Antibacterial and Fluorescence Staining Properties of an Innovative GTR Membrane Containing 45S5BGs and AIE Molecules In Vitro

Abstract

This study aimed to add two functional components-antibacterial 45S5BGs particles and AIE nanoparticles (TPE-NIM⁺) with bioprobe characteristics-to the guided tissue regeneration (GTR) membrane, to optimize the performance. The PLGA/BG/TPE-NIM⁺ membrane was synthesized. The static water contact angle, morphologies, and surface element analysis of the membrane were then characterized. In vitro biocompatibility was tested with MC3T3-E1 cells using CCK-8 assay, and antibacterial property was evaluated with Streptococcus mutans and Porphyromonas gingivalis by the LIVE/DEAD bacterial staining and dilution plating procedure. The fluorescence staining of bacteria was observed by Laser Scanning Confocal Microscope. The results showed that the average water contact angle was 46°. In the cytotoxicity test, except for the positive control group, there was no significant difference among the groups (p > 0.05). The antibacterial effect in the PLGA/BG/TPE-NIM⁺ group was significantly (p < 0.01), while the sterilization rate was 99.99%, better than that in the PLGA/BG group (98.62%) (p < 0.01). Confocal images showed that the membrane efficiently distinguished G⁺ bacteria from G^- bacteria. This study demonstrated that the PLGA/BG/TPE-NIM⁺ membrane showed good biocompatibility, efficient sterilization performance, and surface mineralization ability and could be used to detect pathogens in a simple, fast, and wash-free protocol.

Keywords: aggregation-induced emission nanoparticles; antibacterial; antibacterial differentiation; bioactive glass; bioprobe; guided tissue regeneration (GTR).

Scheme 1

Molecular structures of TPE-NIM⁺ and a general representation of their adsorption onto the surfaces of G⁺ bacterium. Note that the TPE-NIM⁺ and the bacteria are not drawn in real scales.

Scheme 2

Synthetic routes of TPE-NIM⁺. Reagents and conditions: (i) Zn, TiCl₄, tetrahydrofuran (THF), 0–25 °C, 12 h, (ii) n-Butyllithium solution, trimethyl borate, THF, –78 °C, 3 h, (iii) C₂H₅OH, reflux, 2 h, and (iv) Pd(PPh₃)₄, Na₂CO₃, mixed solvent (toluene: ethanol: water), 90 °C, 12 h.

Figure 1

The Proton NMR of TPE-NIM⁺.

Figure 2

Images of the surface of the PLGA/BG/TPE-NIM⁺ membrane after putting a drop of distilled water on it. Measured water contact angle values and captured images at 0 s, 30 s, and 60 s.

Figure 3

SEM images of composite membranes before and after immersion in SBF. (a,b) SEM images of PLGA/10%BG/4%TPE-NIM⁺ membrane before immersion in SBF. White arrows show the exposed 45S5BGs particles. (b) Three fixed points (Spectrum 28.29.31) were selected randomly for EDS analysis. (c,d) SEM images of PLGA/10%BG/4%TPE-NIM⁺ membrane after immersion in SBF for three days: (c) White circles showed the embedded 45S5BGs particles. Yellow arrows showed the hollow structure; and (d) Yellow arrows show the hollow structure. Three fixed points (Spectrum 44.47.48) were selected randomly for EDS analysis.

Figure 4

EDS analysis of composite membranes before and after immersion. (a–c) EDS images of PLGA/10%BG/4%TPE-NIM⁺ membrane without immersion in SBF. (d–f) EDS images of PLGA/10%BG/4%TPE-NIM⁺ membrane after three days of immersion in SBF.

Figure 5

EDS Elemental Analysis with ColorSEM Technology of the PLGA/10%BG/4%TPE-NIM⁺ membrane without immersion in SBF. (a) EDS image with all elements superimposed.

Figure 6

EDS Elemental Analysis with ColorSEM Technology of the PLGA/10%BG/4%TPE-NIM⁺ membrane after three days of immersion in SBF. (a) EDS image with all elements superimposed.

Figure 7

Cytotoxicity of control medium, PLGA/BG/TPE-NIM⁺ composite membranes, respectively, containing 10%, 20%, or 30% BG toward MC3T3-E1.

Figure 8

Cytotoxicity of control medium, PLGA/BG membrane and PLGA/BG/TPE-NIM⁺ membrane toward MC3T3-E1. (* p > 0.05 ** p < 0.05 vs. blank control group).

Figure 9

Live and Dead bacterial staining. (a) S. m bacteria liquid without the treatment with PLGA/BG/TPE-NIM⁺ composite membrane. (b) S. m bacteria liquid with the treatment with PLGA/BG/TPE-NIM⁺ composite membrane for 24 h.

Figure 10

Confocal images of P. gingivalis (left: A–C) and S. mutans (right: A–C) after treatment with PLGA/10%BG/4%TPE-NIM⁺ membrane extract (without 45S5BGs) for 15 min.

Figure 11

Confocal images of S. mutans (A–C) and P. gingivalis (D–F) after treatment with PLGA/10%BG/4%TPE-NIM⁺ membrane extract (without 45S5BGs) for 15 min. (G–L) Selective staining of S. mutans by TPE-NIM⁺. White arrow indicates stained S. mutans and yellow arrows indicate non-stained P. gingivalis.

Figure 12

Confocal images of S. mutans (A–C) and P. gingivalis (D–F) after treatment with 45S5BGs and PLGA/10%BG/4%TPE-NIM⁺ membrane extract (with 45S5BGs), each for 15 min.