Characterization of glycidyl methacrylate - crosslinked hyaluronan hydrogel scaffolds incorporating elastogenic hyaluronan oligomers

Abstract

Prior studies on two-dimensional cell cultures suggest that hyaluronic acid (HA) stimulates cell-mediated regeneration of extracellular matrix structures, specifically those containing elastin, though such biologic effects are dependent on HA fragment size. Towards being able to regenerate three-dimensional (3-D) elastic tissue constructs, the present paper studies photo-crosslinked hydrogels containing glycidyl methacrylate (GM)-derivatized bio-inert high molecular weight (HMW) HA (1 × 10(6)Da) and a bioactive HA oligomer mixture (HA-o: MW ∼0.75 kDa). The mechanical (rheology, degradation) and physical (apparent crosslinking density, swelling ratio) properties of the gels varied as a function of incorporated HA oligomer content; however, overall, the mechanics of these hydrogels were too weak for vascular applications as stand-alone materials. Upon in vivo subcutaneous implantation, only a few inflammatory cells were evident around GM-HA gels, however their number increased as HA-o content within the gels increased, and the collagen I distribution was uniform. Smooth muscle cells (SMC) were encapsulated into GM hydrogels, and calcein acetoxymethyl detection revealed that the cells were able to endure twofold the level of UV exposure used to crosslink the gels. After 21 days of culture, SMC elastin production, measured by immunofluorescence quantification, showed HA-o to increase cellular deposition of elastic matrix twofold relative to HA-o-free GM-HA gels. These results demonstrate that cell response to HA/HA-o is not altered by their methacrylation and photo-crosslinking into a hydrogel, and that HA-o incorporation into cell-encapsulating hydrogel scaffolds can be useful for enhancing their production of elastic matrix structures in a 3-D space, important for regenerating elastic tissues.

Fig. 1

Crosslinking chemistry of GM HA. GM–HA hydrogels were prepared by a two-step process. HA was initially incubated with GM, a chemical catalyst (TEA) and phase-transfer catalyst (TBAB) in a batch reaction combining the glycidyl units of GM and hydroxyl units of HA to create the uncrosslinked GM–HA product. A GM–HA solution was then combined with a photoinitiator (I2959) and treated with UV to stimulate the radical reaction of the methacrylate units of GM–HA.

Fig. 2

FTIR-ATR spectra of (A) HA, (B) uncrosslinked GM–HA and (C) crosslinked GM–HA. The initial addition of GM to HA resulted in an absorption band at 936.12 cm⁻¹ identifying the carbon-to-carbon double bond of GM methacrylate. This absorption band disappeared upon UV exposure, signifying the successful crosslinking between the methacrylate groups.

Fig. 3

Swelling ratios of GM–HA gels. The incorporation of HA-o within the GM–HA hydrogel mildly increased their swelling capacity. These increases were, however, not statistically significant except at high HA-o content. *Denotes p < 0.05 in comparison with 0%.

Fig. 4

Viscoelastic properties of GM–HA. The storage moduli (G′) in all cases was greater than the loss moduli (G″). The addition of HA-o reduced the storage moduli, suggesting lowered gel stiffness of the hydrogels.

Fig. 5

Degradation of GM–HA in vitro. Increasing the concentration of HA-o within the gels reduced their resistance to degradation by testicular hyaluronidase and enhanced the degradation rate. GM–HA hydrogels containing oligomers were completely degraded after 6 h exposure to the super-physiological concentration of the enzyme.

Fig. 6

Interior morphology of GM–HA. The addition of HA oligomers into GM–HA gels (A, 0%; B, 5%; C, 10%; D, 20%) resulted in enhanced porosity of the gels.

Fig. 7

H&E staining of GM–HA. Microscopic images show that fewer inflammatory cells (see arrows) surrounded the GM–HA implants (A–D) than the matrigel control (E), but their density appeared to increase slightly with HA oligomer concentration. Macroscopic images depict a significant amount of tissue infiltration (see arrows) within GM–HA (F–I) implants similar to that of the control (J), and did not appear to be affected by HA oligomer content.

Fig. 8

Immunofluorescence analysis collagen I surrounding GM–HA implants. The cellularity (blue) in the region immediately surrounding implants increased slightly with a greater HA oligomer content, and the distribution of collagen I (green) surrounding the defect region was fairly homogeneous.

Fig. 9

SMC survival of UV crosslinking. Most of the embedded cells survived all exposure times of UV radiation, indicated by the overlaying of nuclei (blue) and live cell (green).

Fig. 10

SMC elastin production within GM–HA. The elastin content (green) within GM–HA gels containing HA oligomers was higher than the hydrogels without HA oligomers. The nuclei of the embedded SMC appeared elongated and, therefore, may have attained a natural spread morphology.

Fig. 11

Quantification of volumetric fluorescence intensities due to elastin synthesized by SMC cultured within GM–HA. The fluorescence intensity due to elastin was enhanced by the addition of HA oligomers into the GM–HA gel. The amount of elastin synthesized, however, was independent of the concentration of HA oligomers within the GM–HA gels. *Denotes p < 0.05 in comparison with 0%.