Biocompatibility of hyaluronic acid: From cell recognition to therapeutic applications

28 Biocompatibility of hyaluronic acid: From cell recognition to therapeutic applications K. G H O S H, Children’s Hospital and Harvard Medical School, USA

28.1

Introduction

Hyaluronic acid (HA), also known as hyaluronan, is a ubiquitous, naturallyoccurring, polyanionic, glycosaminoglycan that consists of repeating nonsulfated disaccharide units (α-1,4-D-glucuronic acid and β-1,3-N-acetyl-Dglucosamine) of variable sizes, appearing in molecular weights ranging from 0.1 to 10 million Daltons (Fraser et al., 1997). HA was initially known to exhibit only unique physicochemical properties that help to maintain tissue viscoelasticity. However, subsequent studies revealed that HA also exerts important biological effects by binding to specific cell surface receptors and other extracellular matrix (ECM) molecules, which initiates intracellular signaling cascades that modulate key functions such as adhesion, migration and proliferation (Aruffo et al., 1990; Entwistle et al., 1996; Toole, 2004). Together, the complex biological and physicochemical properties of HA influence key developmental processes such as embryogenesis, morphogenesis and wound repair (Chen and Abatangelo, 1999; Toole, 2001). As a result, HA has attracted huge interest for use in various therapeutic applications (Vercruysse and Prestwich, 1998; Allison and Grande-Allen, 2006; Balazs and Denlinger, 1989). The purification of the non-inflammatory fraction of HA over three decades ago initiated a host of therapeutic trials that involved supplementation of unmodified HA into the site of defect (Balazs and Gibbs, 1970). Early results showed that HA was effective in protecting retinal damage during ophthalmic surgery, reducing wound scarring, preventing post-operative adhesions, and reducing pain while increasing mobility in arthritic joints (Denlinger and Balazs, 1980; Denlinger et al., 1980; Balazs and Denlinger, 1989); however, these effects were short-lived due to the rapid degradation of native HA by the HA-specific enzyme, hyaluronidase. To increase its residence time in vivo, HA has since been chemically modified and subsequently crosslinked using myriad approaches (Campoccia et al., 1998; Prestwich et al., 1998; Park et al., 2003). To further enhance their biological activity or produce 716

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tailor-made tissues, chemically-modified HAs have been derivatized with various ECM-derived peptides or protein fragments (Ghosh et al., 2006; Shu et al., 2004). Such HA derivatives have found great use as biomaterials in several medical applications such as drug delivery, wound repair and tissue engineering (Allison and Grande-Allen, 2006; Ghosh et al., 2006; Horn et al., 2007; Kim et al., 2007; Luo et al., 2000; Nettles et al., 2004; Shu et al., 2004). This chapter will discuss how the vast knowledge about the key role of HA in tissue development, homeostasis and repair has been leveraged to develop novel and potent therapeutic applications and highlight recent studies that implicate the use of HA in regenerative medicine.

28.2

Native hyaluronan

28.2.1 Occurrence Hyaluronan is found in various mammalian tissues across all vertebrates. In terms of net amount, almost half of the total HA per organism can be found in skin, with the musculo-skeletal system accounting for another quarter fraction of the total quantity. In terms of concentration per tissue, it is the highest in typical connective tissues such as synovial fluid and umbilical cord (~3 mg/ml), while the skin and vitreous humor (eye) also containing moderate concentrations of HA (~0.2 – 0.5 mg/ml). Detailed analyses of HA distribution in mammalian tissues has been published elsewhere (Reed et al., 1988; Laurent, 1981; Engstrom-Laurent et al., 1985; Tengblad et al., 1986; Laurent et al., 1996; Fraser et al., 1993; Laurent et al., 1995) and summarized by Fraser et al. (1997). HA can also be found in lung and kidney, while the lowest concentration has been reported in plasma. Interestingly, the highest concentration of HA in a mammalian tissue is found in rooster comb (7.5 mg/ml), which has long served as an important source for HA isolation (Manna et al., 1999; Swann, 1968; Swann and Caulfield, 1975). Importantly, the source of HA isolation should be carefully determined since HA obtained from different tissues and species contains varying amounts and types of contaminants that may alter its function both in vitro and in vivo (Shiedlin et al., 2004).

28.2.2 Biosynthesis HA is synthesized in the plasma membrane by a specialized enzyme called hyaluronan synthase, with the nascent chains being directly secreted into the extracellular space (Fraser et al., 1997; Prehm, 1983a; Watanabe and Yamaguchi, 1996). The enzyme alternately adds the sugar units from the activated nucleotide precursors (UDP – glucuronic acid and UDP-Nacetlyglucosamine) to the reducing end of the growing chain (Mian, 1986;

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Prehm, 1983b), which is in marked contrast with other glycosaminoglycans that grow at the non-reducing ends. These newly-synthesized HA chains can contain up to 10 000 repeat disaccharide units or more, with the molecular weights reaching up to and beyond four million Daltons. Previous studies have suggested that the final chain size is regulated by thermodynamic parameters, where the decrease in entropy during macromolecular synthesis is balanced by release of free energy during cleavage of repeat disaccharide units and subsequent chain organization (Nickel et al., 1998; Philipson et al., 1985). The secreted HA chains are very flexible and are usually found in the ECM in a randomly-coiled configuration; when stretched from end to end, these chains can extend up to ~10 µm. HA is synthesized by most tissue cells during their original life-span, although mesenchymal cells exhibit the strongest expression (Lee et al., 1993; Nishida et al., 1999; Evanko et al., 1999; Asplund et al., 1993; Heldin and Pertoft, 1993).

28.2.3 Physicochemical and structural properties HA is polyanionic at extracellular pH, which results from oxidation of the carboxylic group on HA. This property allows it to bind cations (e.g. Na+, Ca2+), leading to an increase in osmotic gradient that, in turn, attracts and binds water within the HA polymeric network (Laurent and Fraser, 1992; Comper and Laurent, 1978; Gribbon et al., 2000). As a result, the long HA chains swell and occupy enormous extracellular space. The bound water is largely immobilized, which causes steric exclusion by restricting free diffusion of fluids and other ECM molecules (Ogston and Sherman, 1961; Ogston and Phelps, 1961). In addition, despite being unipolar, HA chains interact with themselves through the creation of distinct hydrophobic patches along their backbones (Scott and Heatley, 1999; Mikelsaar and Scott, 1994). At higher concentrations, such chain-chain interactions form an entangled network, which confers to HA its unique viscoelastic property by its ability to resist (elastically) rapid, short duration fluid flow while undergoing partial realignment and viscous movement in response to show longer duration fluid flow (Furlan et al., 2005; Falcone et al., 2006). Such hygroscopic nature and unique biomechanical function of HA makes it an indispensable component of the vitreous humor and the ECM of cartilage and other skeletal joints (Weiss, 2000). In addition to its unique physicochemical functions, HA also provides important structural support to the ECM. Hyaluronan-binding proteins, called hyaladherins, mediate its interaction with various extracellular components, including proteoglycans, collagen and fibrin, that stabilizes both HA and the ECM (Toole, 2001; Chen et al., 1994). In cartilage, for example, the link protein promotes HA-aggrecan association that is crucial for HA stabilization and, together with its association with collagen fibrils, the resulting aggrecan-

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HA complex provides structural stability to the entire connective tissue (Nishida et al., 1999; Hardingham, 1981). Versican, an aggregating proteoglycan, also associates with HA and retains it in tissues through complex interactions involving fibronectin and collagen (Sorrell et al., 1999; Evanko et al., 1999). Another interesting manifestation of HA’s structural role is the formation of a pericellular coating seen (indirectly) around most cells of mesodermal origin through exclusion of cells and other particles (Lee et al., 1993; Knudson and Knudson, 1993; Knudson et al., 1993). Besides creating a local cellular microenvironment, the HA coating helps in fending off attacks by immune cells and viruses.

28.2.4 Biological function Role in inflammation In addition to exhibiting distinct physicochemical properties, HA also plays an important biological role through its ability to modulate inflammation following tissue injury, which imparts upon HA its superior biocompatibility. Tissue damage causes HA degradation into ‘active’ lower molecular weight (LMW) fragments, which can occur either through the action of hyaluronidase or due to non-enzymatic activities such as mechanical impact or free radical activity (Laurent and Fraser, 1992; Noble, 2002). The LMW HA activates pro-inflammatory cytokines and stimulates tissue cell proliferation, migration and angiogenesis that, collectively, promote tissue repair. Specifically, LMW HA causes toll-like receptor 4 (TLR 4)-mediated activation of dendritic cells and capillary endothelial cells, which secrete inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, and IL-8 (Termeer et al., 2000; Termeer et al., 2002; Taylor et al., 2004). Importantly, this proinflammatory activity of HA is observed exclusively at LMW as improper degradation of HA leads to incomplete tissue repair (Termeer et al., 2000; Termeer et al., 2002; Noble, 2002). The inflammatory cytokines cause capillary endothelial cells to increase expression of HA, which interacts with the HA-specific CD44 receptors on lymphocytes to promote their recruitment to the site of inflammation (Siegelman et al., 1999; Mohamadzadeh et al., 1998). The CD44/HA interaction is a critical determinant of successful tissue repair; CD44-knockout (CD44-KO) mice are unable to repair bleomycin-induced lung damage and eventually die within two weeks (Teder et al., 2002). Detailed analyses showed that unlike wild-type mice, the CD44-KO mice had persistently high levels of inflammation and HA oligosaccharides, further supporting the role of LMW HA fragments in tissue inflammation. LMW HA fragments are also abundant in other pathologic conditions marked by chronic inflammation such as rheumatoid arthritis and chronic colon inflammation, among others (de la Motte et al., 2003; Laurent and Fraser, 1992; Laurent et al., 1995).

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The higher MW (HMW) HA, on the other hand, exerts a contrasting effect on the reparative process by inhibiting both inflammation and angiogenesis (Day and de la Motte, 2005; Chen and Abatangelo, 1999). Recent data suggests that HMW HA chains form intermolecular crosslinks to create robust fibrils that organize into complex molecular scaffolds, which strongly bind leukocytes and sequester them from the underlying proinflammatory tissue cells, thereby limiting tissue inflammation (Day and de la Motte, 2005). This spontaneous crosslinking of HMW HA chains is mediated by four proteins, viz. inter-α-inhibitor (IαI), pre-α-inhibitor (PαI), pentraxin 3 (PTX3) and TSG-6 (a 35 kDa-secreted product of the tumor necrosis factor-stimulated gene-6), all of which are present at sites of inflammation, and involves the covalent attachment of heavy chains of IαI and PαI to the HMW HA molecules (Day and de la Motte, 2005; Zhuo et al., 2004). Free HA molecules fail to bind monocytes (de la Motte et al., 2003), suggesting that the binding of leukocytes to crosslinked HMW HA scaffolds occurs specifically through either induction of CD44 clustering or engagement of other co-receptors by the various molecules that adorn these HA cables. Importantly, the CD44-mediated leukocyte binding to HA scaffolds prevents their activation by controlling their ICAM-1-mediated interaction with the endothelium (Zhang et al., 2004; Selbi et al., 2004). A more recent study suggests that leukocyte interaction with HMW HA cables may actively induce growth factor and ECM secretion that promote tissue repair (Day and de la Motte, 2005). The crosslinked molecular network of HMW HA chains, typically found within joint tissues such as the articular cartilage surface of osteoarthritic knees, is also likely to prevent excessive loss of ECM and simultaneously guide the organization of new matrix (Milner and Day, 2003; Szanto et al., 2004). The crosslinked HMW HA scaffolds may also act as a reservoir for free radicals, thereby limiting excessive tissue damage (Zhuo et al., 2004; Rugg et al., 2005). This size-dependent effect of HA on tissue inflammation may explain, at least in part, the differences observed between fetal and adult tissue repair. Unlike adult wounds, early-gestation fetal wounds undergo scarless repair, which has been linked to the lack of an inflammatory response and consistently elevated levels of HMW HA resulting from increased synthesis by fetal fibroblasts (Chen et al., 1989) and decreased HAdase activity (West et al., 1997); exogenous addition of HAdase to such fetal wounds induces scar formation (Iocono et al., 1998). This, in accordance with the foregoing discussion, suggests that the HA fragments (LMW HA) in fetal wounds triggers inflammation through a TLR 4-mediated mechanism while the HMW HA likely exerts its conciliatory effect by attenuating inflammation via CD44mediated signaling.

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Interaction with tissue cells HA also exerts a strong biological effect by interacting with the CD44 and RHAMM (receptor for hyaluronan-mediated motility) receptors expressed on tissue cells. The binding of HA to these cellular receptors initiates downstream signaling that involves activation of protein phosphorylation cascades and cell cycle proteins, cytokine release and signal transduction to the cytoskeleton and nucleus that, together, regulate key cell functions such as adhesion, proliferation and migration (Entwistle et al., 1996; Toole, 2004). Importantly, tissue synthesis of HA is increased dramatically during important physiological processes such as morphogenesis and tissue repair. These physiological events involve proliferation and en masse movement of tissue cells, which is promoted by both HA-receptor interactions and the ability of HA to create large extracellular spaces required to accommodate huge cell population (Chen and Abatangelo, 1999; Toole, 1997). The expression of both HA and its receptors can be modulated by a variety of ECM signals, suggesting that the biological activity of HA is a tightly controlled phenomenon. In addition to facilitating cell migration and proliferation, HA also promotes matrix remodeling and prevents or minimizes wound scarring, likely due to combined cell signaling and physicochemical effects (Laurent et al., 1986a; Iocono et al., 1998). HA also interacts with CD44 and RHAMM receptors on endothelial cells and promotes angiogenesis, the process of new blood vessel formation. However, the overall effect depends on its molecular weight (MW); while the lower MW HA (oligosaccharide) promotes angiogenesis and new collagen deposition, higher MW HA inhibits new vessel formation (Dvorak et al., 1987; West and Kumar, 1989a; West and Kumar, 1989b; Lees et al., 1995). Although the exact mechanism for this MW regulation is not very clear, increased angiogenesis is accompanied by an increase in the levels of the HA-degrading enzyme, hyaluronidase (HAdase) (Liu et al., 1996; West and Kumar, 1989a).

28.3

Therapeutic implications of native hyaluronan

The excellent biological and physicochemical properties of native HA advocated its use in biomedical applications, although the early attempts exploited mostly its distinct viscoelastic behavior (Balazs and Denlinger, 1989; Weiss, 2000). One of the most common and successful applications of HA has been in the treatment of osteoarthritis, a pathological condition which is characterized by cartilage degeneration and subsequent loss of lubrication at the joints. Supplementation of exogenous HA to arthritic knees improves joint function and stability through increased retention of cartilage ECM molecules such as proteoglycans, which improves the viscoelastic

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properties of synovial fluid and suppresses cartilage degradation (Peyron, 1993; Ronchetti et al., 2000). The underlying mechanisms that mediate this therapeutic benefit of HA are not yet fully understood. Unmodified HA has also been used as an aid for ophthalmic surgery where its unique hygroscopic and viscoelastic properties help in creating large operative spaces and protecting the delicate corneal endothelium from physical damage (Laurent, 1981; Denlinger and Balazs, 1980). Although the initial therapeutic benefits were promising, it soon became clear that exogenous HA was not stable in the tissue for longer durations. This short residence time of unmodified HMW HA results from its spontaneous and rapid degradation by HAdase, which specifically cleaves the molecule at the β1,4 glycosidic bond (Stair-Nawy et al., 1999; Stern and Jedrzejas, 2006). HAdases are widely expressed in human tissues and degrade large HA chains to short oligos that are then metabolized by the surrounding tissue cells, which ensures proper turnover of tissue HA. Cartilage is a particularly dynamic tissue in terms of HA turnover, where the chondrocytes continuously synthesize and catabolize hyaluronan, with the typical half life of a hyaluronan molecules being ~2-3 weeks (Morales and Hascall, 1988; Ng et al., 1992; Flannery et al., 1998). Tissues that have access to lymph vessels, such as the skin and knee joint capsule, drain out excess HA through the lymphatic pathway, where the lined reticulo-endothelial cells actively eliminate the majority of HA, with the remainder catabolized by liver endothelial cells (Fraser et al., 1996; Fraser et al., 1997; Laurent et al., 1986b; Reed and Laurent, 1992); the half-life of HA in the bloodstream is only a few minutes (Fraser et al., 1984). The vigorous nature of HAdase activity is apparent from reports that estimate that almost one-third of the total hyaluronan in human tissues undergoes complete metabolic turnover during a single day. In addition to rapid degradation in vivo, unmodified HMW HA also lacks appropriate mechanical strength required to withstand mechanical loads that tissues such as cartilage commonly experience. This poor biomechanical property also makes handling HA very difficult.

28.4

Engineered hyaluronan

28.4.1 Chemical modification To improve biomechanical properties, increase residence time and allow easy handling, HA has been chemically modified and subsequently crosslinked to obtain a variety of stable derivatives. Importantly, although the HA derivatives exhibit improved physicochemical properties, they exhibit biocompatibility similar to the native HA; this makes HA a unique biomaterial for therapeutic applications since it combines the advantages of both naturally-occurring and synthetic materials (Allison and Grande-Allen, 2006; Vercruysse and

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Prestwich, 1998; Thierry et al., 2004). The carboxylic and hydroxyl groups on the HA backbone are the preferred targets for chemical modification. Esterification and carbodiimide-mediated reactions are the most common schemes that utilize the carboxylic group. The more commonly used HA esters, or HYAFF, are produced from the action of ethyl and benzyl alcohols on the carboxyl group, where the degree of hydrophilicity scales inversely with the degree of esterification (Campoccia et al., 1998). The carbodiimide reactions, on the other hand, involve activation of the carboxylic group at low pH (4.75) such that it couples efficiently to the multivalent dihydrazide groups such as adipic dihydrazide (ADH) and dithiobis propanoic dihydrazide (DTP), where the pendant hydrazide moieties can be further conjugated to other crosslinking or therapeutic agents (Prestwich et al., 1998; Vercruysse and Prestwich, 1998; Vercruysse et al., 1997). Typical derivatizations at the hydroxyl group include sulfation, esterification, and isourea coupling (Campoccia et al., 1998; Zhang and James, 2004; Abatangelo et al., 1997; Barbucci et al., 2000; Mlcochova et al., 2006). In addition to the carboxylicand hydroxyl-group modifications, HA can also be derivatized at its reducing end as well as the deacylated glucosamine groups, although these methods are not very popular due to low yield (Ruhela et al., 2006; Asayama et al., 1998; Dahl et al., 1988). Once HA is derivatized, it is crosslinked to produce robust biomaterials that exhibit the physical and degradation profiles desired for a specific biomedical application. Several crosslinking schemes have been developed that pair up with the appropriate derivatization method. They include, but are not limited to, diacrylate or divinylsulfone crosslinking, crosslinking via internal esterification, light, glutaraldehyde, carbodiimides and disulfides (Ghosh et al., 2006; Zheng Shu et al., 2004; Shu et al., 2002; Tomihata and Ikada, 1997; Park et al., 2003; Baier Leach et al., 2003; Crescenzi et al., 2003; Bakos et al., 2000; Campoccia et al., 1998; Sannino et al., 2004). Although intermolecular crosslinking is more common, intramolecular crosslinking is also possible, as seen in the autocross-linked hyaluronan (ACP™, Fidia), which is an ester derivative containing both inter- and intramolecular links between the hydroxyl and carboxyl groups (Mensitieri et al., 1996). Regardless of the approach, the covalent crosslinking of HA reduces its solubility in water such that addition of water causes the network to swell up to an equilibrium point where the osmotic swelling forces are balanced by the elastic forces of the internal atomic bonds. The strength and degradability of the HA derivative can be controlled by modulating both the degree of crosslinking and nature of the crosslinker and, therefore, it becomes possible to tailor these biomaterials for tissue-specific applications. For example, in osteoarthritic applications, the final product must be resilient and durable enough to withstand continuous cyclic mechanical forces, while biodegradation is a more crucial design parameter for cutaneous applications (Barbucci

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et al., 2002; Milas et al., 2001; Price et al., 2006). These HA-based biomaterials can subsequently be processed into myriad physical forms such as hydrogels, foams, films, fibers and microspheres, the final form depending on the nature of its eventual application (Figallo et al., 2007; Ji et al., 2006; Tang et al., 2007; Ghosh et al., 2006; Bae et al., 2006; Shu et al., 2002). The crosslinked HA network can also be coupled with various therapeutic agents that can be released at a local tissue site at a rate controlled by the degradation profile of the HA biomaterial (Lee et al., 2001; Yerushalmi et al., 1994; Wieland et al., 2007; Vercruysse and Prestwich, 1998).

28.4.2 Biological derivatization To promote tissue repair or regeneration, HA derivatives must activate tissue cells and induce them to migrate, proliferate and differentiate. Interestingly, although HA is known to interact with specific cellular receptors (CD44 and RHAMM), the HA-based biomaterials fail to support tissue cell adhesion and spreading, primarily due to the extreme hydrophilicity of HA that binds water layers on its surface and prevents protein deposition (Jackson et al., 2002; Sawada et al., 1999; Sawada et al., 2001). This issue has been addressed through biological derivatization of the chemically-modified HA. To do so, HA is first chemically modified such that multiple pendant groups are available for subsequent covalent coupling of biologically-derived cell-recognition peptides or proteins. The Arginine-Glycine-Aspartic acid (RGD) tripeptide sequence is the shortest biological motif used for HA modification since several cell-surface integrin receptors interact with the ECM via this peptide sequence. These HA-RGD hydrogels support extensive 3T3 fibroblast attachment, spreading and proliferation in vitro, and when these cells are encapsulated in the hydrogels and implanted in murine cutaneous wounds, they promote granulation tissue formation (Shu et al., 2004; Glass et al., 1996). Importantly, acellular HA-RGD hydrogels fail to support adult dermal fibroblast spreading and proliferation in vitro and fail to promote fibroblast invasion in vivo, suggesting that these hydrogels have good inductive but poor conductive properties (Ghosh et al., 2006; Shu et al., 2004). This limitation has been overcome by coupling more potent FN functional domains that simultaneously engage multiple cellular receptors (Ghosh et al., 2006). Compared to the HA-RGD hydrogels, these FN-modified hydrogels produced significant enhancement in primary adult human dermal fibroblast spreading, migration and proliferation, and more recent findings indicate that they also produce marked accentuation in porcine cutaneous wound repair. In addition to these FN-derived peptides, HA has also been modified by other polypeptides such as poly-L-lysine, poly-D-lysine, glycine or glutamine, and these derivatives show significant improvement in fibroblast adhesion and proliferation (Hu et al., 1999).

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To enhance bioactivity, HA has also been combined with larger adhesive proteins such as collagen and gelatin, with collagen receiving higher preference owing to its greater physiological significance and unique polymerization capability, which also improves the mechano-structural properties of the resulting blends. To improve stability in vivo, these blends are typically crosslinked; for example, composites have been developed by preparing HA-collagen coagulates, which are then crosslinked with starch dialdehyde and glyoxal to produce a collagenase-resistant and cell-interactive biomaterial (Rehakova et al., 1996). Other crosslinkers such as polyethylene oxide and hexamethylene diisocyanate can also be used (Soldan and Bakos, 1997). Similarly, gelatin has also been incorporated into HA solutions and the resultant mix crosslinked using carbodiimide (EDCI) chemistry; this blend subsequently promoted epidermal healing in vivo (Choi et al., 1999). In a more recent study, both HA and gelatin were identically derivatized using an EDCI chemistry that attached free pendant thiol groups to their backbones (Shu et al., 2003). When blended together, the thiol groups on HA and gelatin derivatives underwent spontaneous air-induced crosslinking to form stable, disulfide-linked composites that promoted extensive cell spreading and proliferation in vitro. Incidentally, although ECM-derived peptides are often required to promote cell adhesion and spreading on HA scaffold surfaces, no such biological derivatization seems to be necessary for three-dimensional (3D) cultures. For instance, when chick dorsal root ganglia were cultured in 3D hydrogels obtained by cross-linking thiol-derivatized HA, cultures produced robust neurite extension, which remained stable for up to eight days (Horn et al., 2007). A separate study showed that encapsulation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves, within crosslinked HA hydrogels maintained cell viability and promoted significant production of elastin over a period of six weeks (Masters et al., 2005). Furthermore, photo-crosslinked HA hydrogels have been shown to support chondrocyte viability, maintain the cells’ spherical shape, and promote extensive synthesis of cartilaginous matrix (Nettles et al., 2004). What regulates this difference in bioactivity between 2D and 3D cultures is not fully known although it may likely be due to enhanced intracellular signaling resulting from greater cell-HA interaction in a 3D environment. Just as various ECM-derived peptides or proteins are added to HA to improve its biological activity, HA is also added to a variety of polymeric materials to produce composites that retain the unique material properties of the synthetic polymer while exhibiting greater biological affinity. For example, HA-alginate composites can be formed in the presence of calcium, which facilitates gelation of alginate solution. The resulting gel exhibits both stable mechanical properties (due to alginate) and greater cell recognition (due to hyaluronan), which may together contribute towards increased ECM synthesis by encapsulated chondrocytes (Gerard et al., 2005; Oerther et al., 1999).

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Similarly, HA has also been blended with carboxymethylcellulose, an anionic polymer, and subsequently crosslinked to produce a robust biomaterial for prevention of postsurgical adhesions (Burns et al., 1996). In yet another variation, HA was added to hydroxyapatite-collagen mix that resulted in a biocompatible and mechanically robust material for use as a bone filler (Bakos et al., 1997).

28.4.3 Hemocompatibility In addition to being used as a scaffolding material for engineered tissues or an encapsulating material for drug delivery, HA is also preferred in applications that require continuous contact with blood. This is largely due to its ability to inhibit platelet adhesion and activation and delay both intrinsic and extrinsic coagulation pathways. These nonimmunogenic properties remain unaltered despite chemical modification of the HA network, which is often necessary to elicit greater response from vascular cells. For example, UV crosslinking of hylan or HA-divinyl sulfone (HA-DVS) gels renders them highly conducive to smooth muscle cell ingrowth without compromising their hemocompatibility (Amarnath et al., 2006; Ramamurthi and Vesely, 2005). The anticoagulant property of HA has been exploited in its use as a coating material for cardiovascular stents where it serves a dual role of: (a) increasing the stent’s hemocompatibility; and (b) releasing an encapsulated drug at a sustained rate. For instance, covalent immobilization of HA-heparin nanolayers on stainless steel cardiovascular stents not only improved the stent’s hemocompatibility but also promoted controlled release of a drug encapsulated within the HA-heparin complex (Huang and Yang, 2006). In another study, a HA-diethylenetriamine pentaacetic acid (DTPA) conjugate (HA-DTPA) was complexed with radionuclides yttrium and indium and used for coating stents and catheters during endovascular radiotherapy (Thierry et al., 2004). The resulting stents not only demonstrated significantly less fibrinogen adsorption and clotting but also maintained drug stability and release for over two weeks. It is important to note that although the chemical modifications of HA in these instances is performed solely to facilitate better binding to the stent, HA can also be derivatized such that it elicits differential anti-coagulant activity depending on the degree of modification. For example, Magnani et al. (1996) showed that an increase in the degree of sulfation of the hydroxyl group on HA disaccharide unit produces an increased resistance to the activation of factor Xa and thrombin, the components that trigger blood clotting cascade. Interestingly, the level of platelet aggregation follows an opposite trend, increasing with increasing degree of sulfation although even the highest aggregating effect is comparable with that of heparin (Barbucci et al., 1998).

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Implications for regenerative medicine

Stem cells, the primitive, multipotent cells that reside in the bone marrow and most adult tissues, undergo self-renewal and differentiation into multiple lineages, which together contribute to tissue homeostasis and repair (Weissman et al., 2001). These intrinsic properties of stem cells have led several groups to investigate their use in tissue engineering and regeneration applications. Notably, HA is being increasingly used as a scaffolding material for in vitro culture of stem cells prior to engraftment in the body. This is because: (a) in addition to being present in adult ECM, HA is also found in the bone marrow stroma (Wight et al., 1986) where it supports key functions of the resident mesenchymal stem cells (MSCs), including localization, proliferation and differentiation (Lee and Spicer, 2000); (b) HA is also found at high concentrations in the early embryonic ECM where it promotes gene expression and signaling, proliferation, migration and morphogenesis of embryonic stem cells (ESCs) (Toole, 2004); and (c) these stem cells express either one or both of the major HA receptors, viz. CD44 and CD168 (RHAMM) (Pilarski et al., 1999; Poulsom, 2007; Zhu et al., 2006). Therefore, HA is likely to elicit key stem cell functions necessary to maintain their therapeutic potential. Indeed, HA scaffolds have been shown to promote chondrogenic and osteogenic differentiation of MSCs both in vitro and in vivo when cultured in the presence of appropriate cytokines (Gao et al., 2001; Zavan et al., 2007; Facchini et al., 2006; Lisignoli et al., 2005; Kim et al., 2007). More detailed studies at the molecular level have shown that MSCs interact with HA scaffolds via a CD44-mediated mechanism and that this interaction causes differential expression of various chemokines and their receptors (e.g. upregulation of CXCR4, CXCL13 and MMP-3 while downregulation of CXCL12, CXCR5, MMP-13) that are involved in inflammation and matrix degradation (Lisignoli et al., 2006), the two processes that determine the outcome of tissue repair and regeneration. HA has also been shown to promote MSC adhesion and migration through a CD44-dependent pathway (Zhu et al., 2006), which has important implications in the design of regenerative strategies aimed at stimulating MSC homing to injury sites. Furthermore, HA hydrogels also provide a biocompatible environment for encapsulated human ESCs by maintaining the cells in an undifferentiated state while conserving their differentiation capacity under appropriate signals, as judged by their ability to form embryoid bodies in vitro (Gerecht et al., 2007). It is also likely that the bioactivity and compatibility of HA scaffolds can be further accentuated through systematic derivatization of the HA backbone, as is commonly performed for the more typical tissue engineering applications, as discussed earlier.

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Natural-based polymers for biomedical applications

Conclusion

HA is an important ECM component that plays a crucial role at various stages of a mammalian life span, from early embryogenesis to adult tissue hemostasis and repair. HA demonstrates unique viscoelastic properties, interacts with specific cell surface receptors that induce distinct intracellular signaling cascades, and modulates inflammation during tissue repair. In addition, HA can be easily modified to obtain more stable derivatives that are more resistant to enzymatic or hydrolytic degradation. Together, these properties have led to its widespread use in a variety of biomedical applications ranging from viscosupplementation to tissue engineering. Another striking feature of HA is its ability to resist activation of the blood clotting cascade, which renders it useful as a coating material for cardiovascular stents. That it influences both embryonic and mesenchymal stem cell function suggests that HA may likely play a vital role in regenerative medicine.

28.7

Future trends

Future success of HA-based therapies will depend on our ability to engineer smarter systems that address the complex, dynamic and reciprocal cell-ECM interactions that occur within the tissues. For example, LMW HA induces inflammation and angiogenesis that are essential for tissue repair while HMW HA inhibits both processes, which is important for controlling the reparative process. Therefore, if one were to deliver specially-derivatized LMW fragments that first initiate tissue repair and then, over time, form crosslinks to build HMW HA cables, it would be possible to modulate inflammation at a rate commensurate with tissue repair. Another approach could be to identify and utilize the cues from the wound to biologically and physiochemically modify HA such that following intravenous delivery at a remote site, it homes specifically to the injury site, builds a robust crosslinked scaffold and simultaneously activates the resident tissue cells to promote en masse cell ingrowth that is necessary for effective wound repair. To induce tissue regeneration, however, it would be desirable to develop HA derivatives that simultaneously activate and recruit stem and progenitor cells to the wound site. Recent reports that show that HA specifically interacts with stem cells via a CD44-mediated mechanism and promotes their migration and differentiation should serve as a platform for the development of such novel regenerative tools.

28.8

References

Abatangelo G, Barbucci R, Brun P and Lamponi S (1997), Biocompatibility and enzymatic degradation studies on sulphated hyaluronic acid derivatives, Biomaterials, 18, 1411– 15.

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