2.2. Alginate Hydrogels

2.2.1. Ionic Gelation
Alginates have high affinity for alkaline earth metals and ionic hydrogels can be formed in the presence of divalent cations (except Mg2+) [21,24,25,26,27]. Chelation of the gel-forming ion occurs between two consecutive residues (Figure 2A) and an intermolecular gel network is formed as a result of a cooperative binding of consecutive residues in different alginate chains (Figure 2B). The G-blocks are the key structural elements in alginate hydrogels, but also alternating blocks may contribute to gel formation [27]. The different junction zones in an alginate gel are presented in Figure 2C. Alginates have different affinity for divalent cations in an increasing manner as Ca2+<Sr2+<Ba2+ [28]. The selection of type and amount of gel forming ion will influence the resulting properties of the hydrogels and can be utilized as an important tool to optimize elasticity, swelling and stability. Additionally, gel properties can be tuned by the selection of type and concentration of alginate. In general, gel elasticity, porosity and stability increases by increasing G-content, length of G-blocks and molecular weight [27,29].
The nanoscale porosity of an alginate gel network is tunable and in the range of 5–200 nm [29]. This will enable cellular access to nutrients and removal of waste products and synthesized products such as insulin, dopamine, endostatin and nerve growth factors [30,31].
Ionically gelled alginate can be dissolved by treatment with chelating agents for divalent cations such as citrate and ethylenediaminetetraacetic acid (EDTA) or hexametaphosphate [14]. This enables gentle and fast release of cells entrapped in alginate hydrogels for further downstream processing such as flow cytometry.
Ionic cross-linking between multivalent cations and alginate takes place instantaneously. Gels are, therefore, not easily prepared by mixing these two components directly. Two main techniques have been developed to provide controlled introduction of gelling ions to alginates with a subsequent formation of a gel. The techniques are referred to as diffusion gelation and internal gelation.
Diffusion gelation is characterized by allowing gelling ions to diffuse from a large outer reservoir into an alginate solution. This is commonly used for preparation of hydrogel beads which will be formed instantaneously when an alginate solution is dripped into a solution containing gelling ions (usually CaCl2) [32]. This technique is described in more detail in Section 3.1.
Internal gelation is characterized by a controlled release of gelling ions from an inert source that is dissolved or suspended within the alginate solution. Examples of sources of gel forming ions include carbonate salts (SrCO3 or CaCO3), calcium EDTA, calcium citrate, calcium sulfate (CaSO4), calcium alginate (Ca-alginate) and calcium gluconate [27,33,34,35,36]. Release of gel forming ions can be induced by a change in pH by, for example, incorporating the slowly hydrolyzing glucono-δ-lactone (GDL), limited solubility of a calcium salt source (Ca-alginate, CaSO4) and/or presence of chelating agents [37]. Compared to the method of diffusion gelation, the gelling ions are here released in a controlled fashion and over a period of time from sources distributed throughout the alginate. A molar ratio between GDL and SrCO3/CaCO3 of 2:1 will provide neutral gels [38]. Examples where this technique is utilized are described in more detail in Section 3.2 and Section 3.3.
Figure 2  Four consecutive G-residues from two chain segments of one or two alginate molecules creating a binding site for divalent cations (A). Formation of an intermolecular network of alginate molecules formed in presence of gelling ions such as Ca2+ (B). Gelling ions organized in alternative junction zones (C) [18].

2.2.2. Covalent Gelation
Covalently cross-linked hydrogels can be prepared from chemically modified alginates [39,40]. To better control the physical properties of alginate gels, covalent cross-linking has been broadly investigated [41]. A covalently cross-linked hydrogel is chemically stable and can provide different modes of stress relaxation. By covalently conjugating methacrylate groups onto the alginate backbone, covalently cross-linked hydrogels can be prepared in the presence of a photoinitiator and UV light. Cells will be evenly distributed throughout the hydrogel if the cells are suspended in the alginate solution prior to photocrosslinking [17,42]. Photocrosslinking has a number of different advantages. By comparison with ionic gelation, photocrosslinking allows formation of more stable alginate hydrogels independent of the level of gel forming ions and non-gelling ions [42]. Mechanical properties and biodegradation rates of a hydrogel can be adjusted by varying the degree of alginate methacrylation [43].