A viscoelastic alginate-based hydrogel network coordinated with spermidine for periodontal ligament regeneration

Abstract

Periodontitis can cause irreversible defects in the periodontal ligament (PDL), the regeneration of which is the major obstacle to the clinical treatment of periodontitis. Implanting hydrogel for releasing anti-inflammatory drugs is a promising treatment to promote PDL regeneration. However, existing hydrogel systems fail to mimic the typical viscoelastic feature of native periodontium, which may have been shown as an important role in tissue regeneration. Meanwhile, the synergistic benefits of mechanical cues and biochemical agents for PDL regeneration remain elusive. In this study, we developed a bi-crosslinking viscoelastic hydrogel (Alg-PBA/Spd) by integrating phenylboronic acid-modified alginate with anti-inflammatory agent (spermidine) through borate ester and B-N coordination bonds, where spermidine will be released with the degradation of the hydrogel. Alg-PBA/Spd hydrogel is biocompatible, injectable and can quickly adapt to complex periodontal structures due to the dynamic crosslinking. We demonstrated in rat models that the viscoelastic Alg-PBA/Spd hydrogel significantly promotes the deposition of periodontal collagen and accelerates the repair of periodontal damage. Our results suggest that the viscoelastic Alg-PBA/Spd hydrogel would be a promising mechano-biochemically synergistic treatment for periodontal regeneration.

Keywords: mechanical microenvironment; mechano-biochemically synergistic treatment; periodontal regeneration; viscoelastic hydrogel.

Graphical abstract

Figure 1.

Schematic illustration of Alg-PBA/spd hydrogel. The chemical structure of (A) Alg-PBA, (B) Spermidine, (C) boronic bond and (D) B–N coordination. (E) Scheme of the preparation process of forming Alginate-PBA/spermidine hydrogel. (F) Optical photographs of the Alginate-PBA/spermidine mixture solution and the formed Alg-PBA/spd hydrogel.

Figure 2.

Mechanical properties and structure of Alg-PBA/spd hydrogel. (A) Variations of storage modulus and loss modulus (G′ and G″) of Alg-PBA/spd hydrogel with different Alg-PBA concentrations versus angular frequency (0.1–10 rad/s) and 1% strain. (B) Storage modulus of Alg-PBA/spd hydrogel with different Alg-PBA concentrations at 1 rad/s frequency and 1% strain. Values are exhibited as mean ± SD. ***P < 0.001. (C) Representative SEM images of the Alg-PBA/spd hydrogel. Scale bar = 50 μm. (D) Porosity of Alg-PBA/spd hydrogel with different Alg-PBA concentrations. Values are exhibited as mean ± SD. *P < 0.05, ***P < 0.001. (E) Loss tangent of the Alg-PBA/spd hydrogel with different spermidine concentrations versus angular frequency (0.1–10 rad/s) and 1% strain. (F) Stress relaxation curves of Alg-PBA/spd hydrogel at different strains.

Figure 3.

Injectability, self-healing, tensile properties, remodeling, degradability and drug release of Alg-PBA/spd hydrogel. (A) Self-healing capacity of Alg-PBA/spd hydrogel by testing the G′ and G″ at alternating strain cycles of 5% and 500%. (B) Two pieces of cracked Alg-PBA/spd hydrogels are in contact with each other and the healed hydrogel can support its own weight. (C) Monitoring of the self-healing process of a scratch made on an Alg-PBA/spd hydrogel film by optical microscopy. Scale bar = 500 μm. (D) Viscosity of Alg-PBA/spd hydrogel with the shear rate from 0.1 to 5 1/s. (E) Photographs of the continuous injection of Alg-PBA/spd hydrogel through the 12# needle into any custom-designed shape. (F) Photographs of Alg-PBA/spd hydrogel with good tensile properties. (G) Photographs of Alg-PBA/spd hydrogel that can be remolded into various shapes. (H) Swelling and degradation curves of Alg-PBA/spd hydrogel in PBS. (I) The curve of cumulative spd release in PBS and 5 mM H₂O₂.

Figure 4.

Biocompatibility and anti-inflammation of Alg-PBA/spd hydrogel. (A) Cytotoxicity of Alg-PBA/Spd through CCK-8 assay of PDLFs viability after co-culture with different hydrogels mass fraction at specific time points (Day 3). (B) CCK-8 assay of PDLFs proliferation after co-culture with different hydrogel mass fraction for Days 1, 3, 5. PDLFs, periodontal ligament fibroblast cells; CCK-8, cell counting kit-8. (C) Live/dead staining of PDLFs at specific time points (Days 1, 3, 5) in 1 week of co-culture. Calcein AM staining (green) shows the high viability of cells. Scale bar =100 μm; 0.50%, 0.25%, 0, the mass fraction of Alg-PBA/spd hydrogel. (D) Live cell counts of the live/dead staining of PDLFs in (C). (E) ELISA analysis of IL-1β secretion in different spd concentrations. (F) ELISA analysis of IL-6 secretion in different spd concentrations. Data are shown as mean ± SD and compared using one-way ANOVA followed by Bonferroni’s post hoc test. ns, *, ** and *** indicate P > 0.05, P < 0.05, P < 0.01 and P < 0.001, respectively.

Figure 5.

Alg-PBA/Spd hydrogel promotes PDL tissue regeneration in vivo. (A) Schematic diagram of periodontal defect modeling in rats. (B) The mucoperiosteal flap was elevated to expose the alveolar bone on the lingual side of the first maxillary molar. The alveolar bone covering the root surface is removed, creating a periodontal window defect. The Alg-PBA/spd hydrogel is injected at the defect site. After the hydrogel completely filled the defect, the gums were sutured. (C) Masson staining images of the periodontal tissues at specific time points (Weeks 1, 2, 3) after different treatments. Scale bar = 200 and 50 μm. PDL, periodontal ligament; B, alveolar bone; C, cementum. (D) The collagen volume fraction of the Masson staining images (a). *P < 0.05, **P < 0.01, ***P < 0.001. (E) H&E staining images of the periodontal tissues at specific time points (Week 3) after different treatments. Scale bar = 100 and 50 μm.