Interpenetrating Low-Molecular Weight Hyaluronic Acid in Hyaluronic Acid-Based In Situ Hydrogel Scaffold for Periodontal and Oral Wound Applications

Abstract

Tissues engineering has gained a lot of interest, since this approach has potential to restore lost tooth-supporting structures, which is one of the biggest challenges for periodontal treatment. In this study, we aimed to develop an in situ hydrogel that could conceivably support and promote the regeneration of lost periodontal tissues. The hydrogel was fabricated from methacrylated hyaluronic acid (MeHA). Fragment/short-chain hyaluronic acid (sHA) was incorporated in this hydrogel to encourage the bio-synergistic effects of two different molecular weights of hyaluronic acid. The physical properties of the hydrogel system, including gelation time, mechanical profile, swelling and degrading behavior, etc., were tested to assess the effect of incorporated sHA. Additionally, the biological properties of the hydrogels were performed in both in vitro and in vivo models. The results revealed that sHA slightly interfered with some behaviors of networking systems; however, the overall properties were not significantly changed compared to the base MeHA hydrogel. In addition, all hydrogel formulations were found to be compatible with oral tissues in both in vitro and in vivo models. Therefore, this HA-based hydrogel could be a promising delivery system for low molecular weight macromolecules. Further, this approach could be translated into the clinical applications for dental tissue regeneration.

Keywords: drug delivery system; hyaluronic acid; hydrogels; periodontal ligament stem cells; tissue engineering.

Figure 1

¹H NMR spectra of methacrylated hyaluronic acid with 80–100% degrees of grafting. The peaks no. 1, 2, and 3 represent 2 protons on methacrylate alkene, 3 protons of the methyl group on the methacrylate, and another 3 protons of the methyl group of HA.

Figure 2

Mechanical profiles of hydrogels, (a–d) show the mechanical profiles of hydrogel formulations containing 0.0%, 0.5%, 1.0%, and 1.5% w/v of sHA, respectively. The relationship between tan δ and % strain is shown as in (e).

Figure 3

Physicochemical profiles of hydrogels including (a) swelling profiles (for 21 days), (b) degradation profiles (for 21 days), and (c) glucuronic acid release profiles for 2 days.

Figure 4

Microstructure images of freeze-dried hydrogels captured by using SEM technique. The scale bars of 2000× magnification are 100 µm.

Figure 5

Wound healing activity of hydrogels on PDLs; the hydrogels were tested for their cytocompatibility, indirectly, before being utilized in the other in vitro cell studies (a). (c) shows the capability of the hydrogels when performing the transwell migration test (for 2 days), serum-free media and serum-contained media were used as a blank and positive control in this study. The significant level of the p value was marked with *, ** and *** which refer to p value ≤ 0.05, 0.01 and 0.001, respectively. The performance of the hydrogels on promoting the proliferation of PDLs (for 7 days) is shown in (b,d). (b) presents the viability of PDLs, which was indicated and converted into the fluorescence intensity using a resazurin-based solution. (d) illustrates the physical appearance of the cells after being cultured on the gels for 7 days.

Figure 6

The physical appearance of the wounds on the rats’ palates in three groups of intervention at each time point, with scale bars of 2 cm (a). (b) shows the histological sections of the rodents palates, staining with hematoxylin and eosin (H&E) after 1 day of treatment, with 50 µm scale bars of 400× magnification.