**3. Recrystallization in ice**

As the phenomenon of recrystallization has origins in metallurgy, geology and materials a general discussion of this process with reference to these areas is necessary (Section 2.0), followed by a discussion on the structure and properties of ice and ice recrystallization (Section 3.0) and the importance of inhibiting ice recrystallization (Section 4.0). Finally, inhibitors of ice recrystallization and proposed mechanism(s) of action will be addressed, beginning with the first known inhibitors of ice recrystallization, biological antifreezes (Section 5.0), and conclud‐ ing with novel synthetic peptides, glycopeptides, polymers and small molecules (Section 6.0). This chapter will conclude with a summary of the role of ice recrystallization in cryo-injury and a discussion on the cryoprotective ability of compounds that exhibit the ability to inhibit ice recrystallization, with the benefits and/or drawbacks of their use during cryopreservation

As stated in the introduction, the process of recrystallization has been extensively studied and reviewed throughout the metallurgic literature. [1,2] While the mechanism is quite complex, it is generally defined as the thermally induced change in grain structure facilitated by the formation and/or migration of high angle grain boundaries and is driven by the stored energy of deformation. [1] A grain is defined as the microstructure that constitutes metals and alloys. In a metal, each grain consists of an ordered arrangement of atoms (depicted in Figure 1). [3,4] A grain boundary is the interface where two or more grains of different orientations meet and is considered a defect within the crystal structure. A grain boundary contains atoms that are not well aligned with neighboring grains, leading to less efficient packing and a less ordered structure within the grain boundary. [5] Thus, grain boundaries have a higher internal energy than ordered grains. [5,6] At elevated temperatures, atoms within grains are able to transfer

(Section 7.0).

**2. The phenomenon of recrystallization**

178 Recent Developments in the Study of Recrystallization

between grain boundaries and neighboring grains. [3,4]

**Figure 1.** An illustration of grains and grain boundaries in polycrystalline metals and/or alloys.

The process of "plastic deformation" causes a permanent change in the shape of the met‐ al or alloy. During this process, energy is stored mainly in the form of dislocations, ulti‐ Ice has many different polymorphic forms. Individual water molecules in ice can possess different arrangements within three-dimensional space and this is dependent upon tempera‐ ture and pressure. The most common form of ice below 0 °C and atmospheric pressure is the hexagonal ice Ih lattice unit. [11,12] It possesses a regular crystalline structure in which a single oxygen atom is hydrogen-bonded to two hydrogen atoms. The hexagonal ice Ih lattice unit is characterized by four axes, *a*1, *a*2, *a*3 and *c*, and the surface of the hexagonal unit has eight faces. [11-14] Two of these faces are normal to the *c*-axis and are the basal faces, and the remaining six are prism faces. The structure of hexagonal ice is shown in Figure 2. The arrangement of intermolecular hydrogen bonds influences the properties and phases of ice. At 0 °C and atmospheric pressure ice grows most rapidly along the *a*-axis to give hexagonal shaped crystals which grow as sheets. [11-13,15]

approximately one monolayer of water. The effect of temperature and thickness also var‐ ies depending on the face of ice (prism or basal) from which it is calculated, [20,22] and studies have also reported that there is twice as much anisotropy of the water molecules in the QLL for the prism face than the basal face. [34] Light scattering techniques have shown that ice crystals grow into the QLL and not into the bulk water layer. [38,39]

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The recrystallization of ice in polycrystalline aqueous solutions is believed to occur through either grain boundary migration or Ostwald ripening. Grain boundary migration in ice is similar to grain boundary migration in metals and alloys where large ice grains grow larger at the expense of small ice grains. In metallurgy a grain consists of an ordered arrangement of atoms and a grain boundary is the interface where two (or more) grains meet. However, in ice a grain consists of the crystallographic orientation of the water molecules commonly observed in ice Ih (Figure 2). Grain boundaries are therefore the interfaces between different oriented ice grains. [40,41] Grain boundary migration occurs as individual molecules transfer from unfavorably oriented ice grains to favorably oriented ice grains. The boundaries of individual ice grains tend to be curved and the degree of curvature is proportional to the size of the grain. Boundaries of small ice crystals have a higher degree of curvature making them more convex (bulge outwards) and thus have a higher amount of surface energy. Large ice crystals have more concave grain boundaries and have a lower amount of surface energy. Grain boundaries migrate towards their center of curvature to reduce the overall degree of curvature, resulting in ice grains with concave boundaries (larger crystals) growing larger while those with convex boundaries (smaller crystals) decrease in size (depicted in Figure 3). [42,43] Thus, the driving force of grain boundary migration in ice arises from a reduction in grain boundary curvature,

**Figure 3.** Representation of a liquid-layer (shaded) in a curved boundary between two ice grains. Large ice grains with concave boundaries (grain 2) grow larger while small grains with convex boundaries (grain 1) decrease in size to re‐

Grain boundary migration of polycrystalline ice assumes that water molecules are transferred directly from the shrinking ice grain to the growing grain. This assumption often neglects the presence of bulk-water or the QLL in between individual ice grains as the system is treated

duce the overall degree of grain boundary curvature. Arrows indicate the direction of boundary migration.

which results in an overall reduction in the energy of the system.

**Figure 2.** Schematic representation of the hexagonal ice Ih lattice unit illustrating the *a*1, *a*2, *a*3 and *c* axes and the bas‐ al, prism and pyramidal planes.

When ice is in an aqueous solution, the interface between the ice lattice and bulk water is not an abrupt transition. Studies have indicated that a semi-ordered layer exists in between the highly ordered ice lattice and the less ordered bulk water surrounding ice crystals [14,16-22]. This layer has been named the quasi-liquid layer (QLL). While more than 150 years ago Michael Faraday proposed that the surface of ice when near the melting temperature is covered by a thin liquid layer, Fletcher was the first to propose a model for the existence of the QLL in 1962, which was subsequently revised in 1968. [16,17] Important insights on the properties of the QLL was described by Haymet where using molecular dynamic simulations and the TIP4P model of water, the structure and dynamics of the ice/water interface was studied. [18,19] Data from these simulations made it possible to calculate the density profile, molecular orientation and diffusion constants of water molecules in the QLL. The thickness of interface region between ice lattice and bulk water is approximately 10-15 Å thick, but this has been shown to be temperature dependent. [18,19,23] The average density profile, translational and orienta‐ tional order and diffusion constants of water within the QLL interface also vary depending on the face of ice from which they are calculated. Studies have suggested that the QLL is thicker on the basal and prism faces than on the pyramidal and secondary prism planes. [14]

The exact molecular nature and thickness of the QLL interface has been debated through‐ out the literature and a wide variety of techniques have been used to study it including atomic force microscopy, [24] X-ray diffraction, [25] infrared spectroscopy, [26] protonbackscattering, [27] Raman spectroscopy, [28] quartz-crystal microbalance measurements, [29] light scattering techniques, [30-32] photoelectron spectroscopy, [33] optical ellipsome‐ try, [22,34,35] optical reflection [36] and mechanical measurements. [37] Ellipsometric studies measuring the refractive index on the basal and prism faces of ice have suggested that the interface is more water-like in nature, rather than ice-like. [20-22,24,33,34] In con‐ trast, other studies have suggested that the orientation and motion of water molecules in the QLL closely resembles that of ice. [25,27,36] The thickness of the QLL has been shown to be temperature dependent, [29,33] such that at temperatures approaching the melting point of ice (at -0.03 °C) the thickness was 15 nm, corresponding to approximate‐ ly 40 monolayers water. [26] However, below -10 °C the thickness was less than 0.3 nm, approximately one monolayer of water. The effect of temperature and thickness also var‐ ies depending on the face of ice (prism or basal) from which it is calculated, [20,22] and studies have also reported that there is twice as much anisotropy of the water molecules in the QLL for the prism face than the basal face. [34] Light scattering techniques have shown that ice crystals grow into the QLL and not into the bulk water layer. [38,39]

The recrystallization of ice in polycrystalline aqueous solutions is believed to occur through either grain boundary migration or Ostwald ripening. Grain boundary migration in ice is similar to grain boundary migration in metals and alloys where large ice grains grow larger at the expense of small ice grains. In metallurgy a grain consists of an ordered arrangement of atoms and a grain boundary is the interface where two (or more) grains meet. However, in ice a grain consists of the crystallographic orientation of the water molecules commonly observed in ice Ih (Figure 2). Grain boundaries are therefore the interfaces between different oriented ice grains. [40,41] Grain boundary migration occurs as individual molecules transfer from unfavorably oriented ice grains to favorably oriented ice grains. The boundaries of individual ice grains tend to be curved and the degree of curvature is proportional to the size of the grain. Boundaries of small ice crystals have a higher degree of curvature making them more convex (bulge outwards) and thus have a higher amount of surface energy. Large ice crystals have more concave grain boundaries and have a lower amount of surface energy. Grain boundaries migrate towards their center of curvature to reduce the overall degree of curvature, resulting in ice grains with concave boundaries (larger crystals) growing larger while those with convex boundaries (smaller crystals) decrease in size (depicted in Figure 3). [42,43] Thus, the driving force of grain boundary migration in ice arises from a reduction in grain boundary curvature, which results in an overall reduction in the energy of the system.

**Figure 2.** Schematic representation of the hexagonal ice Ih lattice unit illustrating the *a*1, *a*2, *a*3 and *c* axes and the bas‐

When ice is in an aqueous solution, the interface between the ice lattice and bulk water is not an abrupt transition. Studies have indicated that a semi-ordered layer exists in between the highly ordered ice lattice and the less ordered bulk water surrounding ice crystals [14,16-22]. This layer has been named the quasi-liquid layer (QLL). While more than 150 years ago Michael Faraday proposed that the surface of ice when near the melting temperature is covered by a thin liquid layer, Fletcher was the first to propose a model for the existence of the QLL in 1962, which was subsequently revised in 1968. [16,17] Important insights on the properties of the QLL was described by Haymet where using molecular dynamic simulations and the TIP4P model of water, the structure and dynamics of the ice/water interface was studied. [18,19] Data from these simulations made it possible to calculate the density profile, molecular orientation and diffusion constants of water molecules in the QLL. The thickness of interface region between ice lattice and bulk water is approximately 10-15 Å thick, but this has been shown to be temperature dependent. [18,19,23] The average density profile, translational and orienta‐ tional order and diffusion constants of water within the QLL interface also vary depending on the face of ice from which they are calculated. Studies have suggested that the QLL is thicker

on the basal and prism faces than on the pyramidal and secondary prism planes. [14]

The exact molecular nature and thickness of the QLL interface has been debated through‐ out the literature and a wide variety of techniques have been used to study it including atomic force microscopy, [24] X-ray diffraction, [25] infrared spectroscopy, [26] protonbackscattering, [27] Raman spectroscopy, [28] quartz-crystal microbalance measurements, [29] light scattering techniques, [30-32] photoelectron spectroscopy, [33] optical ellipsome‐ try, [22,34,35] optical reflection [36] and mechanical measurements. [37] Ellipsometric studies measuring the refractive index on the basal and prism faces of ice have suggested that the interface is more water-like in nature, rather than ice-like. [20-22,24,33,34] In con‐ trast, other studies have suggested that the orientation and motion of water molecules in the QLL closely resembles that of ice. [25,27,36] The thickness of the QLL has been shown to be temperature dependent, [29,33] such that at temperatures approaching the melting point of ice (at -0.03 °C) the thickness was 15 nm, corresponding to approximate‐ ly 40 monolayers water. [26] However, below -10 °C the thickness was less than 0.3 nm,

al, prism and pyramidal planes.

180 Recent Developments in the Study of Recrystallization

**Figure 3.** Representation of a liquid-layer (shaded) in a curved boundary between two ice grains. Large ice grains with concave boundaries (grain 2) grow larger while small grains with convex boundaries (grain 1) decrease in size to re‐ duce the overall degree of grain boundary curvature. Arrows indicate the direction of boundary migration.

Grain boundary migration of polycrystalline ice assumes that water molecules are transferred directly from the shrinking ice grain to the growing grain. This assumption often neglects the presence of bulk-water or the QLL in between individual ice grains as the system is treated below -10 °C. [42] However, Ostwald ripening of polycrystalline ice in an aqueous solution considers the whole ice crystal/liquid water system and thus accounts for the presence of bulkwater and the QLL. In ice, Ostwald ripening is the thermodynamically driven process whereby large ice crystals grow larger at the expense of small crystals, resulting in an overall reduction in energy of the ice crystal/bulk-water interface. [44-46] Throughout the Ostwald ripening process a constant ice volume is maintained. Smaller ice crystals have a higher surface area to volume ratio, giving them higher surface free energy since water molecules on the surface are less stable than the water molecules within the ice crystal. [44,45] However, larger ice crystals have a greater volume to surface area ratio and thus are thermodynamically more stable than small ice crystals. As the total overall volume of ice remains constant during the Ostwald ripening process, water molecules transfer from the surface of smaller ice crystals to bulk-water and then are transferred onto the surface of larger ice crystals. The net result is an increase in the average ice crystal size and a decrease in the total number of ice crystals at a constant total ice volume, resulting in an overall reduction in the free energy of the system. [46]

deal of interest in the scientific and industrial communities. [56] Biological antifreezes are a complex class of compounds with dramatically different structures, making it difficult to understand how they inhibit ice recrystallization. Nevertheless, this important class of compounds is the foundation upon which all "rationally designed" novel ice recrystallization inhibitors are based, including the more recently reported small molecule inhibitors of ice

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In the late 1950s and early 1960s it was observed by Scholander and colleagues that marine teleost fish did not freeze during the winter despite the water temperature being -1.9 °C, over a degree below the freezing point of their blood serum. [51,52] DeVries and Wohlschlag later attributed their survival to the presence of circulating proteins and glycoproteins. [53-55] These proteins later became known as biological antifreezes, specifically antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs). A variety of AFPs and AFGPs have since been identified

There are four classes of structurally diverse fish AFPs that have been identified. These are type I, [13,63-72] type II, [73-81] type III, [73-77,82-87] and type IV AFPs. [88-90] The four types of fish AFPs have a wide variation in their size, which can range from 3-12 kDa, and in their secondary structures, which can be α-helices, β-rolls, random coils and globular structures. AFGPs are also present in fish, and are comprised of a tripeptide repeat of (Thr-Ala-Ala)*n*, in which the secondary hydroxyl group of threonine is glycosylated with the disaccharide β-Dgalactosyl-(1-3)-α-*N*-acetyl-D- galactosamine (structure shown in Figure 4). [15,55,60,61,91-95] In general, AFGPs have a homologous structure and have been separated into eight subclasses, AFGP 1-8, based on their molecular masses which range from 2.6 kDa (n = 4) to 33.7 kDa (n = 50). [55] Minor sequence variations have been identified in AFGPs where the first alanine residue is replaced by proline, or where the glycosylated threonine residue is occasionally replace by arginine. [96-101] The solution structure of AFGPs has been debated in the literature. Early circular dichroism (CD) and nuclear magnetic resonance (NMR) studies suggested AFGPs adopt an extended random coil structure. [102-107] However, studies have also suggested that they adopt an ordered helix similar to a PPII type II helix. [106,108-110] It has also suggested that they adopt an amphipathic helical structure, with a hydrophilic face containing the exposed hydroxyl groups of the disaccharide moiety and a hydrophobic face containing the exposed methyl groups of the amino acid residues. [72] However, the most recent studies have indicated that AFGP 1-5 possess no form of long-range order and that AFGP-8 is predominantly random coil with short segments of localized order. [106-108] A brief summary of the key structural differences between AFPs and AFGPs is provided in Figure 4.

A number of other AFPs have been identified in other organisms. Various insect AFPs have been identified such as those from the spruce budworm moth (*Choristoneura fumiferana*, *Cf*AFP), [111,112] the yellow mealworm beetle (*Tenebrio molitor*, *Tm*AFP), [113,114] the firecoloured beetle (*Dendroides canadensis*, *Dc*AFP), [115] and the snow flea (sfAFP). [116] Plant AFPs have also been identified from carrot (*Daucus carrota*), [117] bittersweet nightshade (*Solanum dulcamara*), [118] perennial ryegrass (*Lolium perenne*), [119-121] Antarctic hair grass

recrystallization. [57-62]

**5.1. Structures of Biological Antifreezes (BAs)**

in a number of different fish, insects, plants and bacteria.
