**2. Hyaluronic Acid**

HA, also referred to as hyaluronan, has a repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosamine (the hexuronic acid and amino sugar, respectively) attached by a beta 1–3 bond, and the disaccharide units are joined by a beta 1–4 bond [1]. [1] It is found in liquid connective tissues such as the synovial fluid of joints and the vitreous humor of the eye where, in conjunction with other charged constituents of the extracellular matrix, it plays a key role in maintaining viscoelasticity via water retention due to both hydrogen bonds and osmotic pressure generated from the high density of anionic groups and accumulation of counter cations. When in water, HA has a gelatin-like consistency. Its viscoelasticity and ability to form matrices that retain water allow it to cushion joints, resist compression and help reduce friction in all joint tissues [3]. It also plays a role in the extracellular matrix of several tissues where it mediates receptor-driven detachment, mitosis, and migration. This control of cell division and cell migration means HA is commonly implicated in tumor development and cancer metastasis [3]. HA is the highest molecular weight GAG [1], presenting with a broad range of molecular masses generally ranging between 10<sup>5</sup> and 10<sup>7</sup> Daltons. This is in stark contrast to other GAGs, which are generally on the order of 103 Daltons [2]. HA's molecular mass plays an important role in its function. Studies have shown that HA fragments of varying lengths may alert the body to trauma and play roles in the progression of wound healing. Degradation of HA increases tissue permeability and HA fragments enhance angiogenesis, promoting tissue healing processes [3]. In contrast, endogenous HA has been shown to promote extracellular matrix secretion, reduce inflammation, and inhibit immune cell migration to maintain homeostasis in healthy tissue [3].

HA has many properties that make it an ideal candidate for tissue engineering scaffolds. It is biodegradable, biocompatible, and resorbable. HA is involved in every step of wound healing in the body [14]. The interplay between its hydrophilicity and control of cell migration allows HA to form a temporary, ideal wound healing environment. Because HA

is hygroscopic, it can control the hydration of tissue during healing, allowing for increased flow of nutrients and effluence of waste products [15]. It also stimulates cells via interactions with CD44, RHAMM, and ICAM-1 cell receptors, which allows it to regulate cell adhesion, motility, inflammation, and differentiation [14]. Despite this, for most cell types it does not support sufficient attachment or spreading and requires chemical modification to support cell growth and survival [16].

In its native form, HA is a weak scaffolding material because it is rapidly degraded in vivo by hyaluronidase and is highly soluble, which can cause dissolution. It must, therefore, be chemically modified and crosslinked or combined with another polymer to form stable, structurally integrated scaffolds that support cell adhesion and proliferation [17]. Encouragingly, HA can be crosslinked under basic, acidic, and neutral pH conditions or combined with other natural and synthetic polymers to confer strength, allowing for diverse applications such as treating difficult to heal wounds, burns, and any form of trauma that requires a space-filling scaffold [14].

Additional support for chemical modification of HA comes from the fact that HA itself does not bond to surrounding tissue when it is used to fill defects, and it is often of sufficiently high molecular weight that it does not diffuse into the surrounding tissue to form an integrated seal when crosslinking it in situ. In addition, while viscous HA gels can be injected, injection of unmodified HA has been shown to cause damage and hemorrhaging in some tissues, such as in the heart and liver. With the goal of overcoming all of these shortcomings, Shin, et al. developed a tissue adhesive HA hydrogel functionalized with the adhesive catecholamine motif from mussel foot protein. This gel was shown to reduce apoptosis, increase viability, and enhance the function of human adipose-derived stem cells and hepatocytes. HA-catecholamine laden with hepatocytes was shown to gel and adhere to the liver of athymic mice within minutes. Further, the gel was still present after two weeks and albumin secreted by the transplanted hepatocytes was detectable in the blood stream 3 days after implantation [18], indicating some recapitulation of endogenous tissue behavior.
