properties. Fibre composition, geometry, content, orientation and interaction with the hydrogel matrix all play a role in determining the final composite properties. For example, Kai et al. ¹⁴² studied the effects of incorporating electrospun poly(ϵ-caprolactone)/gelatine nanofibres into hydrogels and demonstrated increases in Young’s modulus from 3·29±1·02 to 20·30±1·79 kPa and decreases in strain at break from 66·0±1·1% to 52·0±3·0% (Fig. 43). A 1–2 wt-% reinforcement of bacterial cellulose in methacrylate hydrogels led to a 10- to 20-fold increase in storage modulus and also significant increases in loss modulus of the gels. ¹⁴³ In a study by Agrawal et al., ¹⁴⁴ a pultrusion system was used to build three-dimensional patterned fibrous polyurethane structures, which were then impregnated with epoxy–amine hydrogel to form a fibre-reinforced hydrogel composite (Figs. 44a –45d ). They observed that 5 wt-% fibre reinforcement improved the tensile modulus of hydrogel from 0·7 to 2·5 MPa, breaking strength from 0·2 to 1·3 MPa and breaking strain from 30% to around 150%. The influence of the fibres on crack propagation, namely crack blunting, can be seen in Fig. 44e . Generally, as the concentration of fibres in a hydrogel is increased, the stiffness increases and swelling decreases, so these co-dependent properties must be balanced for specific applications. ^145,146 These examples demonstrate the flexibility in tailoring of hydrogel mechanical properties provided by incorporation of fibres.

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43.

Adapted from Ref. 142

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44.

a, b Cross-sectional images of fibre-reinforced hydrogels, c fibre construct prior to impregnating with hydrogel and d final fibre-reinforced hydrogel. e Series of time lapse images showing propagation of a crack, which blunts owing to the presence of fibres

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45.

Relationship between the tensile strength (σf), modulus of elasticity (E), elongation at break (ef) and draw ratio (DR) for cellulose films ¹⁵⁸

Alignment of fibres within a matrix leads to enhanced mechanical strength in the direction of alignment. Anisotropic hydrogels can thus be proposed. In addition, fibre alignment can have benefits for wound healing applications. ⁸⁷ As a consequence, many techniques are employed to align fibres within hydrogel matrices. Controlled ordering of polymer fibres can be accomplished to a degree via application of strong magnetic and electric fields. Preferred orientation has been induced in suspensions of cellulose nano- and microfibrils using both magnetic ^147–149 and electric fields. ^150,151 While cellulose shows orientation perpendicular to an applied magnetic field, ^147–149 orientation is parallel in an electric field. ^151,152

Drawing of polymer fibres and films can change mechanical properties of the material by introducing preferred orientation. Drawing can be performed during consolidation from liquid to solid, or in solid state below the glass transition temperature (cold drawing) or at an elevated temperature. ^153–157 As a result of drawing, the molecular chains in a polymer gradually align with the direction of applied strain, which in turn introduces mechanical anisotropy. Drawing cellulose films in the wet condition and subsequent drying has been employed by Gindl and Keckes. ¹⁵⁸ As shown in Fig. 45, their study resulted in significant increases in tensile strength and modulus of elasticity and decreases in elongation at break of self-reinforced cellulose films in the direction of draw.

Biological functionality

Polymer scaffolds have many different functions in the field of tissue engineering. They are applied as space filling agents, as delivery vehicles for bioactive molecules and as three-dimensional structures that organize cells and present stimuli to direct the formation of a desired tissue. Recently, research focus has been increasing on hydrogels for use in regenerative medicine applications requiring more challenging biological functionalities. ¹¹¹ In native tissues, cells are surrounded by ECM, which is mainly composed of structural fibrous proteins (e.g. collagen, elastin and fibrin) embedded in polysaccharide rich glycans (e.g. GAGs and PGs) (Fig. 52a ). The protein fibrils provide mechanical support as a physical framework for cells and tissues, while the highly negatively charged glycans serve as the ground substrate between protein fibrils to maintain a hydrated state and provide a reservoir for signalling molecules and growth factors.

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52.

a native ECM composed of protein fibrils as physical framework and highly negatively charged glycans as ground substrate, b dense fibre mats produced from a conventional electrospinning technique, c multi-layered fibre–hydrogel composites made from dense fibre mats stacked with hydrogels and d fibre-reinforced hydrogel made from hydrogel embedded, low density fibres fabricated using a novel electrospinning technique ¹⁶⁶

Beyond simple hydrogels, fibres (both micro and nano) can be incorporated into hydrogels to further mimic the ECM. Fibre-reinforced hydrogels have the advantage of mimicking the chemical, biological and physical composition of native ECM for different biomedical applications. Recently, a novel electrospinning technique was developed to fabricate loose polymer fibre matrix with macroscale pores, which allowed engineering of a three-dimensional fibre–hydrogel composite for clinically relevant tissue defects. ¹⁶⁶ Bulk three-dimensional fibre-reinforced hydrogels were studied using PCL fibre mats embedded in PEG hydrogels incorporated with cell adhesive peptides of arginine–glycine–aspartic acid (RGD) (Fig. 52d ). As shown in Fig. 53, the mesenchymal stem cells (MSCs) encapsulated in the composite showed a variety of cell morphologies including extensions, indicating cell interactions with the fibrous scaffolds. In contrast, when using hydrogels alone, the cells exhibited spherical morphology. The fibre density in the composite was found to play an important role in cell matrix production with the greatest amount produced at a fibre density of 10% dry weight. Multi-layered composites were also tested to replicate tissues with layered structures such as skin and cornea (Fig. 52c ). Mesenchymal stem cells encapsulated in this composite demonstrated enhanced chondrogenic differentiation with higher GAG and collagen production. Furthermore, MSCs in this composite showed greater response to mechanical stimulation, which can be attributed to the more efficient force transduction from the composite scaffold to the cells, compared to the pure PEG hydrogel.

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53.

a SEM image of fibres embedded in the hydrogel, b confocal microscopy image of the hydrogel composite with fluorescent stained fibres, c–e in the fibre-reinforced hydrogel, mesenchymal stem cells (MSCs) long extensions and dividing nuclei, f in the pure hydrogel, cells showing a round morphology ¹⁶⁶

Ocular applications for transparent and improved strength hydrogels

Apart from standard contact lens material needs for daily wearers, there is significant opportunity for the development of new and improved hydrogels for corneal patch and corneal prosthesis applications. For example, despite the eye’s seemingly insignificant size, accounting for only 0·1% of the body’s frontal silhouette, the incidence of ocular injury in combat has been shown to be 20–50 times greater than expected by its surface area. ¹⁷⁰ As a function of time in US history, the percentage of eye wounds compared with total reported injuries in various wars and conflicts has been increasing dramatically (Fig. 54). Novel materials for treatment on the battlefield to improve outcomes are needed, along with new materials to serve as corneal prostheses for subsequent eye surgery in a clinical setting once the wounded service person can be evacuated from the theatre of war.

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54.

Adapted from Ari ¹⁷⁰

A novel combination of chondroitin–sulphate polyethylene glycol (CS-PEG) adhesive and vitrified collagen gels (CV) shows promise for the repair of open globe ocular injuries commonly sustained on the modern battlefield or in civilian trauma. ¹⁷¹ Injury models were based on photographs of actual combat injuries and created in porcine cadaver eyes before repair with CS-PEG alone, CS-PEG+CV, or sutures (control), as shown in Fig. 55a . Burst pressure testing was used to assess success of repair, with materials withstanding pressures past 50 mmHg considered successful. These data are summarized in Figs. 55b and c. For linear defects, CS-PEG+CV membrane was comparable to sutures in its ability to seal the eye, but may prove advantageous during the healing process due to the biocompatibility of the materials and the nature of the less invasive repair procedure. Additionally and perhaps more significantly, suturing of circular defects or lacerations with tissue loss is not possible due to the severe astigmatism that would result, but reconstruction with CS-PEG+CV was demonstrated to be successful at repair of large circular injuries with diameters up to 8 mm. The biosynthesized CV membrane holds great potential in its physical and biological properties and drug delivery capability. This new material also may enable repair of previously irreparable ocular injuries.

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55.

Adapted from Mulreany et al. ¹⁷¹

Load-bearing tissue applications for improved strength hydrogels

There are several load-bearing applications in which hydrogels can be utilized. One potentially useful application for hydrogels is as an interface material between bone and implants. Netti et al. ¹⁷² showed that pHEMA hydrogels are promising replacements for bone cement in cases where it is desirable to fix a bone–joint prosthesis interface. The concept is that the hydrogel is placed in the correct position between the bone and the prosthesis in a dehydrated state. The hydrogel subsequently absorbs water from its surroundings and swells until its hydrostatic pressure in the material is equal to the osmotic pressure of water in the system. While Netti et al.’s in vitro pull-out mechanical testing was very promising, the in vivo testing showed some significant variations in results and indicated lower relative pull-out forces. The less promising in vivo test results were attributed to differences in morphology of the animal anatomy from sample to sample. While the mechanical tests in vivo were inconclusive, the histological examination showed that the pHEMA hydrogel had potential as a biomaterial adjacent to bone because there was no evidence of adverse reaction in the bone to the material. For example, new bone was observed to form a shell around the hydrogel 28 days after implant (Fig. 56).

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56.

New bone (B) forming a shell surrounding the hydrogel (H) 28 days after implantation in the metaphysis ¹⁷²

Another application for load-bearing hydrogels is intervertebral disc replacement. Nucore is an injectable protein-based nucleus replacement which thermal cures in situ. ¹⁷³ Since the cured hydrogel is larger than the injection site, a mechanical barrier is formed and the hydrogel, which also has adhesive properties, resists extrusion. This injectable silk elastin polymer and cross-linker combination is undergoing investigational device exemption feasibility study. Intervertebral disc replacement hydrogels that cure via photopolymerization are also under development. ¹⁷⁴

Collagen has also been used in a variety of load-bearing applications. It can be isolated from a variety of sources and is relatively non-immunogenic. ⁴⁹ Wood hemicellulose and cellulose nanowhiskers were also used to successfully prepare a fibre-reinforced hydrogel that has superior toughness, extensibility, and recovery properties compared to conventionally cross-linked hydrogels. ¹⁷⁵ The improved properties along with satisfactory water holding capacity, mechanical properties, and viscoelasticity make the reinforced hydrogel an interesting candidate material for articular cartilage replacement.

Wound management applications for transparent and improved strength hydrogels

Burn wounds and chronic venous ulcer wounds are difficult wounds to treat with dressings because many dressings do not adequately serve the wound. For example, some dressing materials may not facilitate good transfer of oxygen to the wound for healing while others may not be able to donate or absorb water/exudates from the wound or may permit too much moisture loss from the wound site. Sticking to the wound during dressing removal also presents significant pain and re-injury to the patient. One excellent material for such wound treatments is bacterial cellulose. The material is shown in Fig. 57 and has almost all of the above positive attributes (e.g. it has excellent oxygen permeability and can effectively absorb exudate and hydrate dry areas of a wound, which is unique in comparison with other dressings that have only a single function ¹⁷⁶ ) except for the fact that although Czaja et al. ¹² claim that the material is transparent and allows for continuous clinical observation of the healing process, the material still looks rather opaque. A truly transparent dressing with all of the attributes of bacterial cellulose and significant conformability yet high ultimate tensile strength and tear strength would revolutionize the burn wound standard of care.

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57.

Microbial cellulose dressing applied on a wounded hand. The unique physical properties of microbial cellulose allow an excellent moulding, displaying a high degree of adherence even to the moving parts ¹²

Alginate has been used in applications in the food, textile, printing, paper and pharmaceuticals industries. ²⁵ Owing to their non-toxicity and ability to hold a large amount of water, alginate gels, foams and fibrous non-woven dressings have been applied to wound healing. The exchange of calcium ions in alginate fibres with sodium ions in the body causes a release of calcium ions, which act as a haemostatic agent. ²⁵ As calcium alginate fibres convert to sodium alginate fibres, large amounts of exudates are absorbed. ²⁵ In their dry form, these materials absorb wound fluid, whereas in their hydrated form they donate water to the wound. This helps to maintain a moist environment, which promotes quicker migration of the epithelial cells from the edge of the wound to the bulk. ²⁵ It should also be noted that chitin and chitosan are natural polymers that are biodegradable, biocompatible and non-toxic and have an ability to accelerate wound healing. ^32,177

Sensing/device applications for transparent and/or improved strength hydrogels

A novel hydrogel based glucose sensor has recently been developed and reported by Holtz and Asher. ^178,179 The researchers relied on Bragg diffraction for the sensing technique. In creating a photonic crystal by fabricating an array of small polystyrene spheres, functionalizing the spheres with glucose oxidase, and then polymerizing the polystyrene spheres into a hydrogel, the researchers created a visible light sensor that changes colour as the local environment’s glucose concentration varies. For patients with diabetes mellitus, the sensor could essentially be used as part of a contact lens for real-time non-invasive sugar monitoring, as shown in Figs. 58 and 59. A small region of the lens would contain the modified photonic crystal hydrogel patch and the colour of the patch would change with the level of glucose in the tears of the subject. When the sensor is designed with appropriate sensitivity and wavelength range, the colour changes would be quite stark and easily noticeable for the wearer. It should be noted that hydrogel reinforcements, particularly at the edge, for contact lenses or pseudo-contact lenses like the ones discussed here, are expected to improve handling performance for wearers.

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58.

Pictorial representation of use of glucose-sensing material in a contact lens-like application, adapted from Ref. 179

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59.

Effects of glucose concentration on the optical properties of the hydrogel based photonic crystal glucose sensor. a spectral shift trend as a function of glucose concentration; b peak diffraction wavelength trend as a function of glucose concentration, adapted from Ref. 179