The impact of hyaluronic acid oligomer content on physical, mechanical, and biologic properties of divinyl sulfone-crosslinked hyaluronic acid hydrogels

Abstract

In recent studies, we showed that exogenous hyaluronic acid oligomers (HA-o) stimulate functional endothelialization, though native long-chain HA is more bioinert and possibly more biocompatible. Thus, in this study, hydrogels containing high molecular weight (HMW) HA (1 x 10(6) Da) and HA-o mixtures (HA-o: 0.75-10 kDa) were created by crosslinking with divinyl sulfone (DVS). The incorporation of HA-o was found to compromise the physical and mechanical properties of the gels (rheology, apparent crosslinking density, swelling ratio, degradation) and to very mildly enhance inflammatory cell recruitment in vivo; increasing the DVS crosslinker content within the gels in general, had the opposite effect, though the relatively high concentration of DVS within these gels (necessary to create a solid gel) also stimulated a mild subcutaneous inflammatory response in vivo and VCAM-1 expression by endothelial cells (ECs) cultured atop; ICAM-expression levels remained very low irrespective extent of DVS crosslinking or HA-o content. The greatest EC attachment and proliferation (MTT assay) was observed on gels that contained the highest amount of HA-o. The study shows that the beneficial EC response to HA-o and biocompatibility of HA is mostly unaltered by their chemical derivatization and crosslinking into a hydrogel. However, the study also demonstrates that the relatively high concentrations of DVS, necessary to create solid gels, compromise their biocompatibility. Moreover, the poor mechanics of even these heavily crosslinked gels, in the context of vascular implantation, necessitates the investigation of other, more appropriate crosslinking agents. Alternately, the outcomes of this study may be used to guide an approach based on chemical immobilization and controlled surface-presentation of both bioactive HA-o and more biocompatible HMW HA on synthetic or tissue engineered grafts already in use, without the use of a crosslinker, so that improved, predictable, and functional endothelialization can be achieved, and the need to create a mechanically compliant biomaterial for standalone use, circumvented.

Figure 1

Mean Fluorescence Intensities (MFI) due to HA oligomers (HA-o) embedded within DVS-HA gels. The oligomer content within 1:1 w/w DVS-HA (A) appeared to remain constant over the 21 days of incubation whereas the oligomer content of 1:2 w/w DVS-HA (B) decreased slightly before reaching a plateau. However, within both DVS concentration groups, the differences in fluorescence intensities between gels containing different amounts of HA oligomers were maintained across time points [* denotes statistical significance of differences deemed for a p-value < 0.05 in comparison to day 1].

Figure 2

Swelling ratios (mg wet weight/mg dry weight) of DVS-HA hydrogels. Incorporation of HA oligomers within the DVS-HA hydrogels mildly increased their swelling capacity irrespective of their crosslinking density. Irrespective of HA oligomer content, the swelling ratio was dramatically lower for the more crosslinked gels. [* denotes statistical significance of differences deemed for a p-value < 0.05 in comparison to HA oligomer-free gels within the respective groups of gels].

Figure 3

Viscoelastic properties of DVS-HA. The storage moduli (G′) in all cases were greater than the loss moduli (G″). Increasing the concentration of DVS crosslinker within the hydrogels (i.e., 1:1 w/w (A) vs. 1:2 w/w (B) DVS-HA gels), resulted in a greater G′ and overall stiffness of the gels. The addition of HA oligomers, however, reduced the storage moduli indicating lowered gel stiffness.

Figure 4

Degradation of DVS-HA in vitro. Hydrogels containing greater amounts of DVS crosslinker, i.e., the 1:1 w/w DVS-HA gels (A) exhibited greater stability against degradation by testicular hyaluronidase than the less crosslinked gel formulations, i.e., vs. 1:2 w/w (B) DVS-HA. However, increasing the concentration of HA oligomer content within each of these gel formulations enhanced the degradation rate of the gels.

Figure 5

H&E staining of sub-cutaneous DVS-HA gel implants. Light microscopy images (A) showed a distinct, darkened ring of inflammatory cells (see arrows) to surround all gel implants. The thickness of this highly cellular region appeared to be increase with the extent of DVS-crosslinking within the gels. The more crosslinked hydrogels, i.e., the 1:1 w/w DVS-HA gels stimulated an enhanced inflammatory response from the surrounding tissue that was greater than both the less crosslinked gels, i.e., the 1:2 w/w DVS-HA gels, and matrigel plugs. Likewise, gels containing greater HA oligomer (HA-o) content, appeared to incite a greater inflammatory response, though these effects were muted compared to the impact of DVS crosslinking. The macroscopic images (B) show very little tissue infiltration within DVS-HA, as compared to the matrigel control. The defect containing the 1:1 w/w DVS-HA gel implants were consistently void of inward tissue projections, though some tissue projections (see arrows) were observed in those defects that contained the 1:2 w/w DVS-HA gels.

Figure 6

Immunofluorescence analysis of fibrous mass surrounding implants. Gels containing greater crosslinker (DVS) content (1:1 DVS-HA vs. 1:2 DVS-HA) prompted greater cellularity in the region immediately surrounding them though the fibrous mass in this region, shown to be collagen I (green), appeared far less dense than in regions further afield. This suggested that the cells near the implant are likely not collagen-synthesizing fibroblasts, but rather inflammatory cell recruits. The recruitment of the inflammatory cells (blue) appeared to be enhanced by increasing HA oligomer (HA-o) content.

Figure 7

Matrigel adsorption onto DVS-HA gels. A drop in protein content within the bulk coating suspension was observed indicating protein adsorption onto the gel surfaces. Calculations revealed that the density of adsorbed protein was the same on all DVS-HA gels, regardless of crosslinker density or HA oligomer content. [* denotes statistical significance of difference vs. no gel condition, deemed for a p-value of < 0.05].

Figure 8

Morphology and proliferation of ECs cultured on matrigel-adsorbed DVS-HA gels. The incorporation of HA oligomers within the gels enhanced EC adherence onto their surfaces. The ECs appeared to spread and exhibited natural cobblestone morphology similar to cells cultured on fibronectin (A), but did not form tubular projections as did ECs cultured on matrigel alone (B). The ECs formed isolated clusters atop 1:2 w/w DVS-HA gels (C-F), while they were more uniformly distributed atop the more crosslinked 1:1 w/w DVS-HA gels (G-J). The 1:1 w/w DVS-HA gels stimulated greater EC proliferation than 1:2 w/w DVS-HA gels, as did increased HA oligomer content within either gel type. In all cases, the surface-embedded HA oligomers were able to interact with ECs to enhance their proliferation. However, EC proliferation levels on these gels were overall lower than those cultured on matrigel and fibronectin substrates. [* denotes statistical significance of differences in proliferation ratios relative to HA-o-free gels within the respective crosslinking groups, deemed for a p-value < 0.05].

Figure 9

ICAM-1 expression of ECs cultured on DVS-HA gels. ICAM-1 expression (green) by ECs (nuclei appear blue) were very similar across the DVS-HA gel formulations. ICAM-1 expression in these cases was also similar to that that expressed by ECs cultured on fibronectin and matrigel substrates, but much lower than that expressed by TNF-α-stimulated ECs.

Figure 10

VCAM-1 expression of ECs cultured on DVS-HA. ECs (nuclei in blue) cultured on the various DVS-HA gel formulations expressed similar levels of VCAM-1 (green), which were much lower than when ECs were stimulated with TNF-α, but were somewhat elevated over EC cultures atop fibronectin and matrigel substrates.

Figure 11

Mean fluorescence intensities (MFI) due to ICAM-1 (A) and VCAM-1 (B) expression by ECs cultured on DVS-HA. The data confirms visual observations that the fluorescence due to EC expression of both CAMs remained the same irrespective of the formulation of the substrate DVS-HA gels. While the intensity of fluorescence due to ICAM-1 expression by ECs cultured on the gels was similar to that expressed by ECs cultured on fibronectin and matrigel, fluorescence intensities due to VCAM-1 expression were slightly elevated. Both ICAM-1 and VCAM-1 expression levels by ECs cultured on the DVS-HA gels (all formulations) were however much lower than ECs stimulated with TNF-α. [* denotes statistical significance of differences of MFI vs. ECs cultured on fibronectin, deemed for a p-value of < 0.05].