Chemical Cross-linking

The most common form of gelling polymers to form hydrogels is through the addition of a bifunctional chemical compound that will react with functional groups on the polymer backbones to cross-link them. This usually involves reactions with carboxyl, amine or thiol groups that branch off the main chain of the polymer. Sometimes heat or agitation is required for the cross-linking to occur. Depending on the concentration of cross-linker and gel precursors in the solution the gelling can take anything from minutes to hours to complete . Some systems require alternate forms of cross-linking, such as alginate, which is cross-linked by the addition of a divalent cation, most commonly Ca ²⁺ . Chemical cross-links are also most common because the chemicals are typically non-toxic, which is necessary for both in vitro and in vivo applications, and allow for a much greater control over the location of the linkages.

Photo-cross-linking

Photo-cross-linking is typically achieved by the addition of an ultraviolet (UV)-sensitive photoinitiator, although some initiators are also susceptible to visible light . The initiator begins cross-linking the hydrogel through a radical polymerization of double bonds in the hydrogel precursors. This cross-linking technique requires the addition of double bonds to the gel backbone in order to function properly. The advantages of photo-cross-linking over chemical cross-linking are that the reaction usually completes on a shorter time-scale, seconds to minutes, and tends to release almost no heat, whereas chemical cross-linking may require heating to proceed . However, radical polymerization tends to be more difficult to control. As a more energetic reactive species, radicals can react at unpredictable places and do not necessarily target the same atom every time. This leads to a more random hydrogel that can have different properties at different locations throughout the gel.

Methacrylated HA (HA-MA) was recently employed in research on cutaneous and corneal wound healing , embryonic stem cell expansion , and drug and growth factor delivery . In addition, photo-cross-linked HA-MA provided a 3D microenvironment suitable for mesenchymal stem cells (MSCs) to differentiate into a chondrogenic phenotype .

Sol-gel Synthesis

Sol-gel synthesis uses a simple phase transition to convert the hydrogel precursors to a stable gel form. This effect is usually pH or temperature dependent and can be very useful for drug delivery applications where an injected hydrogel solution mixed with a drug molecule transitions at or below body temperature to form a gel . The drug molecule then diffuses slowly out of the gel in a time-controlled manner based on the degree of physical cross-linking. Cells can also be encapsulated in this manner, and remain viable .

Cryogelation

This synthesis technique, like sol-gel synthesis, also creates a physically cross-linked hydrogel. When the gel precursors are at room temperature, they form a solution, although a potentially viscous one. When frozen, the ice crystals that form tend to aggregate the polymer chains together. Upon thawing, the chains form non-covalent bonds resulting in a hydrogel . This technique can be used to store molecules or even cells for extended periods. However, the polymers for which this synthesis technique is possible tend to be synthetic and thus do not contain many adhesive sites for cells. This problem is typically overcome by the addition of some other polymer such as collagen or chitosan that contains adhesion sites for the cells .

Spinning

Spinning is a technique where a hydrogel precursor solution is extruded through a needle to form microscaled or nanoscaled fibers. This is typically achieved using electrospinning without a cross-linking agent. In electrospinning, a large voltage is applied to the needle as the solution is pushed through with a syringe pump. As the charged solution exits the needle, a “taylor cone” is formed where the electrostatic repulsion is high enough that a very fine stream of solution erupts from the needle. During flight, the solvent dries out and the charge moves to the surface of the forming fiber. This causes the fiber to whip around owing to the electrostatic repulsion felt over small bends ( Fig. 16.2 D). The fiber is finally collected on a grounded plate , or for aligned fibers, separated collecting plates or a spinning mandrel or disc . In addition to electrospinning, hydrodynamic spinning is a potential fabrication technique . In hydrodynamic spinning, a similar extrusion process is used; however, the addition of a cross-linker is required. The diameter of the fibers formed is determined by the flow rate through the apparatus, and hollow fibers can be formed from the combination of three concentric streams .

Schematic diagrams of various microfabrication techniques. (A) In photolithography, a thin layer of hydrogel precursor is applied to a substrate, typically glass, before being exposed to ultraviolet (UV) light through a photomask. This causes cross-linking in regions exposed to UV light. The remaining hydrogel precursor solution can then be washed away. (B) In micromolding, the poly(dimethyl siloxane) (PDMS) stamp is “inked” with a chemical cross-linker solution before being pressed into a thin layer of hydrogel precursor on a substrate. The PDMS stamp is removed after a short while, leaving behind a cross-linked and molded hydrogel. (C) In emulsification, a rod agitates a solution of hydrogel precursors dissolved in an organic solvent that is mixed with an aqueous phase which contains a cross-linker. This agitation results in small droplets of organic hydrogel precursor in the aqueous cross-linker solution. The cross-linker diffuses into the droplets which form small beads upon gelation. (D) An electrospinning set-up shows the hydrogel solution being extruded through a syringe and collecting on a grounded plate. The “taylor cone” forms at the opening of the needle as electric charges built up in the hydrogel force it into a thin stream which will dry to a final thickness of nanometers to micrometers. (E) Microfluidics shows two precursor solutions being pumped into a T junction which then mix to form cross-linked hydrogel microspheres.

Bioprinting

The technique of bioprinting consists of two components. First, cell aggregates, cellularized sECM hydrogels or cell-seeded microspheres comprise the “bioink.” The cell-free polymers that provide the substratum for the bioink are the “biopaper.” Bioprinting consists of stepwise assembly of bioink and biopaper components into an organ-appropriate 3D architecture using a three-axis printer . For example, vascular networks can be printed by a “scaffold-free” process involving automated deposition of sausage-like cell aggregates and agarose tubes . In each case, computer-assisted design is used to guide the deposition of cells in geometries that recapitulate the structure of the target tissue or organ . After printing, the engineered construct is allowed to mature and gain functionality in a bioreactor or in vivo environment .

Centrifugal Casting

Living polymers, such as the sECMs described below, offer the unique opportunity of forming a wide variety of tubular structures in the body, including potentially kidney nephrons, by centrifugal casting . This technique was first validated by sandwiching a layer of fluorescently labeled endothelial cells between two layers of an in situ cross-linkable sECM by axial centrifugation at approximately 11 × g . After centrifugally coating the inside of a Dacron vascular prosthesis with the sECM, a cell suspension was applied and centrifugally cast as the gel cross-linked during centrifugation. The heavier cells were entrapped between the two layers, remained viable and remodeled the cross-linked HA–gelatin sECM. Cells in the sECM suspension can also be applied to laser-machined micropores of a sheet of small intestinal submucosa (SIS), which affords a robust construct that retains the cell-seeded hydrogel and permits rapid biofabrication of a tubular tissue in a bioreactor-free fashion .

Microfabrication

Complex patterns can also be generated in hydrogels using various microfabrication techniques, typically by reshaping a preformed hydrogel. Microspheres of hydrogels can be formed through the use of emulsification polymerization . Here a mechanical agitation of the hydrogel precursors in an organic phase causes microspheres to form which can then be gelled through a variety of cross-linking methods ( Fig. 16.2 C). Size can be controlled through the addition of surfactant molecules and the degree of agitation .

Photolithography is another useful technique for the fabrication of complex patterns . A thin layer of hydrogel precursor is applied to a substrate. Then a photomask with the desired pattern is placed over the hydrogel and the entire assembly is exposed to UV light, which causes gelation of the pattern ( Fig. 16.2 A). The remaining hydrogel can then be washed away. Photolithography is capable of producing complex features on a submicrometer to millimeter scale. However, photolithography is essentially a 2D approach and multiple steps may be required to achieve a 3D structure.

Microfluidics can also be used to create hydrogels . Here, small channels are formed in a polymer such as poly(dimethyl siloxane) (PDMS) by molding it from a master. The device is then sealed by placing the PDMS mold onto a piece of glass, or a stronger seal can be formed by air plasma treatment of the PDMS before inversion on glass . Hydrogel precursors can be pumped in through multiple inlets and by controlling the flow rate and design of the channels, various morphologies can be generated ( Fig. 16.2 E). Typically, rods and spheres are formed but hybrid particles can also be created where each side has a different composition . In addition, microfluidics can be combined with photolithography to capture cells in a particular location by cross-linking the surrounding hydrogel .

Finally, micromolding is a simple technique for the fabrication of hydrogels with micrometer-scale features . Micromolding is based on a technique called soft lithography, where a PDMS stamp is molded from a prefabricated silicon wafer ( Fig. 16.2 B). Hydrogel precursors are molded with the PDMS stamp and subsequently gelled by the addition of a cross-linking agent through the mold, by irradiation for photopolymerization or by a sol-gel transition. Micromolding is a fast and efficient technique for generating 3D structures and can support features down to the nanoscale.

Biomimetic Hydrogels

These materials tend to have very similar properties to the implant site, whether they are natural or synthetic, and are usually able to integrate seamlessly with the actual tissue. Collagen/gelatin and HA are two natural compounds that are major components of the ECM. Other ECM proteins such as fibrin, fibronectin and laminin have also been used to fabricate hydrogels . Synthetic hydrogels can be combined with other naturally occurring polymers to create biomimetic implants, such as those used for bone repair , which must be stronger than a natural material on its own.

Biodegradable Hydrogels

Most natural hydrogel materials are biodegradable by enzymes that are naturally produced by the body. Among synthetic hydrogels, those made from polymers of amino acids, such as poly(lactic acid) (PLA) and poly(glycolic acid) (PGA), are easily biodegradable. Others, such as PCL and OPF, are polyesters that can be slowly degraded through hydrolysis. Biodegradable hydrogels can be tuned to degrade at different rates by varying cross-linker concentration and through the addition of other monomers to form block copolymers that may degrade at a faster or slower rate .

Bioinert Hydrogels

The last class of hydrogels is bioinert hydrogels. These elicit essentially no immune or inflammatory responses when implanted. Poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) are two examples of synthetic bioinert hydrogels ( Fig. 16.1 ). PEG and PVA are very hydrophilic and offer very few attachment sites for cells. These materials can be used to help isolate encapsulated cells from an immune response or serve as long-term repositories for drug delivery . They are also very stable in water because the backbone of PVA is all carbons, and that of PEG is polyether. PEG is commonly used as a surface treatment for other implants because of its ability to be conjugated to other functional groups through terminal alcohol domains.