Treatment of Periodontal Inflammation in Diabetic Rats with IL-1ra Thermosensitive Hydrogel

Abstract

Periodontitis is a chronic inflammatory disease that is considered to be the main cause of adult tooth loss. Diabetes mellitus (DM) has a bidirectional relationship with periodontitis. Interleukin-1β (IL-1β) is an important pre-inflammatory factor, which participates in the pathophysiological process of periodontitis and diabetes. The interleukin-1 receptor antagonist (IL-1ra) is a natural inhibitor of IL-1, and the balance between IL-1ra and IL-1β is one of the main factors affecting chronic periodontitis (CP) and diabetes. The purpose of this study is to develop a drug carrier that is safe and nontoxic and can effectively release IL-1ra, which can effectively slow down the inflammation of periodontal tissues with diabetes, and explore the possibility of lowering the blood sugar of this drug carrier. Therefore, in this experiment, a temperature-sensitive hydrogel loaded with IL-1ra was prepared and characterized, and its anti-inflammatory effect in high-sugar environments in vivo and in vitro was evaluated. The results showed that the hydrogel could gel after 5 min at 37 °C, the pore size was 5-70 μm, and the cumulative release of IL-1ra reached 83.23% on the 21st day. Real-time polymerase chain reaction (qRT-PCR) showed that the expression of IL-1β, Interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) inflammatory factors decreased after the treatment with IL-1ra-loaded thermosensitive hydrogel. Histological evaluation and micro-computed tomography (Micro-CT) showed that IL-1ra-loaded thermosensitive hydrogel could effectively inhibit periodontal inflammation and reduce alveolar bone absorption in rats with diabetic periodontitis. It is worth mentioning that this hydrogel also plays a role in relieving hyperglycemia. Therefore, the temperature-sensitive hydrogel loaded with IL-1ra may be an effective method to treat periodontitis with diabetes.

Keywords: diabetes; injectable and thermosensitive hydrogel; interleukin 1 receptor antagonist protein; periodontitis.

Figure 1

Characterizations of CS/β-GP/gelatin thermosensitive hydrogels. (a) Image of CS/β-GP/gelatin mixed solution transformed into hydrogel after 5 min at 37 °C. (b) Optical microscope images of chitosan/β-GP/gelatin thermosensitive hydrogel. (c) FT-IR spectra for chitosan, β-GP, gelatin, and chitosan/β-GP/gelatin thermosensitive hydrogel. (d) XRD patterns of chitosan, gelatin, and chitosan/β-GP/gelatin thermosensitive hydrogel. (e) Degradation percentage curve of CS/β-GP/gelatin hydrogel. (f) The release percentage curve of IL-1ra from CS/β-GP/gelatin hydrogel.

Figure 2

Biocompatibility and anti-inflammatory factor characteristics of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel. (a) Viability of RAW 264.7 cells cultured with IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogels at different concentrations (10–200 μg/mL) at 24 h and 48 h. (b) Fold change of IL-1β, IL-6, and TNF-α mRNA expression in the blank control, LPS stimulated, High glucose medium+ LPS stimulated, High glucose extract + LPS stimulated, High glucose extract + LPS + IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel groups (n = 3). (c) H&E staining of liver and kidney tissue in the groups I, II, III, IV, V, and VI, scale bar represents 100 μm. (d) Effect of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogels on serum creatinine (Cr), aspartate transaminase (AST), and alanine transaminase (ALT) levels. *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

Figure 2

Biocompatibility and anti-inflammatory factor characteristics of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel. (a) Viability of RAW 264.7 cells cultured with IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogels at different concentrations (10–200 μg/mL) at 24 h and 48 h. (b) Fold change of IL-1β, IL-6, and TNF-α mRNA expression in the blank control, LPS stimulated, High glucose medium+ LPS stimulated, High glucose extract + LPS stimulated, High glucose extract + LPS + IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel groups (n = 3). (c) H&E staining of liver and kidney tissue in the groups I, II, III, IV, V, and VI, scale bar represents 100 μm. (d) Effect of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogels on serum creatinine (Cr), aspartate transaminase (AST), and alanine transaminase (ALT) levels. *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

Figure 3

Anti-inflammatory effect of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel in vivo and its effect on body weight and blood sugar of rats. (a) Fold change of IL-1β, IL-6, and TNF-α mRNA expression in groups I, II, III, IV, V, and VI. (b) Effect of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel administration on rat weight. A black line is used to indicate the 14-day weight change in the I group to make the image results more intuitive. (c) The content of glycosylated hemoglobin in groups I, II, III, IV, V, and VI. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 4

Effect of IL-1ra-loaded CS/β-GP/gelatin thermosensitive hydrogel on alveolar bone in rats. (a) Photos of periodontitis models induced by ligature around maxillary first molars of rats in groups I, II, III, IV, V, and VI. (b) Micro-CT images of maxillary alveolar bone surrounding the maxillary first molars and maxillary second molars four weeks after the treatment: the images with black background are representative sagittal Micro-CT slices; the images with blue background are three-dimensional Micro-CT reconstruction images. The circled area is the subsequent analysis area of alveolar bone parameters. (c) Quantitative analysis of ABL, BV/TV, and Tb. Th determined by Micro-CT images. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 5

Immunohistochemical analysis of periodontal tissue sections in rats. (a) H&E image of the periodontal tissue section. The marked area is the periodontal tissue between the first molar and the second molar of rat maxilla, the part marked 1 in the figure shows more clearly and intuitively the expression of alveolar bone and gingival epithelium between the first molar and the second molar, scale bar represents 500 μm. (b) The results of IHC staining of IL-1β and IL-6 in periodontal tissues of rats in each group after 4 weeks of implantation, scale bar represents 50 μm.