Brief summary

HA is the only non-sulfated glycosaminoglycan, and consists of repeating disaccharide units of N-acetylglucosamine and glucuronic acid. The molecular weight can vary from over 6,000 kDa in umbilical cords and rooster combs to 100 kDa and below in serum.  HA is found in the ECM of all tissues of the body, and is particularly abundant in umbilical cords, embryonic hearts, the eye, synovial fluid, and cartilage. HA occurs primarily as the sodium salt under physiological conditions; collectively, HA, sodium HA other HA salts are often referred to as hyaluronan.  The two major commercial sources of HA are rooster combs for >2,000 kDa HA and bacterial fermentation for 50 – 100 kDa HA, and these are polydisperse.  Recently, a third option, unique to Hyalose, is HA and labeled HA oligosaccharides produced enzymatically to give monodisperse polymers ranging from only a few disaccharide units up to 20 kDa. HA forms lubricious and viscoelastic solutions that can protect eye tissues during ophthalmic surgeries, provide viscosupplementation in joints, reduce post surgical adhesions, and deliver drugs. However, the short residence time in vivo of HA and its poor mechanical properties led to many chemical modifications of HA. In the last two decades, HA-based medical devices and coatings have addressed clinical needs with different levels of success

Chemically-modified HA derivatives can be grouped into two types: monolithic and living. The chemistry and biology of HA is exhaustively reviewed on the Glycoforum website; specifically, the section on HA derivatives is found at http://glycoforum.gr.jp/science/hyaluronan/HA18/HA18E.html. Monolithic derivatives arise when the chemical modification gives a final form that cannot form new chemical bonds in the presence of cells or tissues, and can only be physically fabricated into different forms.  In contrast, living derivatives contain modifications that permit the formation of new covalent bonds in the presence of cells or tissues, enabling a change in physical form during in vivo or in vitro biological use.  For example, an in situ-crosslinkable material for adhesion prevention or cell encapsulation would be a living modification.

2.1 – The Old: Monolithic HA Derivatives

• 2.1.1.   Carbodiimide-mediated reaction products

In water at pH < 5, the carboxylic acid groups of HA can react with a ethyl dimethylaminopropylcarbodiimide (EDCI) to form an intermediate O-acylurea that can be trapped or rearrange to the more stable N-acylurea adduct. Interaction of the cationic aminopropyl moiety with remaining carboxylates can lead to physical gelation. Seprafilm (Genzyme) is the product of EDCI plus HA and carboxymethylcelluose, yielding a bioabsorbable material that is used to prevent post-surgical adhesions in abdominal and gynecological indications. Seprafilm is brittle when dry and sticky when moist and is therefore difficult for surgeons to handle. The use of biscarbodiimides affords chemically crosslinked HA, a technology, found in the post-surgical adhesion product Incert (Anika Therapeutics).

• 2.1.2.   Divinylsulfone crosslinking

Hylans (Biomatrix, Genzyme) are HA derivatives that are crosslinked through the hydroxyl groups, leaving the majority of the carboxylic acids unmodified. Hylan A develops HA-protein crosslinks with formaldehyde, giving HA products with increased molecular weight. In contrast, Hylan B is a highly swollen gel with an infinite network of divinylsulfone crosslinks. Hylans are more viscoelastic than HA and have longer half-lives in vivo. Hylans are used as injectable materials (Synvisc) to treat osteoarthritic knees, as dermal fillers to treat facial wrinkles (Hylaform) and as space-occupying stents in nasal/sinus surgery (Hylasine, Sepragel Sinus).

• 2.1.3.   Esterification

HA esters are prepared by the reaction of quaternary ammonium salt of HA with an alcohol that imparts hydrophobicity, using a non-aqueous solvent such as dimethylformamide for the reaction. For example, the benzyl ester HYAFF-11 (Fidia Advanced Biopolymers) is less susceptible to enzymatic degradation than native HA. HYAFF-11 scaffolds are cell adhesive and are stable in aqueous solution for over 3 weeks. A 3-D HYAFF scaffold supports proliferation of keratinocytes, fibroblasts, chondrocytes, and mesenchymal stem cells. The dermal replacement (Hyalograft 3D) consists of HYAFF-11 with autologous fibroblasts combined with an epidermal replacement (Laserskin) made of a microperforated HYAFF 11 membrane containing autologous keratinocytes. A clinical study showed 59% of the treated pressure ulcers reached complete closure in less than 4 months with this product. Hyalograft C, which consists of autologous chondrocytes grown on a HYAFF 11 scaffold, is used to treat cartilage defects of the knee, and patients improved continuously out to 3 years.

HA carboxyl groups can be activated with 2-chloro-1-methylpyridinium iodide (CMPI) to form internal ester bonds. The so-called autocrosslinked polymer (ACP; Fidia) can be used as a gel (Hyalobarrier, Hyaloglide) was effective in the prevention of adhesions in gynecological and joint surgeries in preclinical model studies.

• 2.1.4. Bis-epoxide crosslinked HAHA that can be crosslinked with a diglycidyl ether under strongly alkaline conditions to give a biocompatible material with in vivo residence times up to 4 weeks.  For example,  Restylane (Q-Med) and Juvederm (Allergan) are used as cosmetic dermal fillers. HA can also be crosslinked twice with the same bis-epoxide, first under basic conditions and then under acidic conditions. Double crosslinking reduced hyaluronidase degradation compared to the single crosslinked intermediate. Puragen (Mentor) is a doubly crosslinked HA gel, and is used as dermal filler in the same manner as Restylane and Juvederm.

2.2 – The New: Living HA derivatives

• 2.2.1.   Reversible crosslinking using carbodiimide-mediated hydrazide chemistry

As described above, HA-derived O-acylureas can undergo further coupling reactions with compounds that are nucleophilic at pH 4-6, e.g., N-hydroxysuccinimide, aminooxy compounds, and hydrazides. Polyvalent hydrazides afford crosslinked HA gels with a range of mechanical properties simply by varying the molar ratios of the reagents. When hydrazides containing disulfide bonds are used, living HA derivatives can be produced. The resulting thiol-modified HA can be crosslinked to itself, or to other synthetic and natural polymers bearing thiol groups.  Moreover, a variety of physical properties and rates of biodegradation can be obtained by controlling several parameters for the biocompatible HA hydrogels, including: (i) molecular weight of starting HA employed; (ii) percentage thiol modification; (iii) concentration of thiol-modified HA; (iv) choice of chemistry for polyfunctional crosslinker; (v) molecular weight of polyfunctional crosslinker; and (vi) ratio of thiols to electrophiles.

The crosslinked can act as a barrier to cell infiltration and migration. Such a material promotes scar-free wound healing and prevents post-operative adhesion formation. This formulation been employed for scar-free mucosal healing following endoscopic sinus surgery, for prevention of post-surgical abdominal adhesions, and to minimize subglottic stenosis in airway stents. Veterinary products from Sentrx Animal Care have been successfully employed for equine and small animal wound care.

• 2.2.2. Electrophilic HA

HA derivatives equipped with thiol-reactive electrophilic esters can react with thiol-modified macromolecules to give “crosslinker-free” hydrogels. HA was converted to the bromoacetate and iodoacetate derivatives. Crosslinker-free sECM hydrogels were prepared by combining the polyfunctional electrophilic HA haloacetates with thiol-modified HA (CMHA-S). The resulting hydrogel was cytocompatible but did not support the attachment of primary fibroblasts. Thus, the HA haloacetates offer an alternative living macromonomer for producing crosslinker-free sECM biomaterials that can function as anti-adhesive barriers or for culture of non-adherent cell types.

• 2.2.3. Photo-crosslinked HA

Methacrylation of HA by modification of the hydroxyl groups produces  macromonomers that can be seeded with cells and photocrosslinked into hydrogels with either UV or visible light plus an initiator. Potential uses for in situ photopolymerized HA hydrogels include prevention of adhesions and tissue regeneration. For example, chondrocytes encapsulated in photocrosslinkable HA methacrylate retained the chondrocytic phenotype and synthesized cartilage matrix in vitro, and accelerated healing in an osteochondral defect model in vivo. Importantly, photocrosslinked HA methacrylates have been used for self-renewal of human embryonic stem cells without differentiation.