An injectable MB/BG@LG sustained release lipid gel with antibacterial and osteogenic properties for efficient treatment of chronic periodontitis in rats

Abstract

Periodontitis is a chronic inflammatory disease characterized by the colonization of pathogenic microorganisms and the loss of periodontal supporting tissue. However, the existing local drug delivery system for periodontitis has some problems including subpar antibacterial impact, easy loss, and unsatisfactory periodontal regeneration. In this study, a multi-functional and sustained release drug delivery system (MB/BG@LG) was developed by encapsulating methylene blue (MB) and bioactive glass (BG) into the lipid gel (LG) precursor by Macrosol technology. The properties of MB/BG@LG were characterized using a scanning electron microscope, a dynamic shear rotation rheometer, and a release curve. The results showed that MB/BG@LG could not only sustained release for 16 days, but also quickly fill the irregular bone defect caused by periodontitis through in situ hydration. Under 660 ​nm light irradiation, methylene blue-produced reactive oxygen species (ROS) can reduce local inflammatory response by inhibiting bacterial growth. In addition, in vitro and vivo experiments have shown that MB/BG@LG can effectively promote periodontal tissue regeneration by reducing inflammatory response, promoting cell proliferation and osteogenic differentiation. In summary, MB/BG@LG exhibited excellent adhesion properties, self-assembly properties, and superior drug release control capabilities, which improved the clinical feasibility of its application in complex oral environments.

Keywords: Antiinflammation; Antimicrobial photodynamic therapy; Periodontal regeneration; Periodontitis; Sustained release.

Graphical abstract

Scheme 1

Scheme of the synthesis process, antibacterial effects, anti-inflammatory effects, and promotion of periodontal regeneration of injectable MB/BG@LG.

Fig. 1

The SEM images of the surface (a) and cross-sectional sides (b) of the lyophilized system of MB/BG@LG (The latter image is a partial magnification of the boxed region in the previous image); (c) The EDS analysis of MB/BG@LG. (d) The particle size distribution of bioglass. (e) The FTIR spectra of BG, MB, LG, MB/BG@LG. (f) The release rate of MB in MB/BG@LG at different times.

Fig. 2

(a) The photograph of MB/BG@LG before and after gelatinization. (b) The injectable property of MB/BG@LG (Movie 1). (c) Experiments simulating the effect of oral saliva flow on MB/BG@LG adhesion (Movie 2). (d)The plasticity property of MB/BG@LG. The viscosity varies and the shear stress changes with the shear rate of MB/BG@LG before gelatinization (e) and MB/BG@LG after gelatinization(f). (g) Time-responsive G′ and G″ modulus changes of MB/BG@LG in the process of sol-gel transition. (h) Frequency -responsive G′ and G″ modulus changes of MB/BG@LG after gelatinization.

Fig. 3

The UV spectra of PBS(a), MB(b), MB/BG@LG(c) under 660 ​nm illumination at different times. The ROS generation rate(d) of Blank, MB/BG@LG, MB.

Fig. 4

Evaluation of the MB/BG@LG in vitro antibacterial properties. The images and quantitative analysis of S. aureus (a, b) or E. coli (c, d) bacterial colony treated with PBS and MB/BG@LG released fluid at different concentrations with and without 660 ​nm light. The SEM photograph of S. aureus (g) or E. coli (h) cultured with PBS and the impregnated solution of MB/BG@LG for 3 ​min with and without 660 ​nm light.

Fig. 5

(a) The Live/Dead staining images of L929 and MC3t3-E1 cultured with MB/BG@LG culture medium on days 1 and 3. The cell viability of L929(b) and MC3t3-E1(c) cultured with MB/BG@LG culture medium on days 1, 3, and 5. (d) The hemolysis rate of Light, LG, MB/BG@LG (-light), and MB/BG@LG (+light) groups. (e)The SEM photographs of MC3t3-E1 cultured with MB/BG@LG culture medium for 24 ​h.

Fig. 6

The ALP staining photograph of MC3t3-E1 cultured with MB/BG@LG culture medium for day 7(a) and day 14(b). The quantitative analysis of alp on day 7 (c) and day14 (d).

Fig. 7

(a) The modeling process in rats with periodontitis. (b) The three-dimensional micro-CT images of alveolar bone in the healthy rats, periodontitis rats and the rats treated with MB/BG@LG with and without 660 ​nm light. (c) CEJ-ABC distance on the buccal sides, (d) TV, (e) BV, (f) BV/TV calculate from micro-CT results. ∗, ∗∗and∗∗∗indicate p ​< ​0.05, p ​< ​0.01 and p ​< ​0.001.

Fig. 8

The H&E staining (a), OPG (b), MMP-9 (c) and CD68 (d) of healthy rats, periodontitis rats and the rats treated with blank, LG and MB/BG@LG with and without 660 ​nm light. The quantitative analysis of OPG (e) and MMP9 (f) expression of healthy rats, periodontitis rats and the rats treated with blank, LG and MB/BG@LG with and without 660 ​nm light. The TNF-α (g) and CD68 (h) expression of healthy rats, periodontitis rats and the rats treated with blank, LG and MB/BG@LG with and without 660 ​nm light.

Fig. 9

The H&E staining of major organs (heart, liver, spleen, lung, and kidney) in healthy rats, periodontitis rats, and the rats treated with blank, LG, and MB/BG@LG with and without 660 ​nm light for 28 days.

Fig. 10

(a)The H&E staining of the skin in the back after subcutaneous implantation of LG and MB/BG@LG for 3, 7, 14, 28, and 56 days. (b) The photograph of the skin in the back after subcutaneous implantation of LG and MB/BG@LG for 3, 7, 14, 28, and 56 days.