Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks

Abstract

Hyaluronic acid is a natural polysaccharide found abundantly throughout the body with many desirable properties for application as a biomaterial, including scaffolding for tissue engineering. In this work, hyaluronic acid with molecular weights ranging from 50 to 1100 kDa was modified with methacrylic anhydride and photopolymerized into networks with a wide range of physical properties. With macromer concentrations from 2 to 20 wt %, networks exhibited volumetric swelling ratios ranging from approximately 42 to 8, compressive moduli ranging from approximately 2 to over 100 kPa, and degradation times ranging from less than 1 day up to almost 38 days in the presence of 100 U/mL of hyaluronidase. When 3T3-fibroblasts were photoencapsulated in the hydrogels, cells remained viable with low macromer concentrations but decreased sequentially as the macromer concentration increased. Finally, auricular swine chondrocytes produced neocartilage when photoencapsulated in the hyaluronic acid networks. This work presents a next step toward the development of advanced in vivo curable biomaterials.

Figure 1

Top: chemical structure of methacrylated hyaluronic acid (MeHA). Bottom: general schematic of the free radical polymerization of MeHA to form crosslinked hydrogel networks. This process involves the formation of radicals from the exposure of the initiator to light, which propagate through the vinyl groups of the MeHA to form kinetic chains (shown as dashed lines). These networks eventually degrade by enzymatic cleavage of the HA backbone.

Figure 2

Equilibrium volumetric swelling ratio (Q_V) for photocrosslinked HA networks with variations in macromer molecular weight and concentration. The swelling ratios are statistically different (denoted by * between two bars) between the different macromer concentrations for each molecular weight MeHA.

Figure 3

Mechanical properties of HA hydrogels. A. Representative stress versus strain plots of hydrogels fabricated from 10 (solid) and 5 (dotted) wt% macromers (50 kDa MeHA). B. Compressive modulus for various HA networks at equilibrium swelling. The compressive moduli are statistically different (denoted by * between two bars) between the different macromer concentrations for each molecular weight MeHA.

Figure 4

Time for complete degradation of HA hydrogels in 100 U hyaluronidase/ml of PBS, where the hyaluronidase was replenished every other day throughout degradation.

Figure 5

Uronic acid measured during the degradation of HA hydrogels. A. Cumulative percentage of uronic acid detected for HA hydrogels formed from 2 (●), 5 (■), and 10 (▲) wt% of the 50 kDa MeHA and degraded in 100 U hyaluronidase/ml. B. Cumulative percentage of uronic acid detected for HA hydrogels formed from 5 wt% 350 kDa MeHA and degraded in both 100 (●) and 10 (■) U hyaluronidase/ml.

Figure 6

Viability of photoencapsulated 3T3-fibroblasts in the HA hydrogels. Absorbance (indicative of encapsulated cell mitochondrial activity and viability) for the various HA networks after 1 day (black) and 1 week (white) of in vitro culture. The MTT solution was diluted 4-fold for all samples to obtain absorbance values in the linear range. The absorbance is statistically different (denoted by * between two bars) between the different macromer concentrations for the 50 kDa MeHA at the 1 week time point.

Figure 7

Neocartilage formation by swine chondrocytes photoencapsulated in HA and PEG hydrogels. A. Histological sections (stained for glycosaminoglycans, bar = 100 μm) of auricular chondrocytes photoencapsulated in 2% 350kDa MeHA (left) and 10% PEGDM (right) hydrogels 6 weeks after implantation in nude mice. B. Glycosaminoglycan content (reported as ng chondroitin sulfate/chondrocyte) immediately after photoencapsulation (black) and after 4 (grey) and 8 (white) weeks of auricular chondrocyte culture in nude mice for 2% 350 kDa MeHA and 10 wt% PEGDM.