Ideally, rationally designed tissue engineering scaffolds promote natural wound healing and regeneration. Therefore, we sought to synthesize a biomimetic hydrogel specifically designed to promote tissue repair and chose hyaluronic acid (HA; also called hyaluronan) as our initial material. Hyaluronic acid is a naturally occurring polymer associated with various cellular processes involved in wound healing, such as angiogenesis. Hyaluronic acid also presents unique advantages: it is easy to produce and modify, hydrophilic and nonadhesive, and naturally biodegradable. We prepared a range of glycidyl methacrylate-HA (GMHA) conjugates, which were subsequently photopolymerized to form crosslinked GMHA hydrogels. A range of hydrogel degradation rates was achieved as well as a corresponding, modest range of material properties (e.g., swelling, mesh size). Increased amounts of conjugated methacrylate groups corresponded with increased crosslink densities and decreased degradation rates and yet had an insignificant effect on human aortic endothelial cell cytocompatibility and proliferation. Rat subcutaneous implants of the GMHA hydrogels showed good biocompatibility, little inflammatory response, and similar levels of vascularization at the implant edge compared with those of fibrin positive controls. Therefore, these novel GMHA hydrogels are suitable for modification with adhesive peptide sequences (e.g., RGD) and use in a variety of wound-healing applications.

Photocrosslinked hyaluronic acid hydrogels: natural, biodegradable tissue engineering scaffolds.

Hyaluronan : preparation, structure, properties, and applications

Viscosity-molecular weight relationships, intrinsic chain flexibility, and dynamic solution properties of guar galactomannan

Since its first isolation in 1934, hyaluronic acid (HA) has been studied across a variety of research areas. This unbranched glycosaminoglycan consisting of repeating disaccharide units of N-acetyl-d-glucosamine and d-glucuronic acid is almost ubiquitous in humans and in other vertebrates. HA is involved in many key processes, including cell signaling, wound reparation, tissue regeneration, morphogenesis, matrix organization and pathobiology, and has unique physico-chemical properties, such as biocompatibility, biodegradability, mucoadhesivity, hygroscopicity and viscoelasticity. For these reasons, exogenous HA has been investigated as a drug delivery system and treatment in cancer, ophthalmology, arthrology, pneumology, rhinology, urology, aesthetic medicine and cosmetics. To improve and customize its properties and applications, HA can be subjected to chemical modifications: conjugation and crosslinking. The present review gives an overview regarding HA, describing its history, physico-chemical, structural and hydrodynamic properties and biology (occurrence, biosynthesis (by hyaluronan synthases), degradation (by hyaluronidases and oxidative stress), roles, mechanisms of action and receptors). Furthermore, both conventional and recently emerging methods developed for the industrial production of HA and its chemical derivatization are presented. Finally, the medical, pharmaceutical and cosmetic applications of HA and its derivatives are reviewed, reporting examples of HA-based products that currently are on the market or are undergoing further investigations.

Hyaluronic Acid in the Third Millennium.

The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity

Molecular weights by osmometry (Mn), light scattering (Mw), and sedimentation-diffusion (MsD) were determined for selected fractions and unfractionated samples of hyaluronic acid in the molecular weight range 3 × 104−1.7 × 106. For the determination of MsD from the sedimentation coefficient s, a calibration procedure based on the data of Laurent, Ryan, and Pietruszkiewicz gave the following relations in 0.2M NaCl: log (s−1)0 = 14.681 − 0.413 log MsD and log D0 = −3.828 − 0.568 log MsD, where subscript, zero refers to zero concentration and D is the diffusion coefficient. The average Mandelkern-Flory parameter β in 0.2M NaCl is 3.0 × 106. Data for the intrinsic viscosity [η] may be fitted for M > 105 by [η] = K′Ma, where K′, a are in 0.2M NaCl, 0.0228, 0.816; in 0.5M NaCl, 0.0318, 0.777; in 0.1 M HCl, 0.0279, 0.763, respectively. Below M = 105, [η] appears to fall below the values calculated, possibly due to non-Gaussian behavior of short chains. The second virial coefficient A2 in 0.5M NaCl approaches the ideal Donnan value, decreasing slightly with increasing molecular weight. The friction parameter P is about 20% smaller than that of uncharged polymers of similar dimensions. The flexible coiling nature of this polymer is confirmed.

Ionic polysaccharides. 3. Dilute solution properties of hyaluronic acid fractions.

Fractionated samples of sodium hyaluronate of low molecular weight were used to calibrate the carbazole method for glucuronyl analsis and to determine the density increment (based on dry weight) of 0.444 (±0.003) mL/g in water and 0.386 (±0.003) mL/g for samples dialyzed against 0.2M NaCl. Weight-average molecular weights obtained by high-speed sedimentation equilibrium were used to calibrate the limiting viscosity number [η] in 0.2M NaCl, which gave [η]/Mw = 0.0028 (±0.0002) mL/g, valid to Mw = 0.0028 (±0.0002) mL/g, valid to Mw = 105. Experimental data from this work and the literature, including viscosity and light- and small-angle x-ray scattering measurements, were compared to theoretical chain models of the Kratky-Porod (KP) wormlike and the helical wormlike (HW) chain, as treated by Yamakawa and collaborators. Although either model could be fitted to experimental data about equally well with consistent parameters, provided those for the HW chain were of weakly helical nature, calculation of the unperturbed meansquare end-to-end distance as a function of chain length from a conformational model favored the KP chain alternative. The parameters that provide the best fit to experimental data for the KP wormlike model are a persistence length of 4.5–5 nm and a diameter of 1.1 nm. The latter is resonable for a hydrated hydrodynamic cylinder in view of the approximate unhydrated value of 0.7 nm estimated from the density increment.

Viscometry and sedimentation equilibrium of partially hydrolyzed hyaluronate: Comparison with theoretical models of wormlike chains

Sodium hyaluronate (NaHy) and sodium carboxymethyl cellulose (NaCMC) behave similarly with respect to concentration.N3 of an added 1 : 1 electrolyte. The second virial coefficient A2 (light scattering) is identical within experimental error at a given.N3. The limiting viscosity number [η] also varies with N3−1/2in similar fashion for samples of similar [η] of the two polymers. Differences in Na+ activity in salt-free solutions are interpreted on the basis of weaker Na+ binding in NaHy, presumably due to the greater charge separation along its chain backbone. Added electrolyte is excluded in dialysis more strongly by NaHy (or its acid form) than by NaCMC. The Flory parameter Φ is smaller in good solvents for NaHy, as for many other polyelectrolytes, than for nonionic polymers.

Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronatc with that of carboxymethyl cellulose

The root-mean-square end-to-end distance has been calculated for a model allowing free rotation about glycoside bonds for the general case of polysaccharides having a disaccharide repeating unit. Numerical estimates are given for several naturally occurring structures based on an idealized pyranose unit in the C1 chair conformation. Extrapolation procedures which make use of the intrinsic viscosity [η] in good solvents to obtain unperturbed dimensions do not represent, data for hyaluronic acid very well, especially at low molecular weights. However, order-of-magnitude estimates suggest that this polymer behaves similarly to other polysaccharides, and probably has stiffer local structure than typical non-ionic synthetic polymers. A double logarithmic plot of the product of [η] and Mw, the weight-average molecular weight, against the degree of polymerization in the range for Mw of 104 to 2 × 104 permits a straight-line fit of available data for all the glycosaminoglycans, including heparin and the chondroitin sulfates, as well as sodium carboxymethyl cellulose. This result suggests similarity of short-chain hydrodynamic behavior of these polymers.

Ionic polysaccharides. IV. Free-rotation dimensions for disaccharide polymers. Comparison with experiment for hyaluronic acid.

The decrease in the limiting viscosity number [η] with temperature T for hyaluronic acid in nonalkaline solution and chondroitin 4-sulfate in neutral and alkaline solutions may be expressed in terms of the temperature coefficient of the Kratky-Porod persistence length a: d ln a/dT = −0.0040 (±0.0005). The result, while numerically somewhat smaller, resembles qualitatively that of cellulose derivatives. As in the latter case, standard conformational calculations underestimate the coefficient, which may be due to neglect of random occurrence of local conformational features of higher energy. In alkaline solution, large decreases in [η] of hyaluronic acid are accompanied by a positive temperature coefficient of [η]. This temperature effect is interpreted as an endothermal shift from the alkaline, low [η] form of the polymer to the neutral, high [η] form with increasing temperature.

Robert L. Cleland

Papers

Ionic polysaccharides. 3. Dilute solution properties of hyaluronic acid fractions.

Viscometry and sedimentation equilibrium of partially hydrolyzed hyaluronate: Comparison with theoretical models of wormlike chains

Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronatc with that of carboxymethyl cellulose

Ionic polysaccharides. IV. Free-rotation dimensions for disaccharide polymers. Comparison with experiment for hyaluronic acid.

Effect of temperature on the limiting viscosity number of hyaluronic acid and chondroitin 4‐sulfate