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Extracellular Matrix Hydrogels from Decellularized Tissues for Biological and Biomedical Applications

Brendan C. Jones1, 2, Nicola Elvassore1, Paolo De Coppi1, 2, and Giovanni G. Giobbe1

1University College London, Great Ormond Street Institute of Child Health, Stem Cell and Regenerative Medicine Section, Developmental Biology and Cancer Research and Teaching Department, 20 Guilford St, London WC1N 1DZ, UK

2Great Ormond Street Hospital, Department of Specialist Neonatal and Paediatric Surgery, Great Ormond Street, London WC1N 3JH, UK

1.1 Introduction to Hydrogels

1.1.1 Definition and Use of Hydrogels in Biomedical Applications

A hydrogel is a hydrophilic three-dimensional (3D) network composed of polymer units that is capable of absorbing large amounts of water relative to the dry weight of the component polymers [1-3]. Interaction between polymer units and a gelation stimulus in a hydrated environment, which in the context of biomedical applications may include physiologic liquids rather than pure water, results in formation of the 3D network. Since pioneering work by Wichterle and Lim [4], hydrogels have been used in numerous biological and biomedical applications including tissue engineering, drug delivery, cell culture, and as biosensors, hemostatic agents, and self-healing materials [1, 3]. Their widespread and growing use is due to numerous advantageous characteristics, such as tunable mechanical properties, ability to add functional groups to polymers that may act as ligands or be responsive to external stimuli, cytocompatibility, and low toxicity [1].

Hydrogels have received particular attention in cell culture and tissue engineering applications. Specific advantages of hydrogels in this field include the ability to encapsulate cells, proven biocompatibility and cytocompatibility, the potential to mimic physiologic environments, and act as cell and drug delivery vehicles [3, 5, 6]. In the context of these applications, some practical problems related with the use of hydrogels remain: the hydrogels are often difficult to handle, relatively fragile, hard to sterilize once gelation has occurred, and many are not currently produced using Good Manufacturing Protocol (GMP) grade processes limiting their translation into human patients [3, 5].

1.1.2 Classification and Properties of Hydrogels

There are numerous methods to classify and describe hydrogels, many of which are discussed in detail elsewhere in this book, and are summarized in Table 1.1 [1, 3, 5-7]. The variables most relevant when considering biomedical applications of hydrogels are the nature of the forces holding the network together (response), the source of the polymers, and the cross-linking stimulus. The network of a reversible hydrogel is held together by molecular entanglements and/or secondary forces (ionic, hydrogen, or hydrophobic bonding), which can be reversed by changes in physical conditions. By contrast, permanent hydrogels are held together by covalent cross-linking [3]. Polymers may be naturally occurring, synthetic, or a hybrid natural-synthetic copolymer and may be tunable, allowing variation of mechanical properties. The stimulus to initiate cross-linking may be a defined range or change in temperature, pH, light, magnetic field, or ion strength/chemical composition of solution, or the addition of a specific cross-linking molecule [1, 2].

Table 1.1 Classification of hydrogels.

Source: Adapted from Chai et al. [1], Hoffman [3], Giobbe et al. [5], Holloway et al. [6], and Capeling et al. [7].

• Ionotropic hydrogel
• Polyelectrolyte complexes

• Copolymerization of polymer and cross-linking molecule
• Cross-linking of water-soluble polymers

Temperature is a useful trigger of the gelation in the context of biological applications. When polymer units and water (or physiologic liquid) are mixed, the polymer network will take on water to swell, followed by gelation in a definable temperature range for a given hydrogel [1, 2, 5]. The lower bound of this range is termed the lower critical solution temperature (LCST) - above the LCST phase separation and gelation occurs, while below the LCST the system is miscible in all proportions - with transition from gel to sol or sol to gel states related to changes in Gibbs free energy [1, 3]. This property can be extremely useful in biological applications. For example, a hydrogel may be in solution at room temperature but undergo gelation at 37 °C.

The amount of water in a hydrogel is termed total bound water, composed of primary bound water (the first water to enter the 3D network and hydrate the most polar groups) and secondary bound water (which is bound to hydrophobic groups exposed by initial swelling), and the proportion of water in a hydrogel is called the volume fraction of water. Osmotic force will draw water into the network toward infinite dilution until an equilibrium is reached with the opposing force of elastic retraction by the network (equilibrium swelling theory) [1, 3]. When equilibrium is reached, water exists in the spaces in the hydrogel between polymers (pores), which are usually inhomogeneous in size, distribution, and connectedness in a given gel. These variables, in combination with solute size, shape, polarity, and solute-matrix interactions, determine the permeation of a solute through a hydrogel [3].

Properties of hydrogels can be studied using a multitude of techniques including histology, immunostaining, electron microscopy, and proteomics by liquid chromatography-tandem mass spectrometry (LC-TMS). Additionally, turbidity (measured by spectrophotometry) is commonly used to determine gelation kinetics, while atomic force microscopy and rheometric techniques are used to study the mechanical properties of hydrogels, for example, storage (G´) and loss (G") moduli [3, 5, 8].

Finally, any hydrogel under consideration for use in biomedical or biological applications must be cytocompatible. Whenever a hydrogel is modified (polymer type, polymer concentration, change in functional groups, etc.), it must be proven to be cytocompatible or it cannot be used in vitro or in clinical applications.

1.1.2.1 Synthetic Hydrogels

Synthetic hydrogels are composed of polymers that have been chemically synthesized and are not found in nature. Examples include poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(co-glycolic acid) (PGA), and poly(vinyl alcohol) [2]. A brief summary of advantages and disadvantages of this broad range of hydrogels will be presented here but is more comprehensively discussed elsewhere in this book.

Potential advantages in the use of synthetic hydrogels for biomedical applications are several. They have well-defined molecular composition and architecture, can be manufactured with low batch-to-batch variability, and avoid the use of xenogeneic material in production process [1, 6]. The monomers are uniform and modular, leading to a relatively predictable relationship between composition and physicochemical properties when polymerized [9]. Therefore, the concentration of monomers can be varied, or the monomers can have functional units added rationally to alter the mechanical and biological signally properties of the resulting hydrogel. These features allow synthetic hydrogels to be easily tuned for specific circumstances [6, 8, 9].

There are several important disadvantages of synthetic hydrogels in biological contexts. Many synthetic hydrogels are covalently cross-linked and resistant to enzymatic degradation (due to lack of proteolytic sensitive domains), such that these gels behave as linear elastic networks. This is of particular relevance in cell culture contexts as these networks can accumulate substantial compressive forces that are then transmitted to encapsulated cells [9, 10]. Furthermore, the breaking of covalent bonds is irreversible, meaning that permanent synthetic hydrogels cannot "self-heal" like reversible gels [9]....