Pauline Yang Wong,Shane Soo,Edmund Soon-Chern Wong,Praveen Praveen,Peta Clode,Murray V. Baker,Victor Haruo Matsubara
A Novel Antimicrobial Hydrogel for the Management of Periodontal Diseases
Antimicrobial agent,Hydrogel,Silver nanoparticles,Chlorhexidine,Tissue scaffolds
Objectives This study aimed to synthesise a drug-delivery system based on a porous polymer hydrogel, with antimicrobial properties against Porphyromonas gingivalis and potential to be used in tissue regeneration. Material and methods 2-Hydroxyethyl methacrylate monomers were polymerised using thermal and photoactivation in the presence of silver nitrate (AgNO3) and/or chlorhexidine digluconate. Poly-2-hydroxyethyl methacrylate (pHEMA) hydrogels containing silver nanoparticles (AgNPs) and/or 0.12% chlorhexidine (CHX) were produced and characterised using cryo-SEM and confocal microscopy. Hydrogel degradation and leaching of AgNP were tested for 1.5 months. The antimicrobial properties were tested against P. gingivalis using broth culture system and disk diffusion tests. Results Our methodology manufactured porous polymeric hydrogels doped with AgNPs and CHX. Hydrogels showed a successful delivery of CHX and sustainable release of AgNPs in a steady hydrogel degradation rate determined based on the weight loss of samples. Hydrogels with AgNPs or CHX had a significant antimicrobial effect against P. gingivalis, with CHX-hydrogels exhibiting a stronger effect than AgNP-hydrogels in the short-term assessment. AgNP-CHX hydrogels showed a compounded antimicrobial effect, whereas control hydrogels containing neither AgNPs nor CHX had no influence on bacterial growth (P < .05). Conclusions The dual-cured pHEMA hydrogel loaded with antimicrobial agents proved to be an efficient drug-delivery system against periodontopathogens, with the potential to be used as a scaffold for tissue regeneration.
A Novel Antimicrobial Hydrogel for the Management of Periodontal Diseases

This study aimed to synthesise a drug-delivery system based on a porous polymer hydrogel, with antimicrobial properties against Porphyromonas gingivalis and potential to be used in tissue regeneration.

2-Hydroxyethyl methacrylate monomers were polymerised using thermal and photoactivation in the presence of silver nitrate (AgNO3) and/or chlorhexidine digluconate. Poly-2-hydroxyethyl methacrylate (pHEMA) hydrogels containing silver nanoparticles (AgNPs) and/or 0.12% chlorhexidine (CHX) were produced and characterised using cryo-SEM and confocal microscopy. Hydrogel degradation and leaching of AgNP were tested for 1.5 months. The antimicrobial properties were tested against P. gingivalis using broth culture system and disk diffusion tests.

Our methodology manufactured porous polymeric hydrogels doped with AgNPs and CHX. Hydrogels showed a successful delivery of CHX and sustainable release of AgNPs in a steady hydrogel degradation rate determined based on the weight loss of samples. Hydrogels with AgNPs or CHX had a significant antimicrobial effect against P. gingivalis, with CHX-hydrogels exhibiting a stronger effect than AgNP-hydrogels in the short-term assessment. AgNP-CHX hydrogels showed a compounded antimicrobial effect, whereas control hydrogels containing neither AgNPs nor CHX had no influence on bacterial growth (P < .05).

The dual-cured pHEMA hydrogel loaded with antimicrobial agents proved to be an efficient drug-delivery system against periodontopathogens, with the potential to be used as a scaffold for tissue regeneration.

Oral diseases, including periodontal and peri-implant diseases, have been classified as major public health issues due to their high prevalence worldwide. The severe form of periodontitis affects approximately 11% of the global population, whilst peri-implantitis appears in 20% of patients in the first 5 to 10 years of implant placement. Despite the significant progress in regenerative periodontal therapies and an increased understanding of the pathophysiology of periodontal diseases, the conventional nonsurgical management of periodontitis has not evolved significantly over the last decade., The long-term effectiveness of scaling and root debridement is affected by regrowth of pathogenic microorganisms and their limited removal in deep subgingival pockets. Therefore, adjunct antimicrobials have been suggested to improve treatment effectiveness, but there are limitations to drug delivery with current topical antimicrobials. Chlorhexidine (CHX) and silver nanoparticles (AgNPs) have shown to be effective antimicrobials against various common periodontal and oral pathogens.9, 10, 11 One product already available in the market is the PerioChip, which is a controlled-release biodegradable gelatin containing chlorhexidine digluconate. Despite effective levels of CHX being maintained in the gingival crevicular fluid over a week without systemic absorption, the clinical benefit of this drug-delivery system remains controversial., Various potential drug-delivery techniques have been tested to deliver drugs directly into the gingival sulcus, including polymer hydrogels such as carboxymethyl cellulose, chitosan, and naturally derived polymers., The hydrogel used in this manner must have favourable properties, including biocompatibility, biodegradability, and good mechanical integrity. Polymer hydrogels have also been used as a scaffold in tissue engineering where the presence of porosities is an essential property for cell adhesion and cell proliferation in tissue growth. A previous study has created a poly(2-hydroxyethyl methacrylate)–based hydrogel doped with AgNPs, which presented a biocompatible porous structure with in vitro antimicrobial effects against Escherichia coli and Staphylococcus aureus. The mechanisms involved in the antimicrobial effect of AgNPs have not yet been fully elucidated. Observations showed that silver ions are able to affect different structures of bacterial cells, increasing, for example, the permeability of membrane that leads to cell lysis. AgNPs can also damage DNA, bacterial proteins, and lipids. AgNPs are considered biocompatible when utilised at an appropriate dosage, with no influence on cell differentiation, but an increase in cell stress and toxicity at higher concentrations. The development of a cost-effective drug-delivery system based on a porous hydrogel, which could also work as a scaffold for tissue regeneration, would be an important advancement in the management of periodontal diseases and peri-implantitis. Therefore, this study aimed to synthesise a novel polymer hydrogel presenting not only antimicrobial activity against periodontal pathogens but also properties required in a scaffold for tissue engineering applications. Antimicrobial activity was achieved by incorporating CHX and AgNPs into the hydrogel structure, and the hydrogel applicability was enhanced through a combination of photo- and thermally induced polymerisation.

2-Hydroxyethyl methacrylate (HEMA; Sigma-Aldrich), 2,2-dimethoxy-2-phenylacetophenone (DPAP; Sigma-Aldrich), AgNO3 (Lab Supply), chlorhexidine digluconate solution (Sigma-Aldrich), potassium persulfate (Unilab, Ajax Chemical Co. Pty Ltd.) were used without further purification. Ultrapure water was obtained from a Milli-Q system and used for sample preparation, storage, and inductively coupled plasma optical emission spectrometry (ICP-OES) studies.

Polymer hydrogels were prepared via both photo and thermal polymerisation of HEMA in aqueous solution as a modification of a previously described method. DPAP was used as a photo activator and potassium persulfate as thermal activator. UV irradiation followed by incubation in water bath at 37 °C temperature was used to initiate the polymerisation reaction (Figure 1). A detailed description of hydrogel preparation is provided as Supplementary Material. Four variations of the hydrogel formula were tested: control hydrogel—plain hydrogel with no addition of chlorhexidine digluconate (CD) or AgNO3; CHX hydrogel—containing CD; AgNP hydrogel—containing AgNO3; and CHX-AgNP hydrogel—containing both CD and AgNO3. Once the polymerisation was completed, the hydrogels were carefully removed from vials (with the aid of a small spatula) and soaked in Milli-Q water for 12 hours to remove unreacted HEMA monomer.

Hydrogel samples were sliced into pieces ∼3 mm wide and ∼0.5 mm thick, blotted on filter paper and mounted onto aluminium pins using optimal cutting temperature (OCT) compound, before being plunged frozen in liquid ethane (−180 °C; Leica, EM GP2). Frozen samples on pins were then loaded into a cryostage under liquid nitrogen and transferred under vacuum to the cryopreparation system (Leica, EM ACE600 + VCT500). Frozen samples were sublimated at –90 °C for 105 seconds to remove surface ice and reveal gel structure and then coated with 4 nm platinum. After coating, the samples were transferred under vacuum to the cryo-SEM (JEOL, IT800 field emission SEM + Leica, VCT500). Imaging was conducted at −155 °C and 10 kV using both secondary and back-scattered electron signals. Qualitative element analysis and mapping of silver was performed at 10 kV using an x-ray detector interfaced to AZtec software (Oxford Instruments, 170 mm2 Ultim® Max SDD EDS detector).

Raman spectra of CHX-containing hydrogels were collected using confocal laser Raman microscopy on a WITec alpha 300RA+ (WiTec) with a 532-nm laser source. The laser was focussed through a 100×/0.9 objective to obtain a spot size of smaller than 1 μm. Spectral acquisitions were obtained with 600 L mm−1 grating and a CCD detector peltier-cooled (−60 °C) with 1024×128 pixels. Laser centring and spectral calibration were performed daily on a silicon chip with characteristic Si Raman band of 520.4 cm−1. Raman maps were acquired with a motorised stage allowing XYZ displacement with precision of better than 1 µm. The WITec Project FOUR software was used to analyse all spectra through background subtraction and peak fitting.

The mass loss of CHX-AgNP hydrogel was measured for 52 days, with samples incubated at 37 °C in aqueous solution at pH 6.8. The hydrogel samples were prepared by cutting the hydrogel with a razor blade into discs (10 mm diameter × 5 mm thick). The hydrogel dry mass was measured every 3 days (starting at day 0) after drying the samples for 40 minutes at 37 °C. At each assessment time point, 4 samples were dried and weighed using a digital laboratory scale.

The leaching of AgNPs from hydrogels was evaluated over a 1.5-month period using an ICP-OES instrument (Agilent Technologies 5100, Agilent). CHX-AgNP hydrogel samples (15 mm thickness and 10 mm diameter) were transferred to centrifuge tubes containing 6 mL of ultrapure water immediately after the 12-hour washing period. Water was collected from each tube and replaced with fresh water (6 mL) every 3 days for 46 days. 70% Nitric acid (HNO3,1 mL) was added to each sample, followed by sufficient ultrapure water to obtain a final volume of 10 mL. All the sample solutions were filtered using nonpyrogenic 0.20-μm filters before injection into the ICP analysis system for Ag+ analysis. Standard solutions with different Ag+ concentrations were prepared from an Ag+ standard solution (1000 ± 3 µg/mL) in 2% HNO3 (from High-Purity Standards) by dilution in water. These solutions were used to generate a linear calibration curve (signal intensity as a function of concentration).

Gram-negative bacteria P. gingivalis (ATCC® 33277) was used as a periodontopathogen. The strain was grown under anaerobic conditions (90% N2, 5% CO2, and 5% H2) at 37 °C on blood agar plates (TSA [Difco Laboratories] enriched with 5% defibrinated sheep blood, 0.5 mg/mL hemin [Sigma-Aldrich], and 1 mg/mL menadione [Sigma-Aldrich]). Enriched Tryptic Soy Broth (eTSB; yeast extract, hemin [5 mg/mL], and menadione [0.5 mg/mL]) was used for overnight growth.

The antibacterial effect of all 4 types of hydrogels doped with CHX and AgNPs was assessed using spectrophotometric semi-quantitative assay. After overnight bacterial growth in eTSB, the P. gingivalis suspension was adjusted to ∼109 CFU/mL and 10 µL of this suspension was inoculated in 15 mL sterile centrifuge tubes with 4 mL of eTSB (2.5 × 106 UFC/mL final concentration). Polymer hydrogels (control hydrogel, CHX hydrogel, AgNP hydrogel, and CHX-AgNP hydrogel) were sliced with a razor blade into discs (10 mm diameter × 2 mm thick) and placed into tubes with P. gingivalis suspension (1 hydrogel type per tube). The mixtures were incubated at 37 °C for 48 hours. Tubes containing polymer discs and bacterial suspension were examined after 24, 36, and 48 hours’ incubation. At each assessment time, the optical density (OD) of each bacterial suspension was measured using a microplate reader (Tecan) at 600 nm. The percentage of P. gingivalis growth inhibition was calculated using the following formula:where Control OD is the absorbance of control hydrogel and Sample OD is absorbance of test hydrogels.

Hydrogels were sliced into discs 10 mm in diameter and 3 mm thick and placed onto blood agar plates inoculated with P. gingivalis and grown under anaerobic conditions at 37 °C for 5 days, followed by a qualitative analysis of the inhibition zones.

Nine samples per group were used for antimicrobial tests. The degradation test had 4 samples per reading. Data were presented as mean ± standard deviation (SD) from 3 independent experiments. One-way analysis of variance (ANOVA) followed by Tukey multiple comparison test was performed. Statistical significance was set at P < .05 (GraphPad Prism Version 6.0c–GraphPad Software).

The methodology developed in this study produced pHEMA hydrogels loaded with CHX and AgNPs using a dual-activation polymerisation reaction (Figure 1A). All hydrogel variations presented with a firm, sponge-like consistency after polymerisation (Figure 1B) except for the CHX hydrogels, which were slightly softer. The colour of the hydrogels varied based on antimicrobials incorporated. The Control and CHX hydrogels were white, the AgNP hydrogels were light brown, whilst CHX-AgNP hydrogels were darker brown.

Cryo-scanning electron micrograph images showed that the pHEMA hydrogels loaded with both AgNPs and CHX presented morphologies based on a network of interconnected pores of dimensions 10 to 30 μm, which were distributed throughout the hydrogel sample (Figure 2). It is possible to identify fewer porosities on the hydrogel surface in contact with the cylindrical quartz vial (Figure 2A). Similar morphology was observed in the hydrogel without AgNPs and CHX. Under higher magnification, it is possible to visualise smaller holes (<10 µm) throughout that are ice crystal damage from freezing (Figure 2B), which is inevitable as freezing is part of the sample processing for cryo-SEM. The back-scattered electron images from cryo-SEM showed abundant silver nanoparticles throughout the hydrogel and they were often clustered, forming larger particles (Figure 2C). When looking at isolated nanoparticles, it is possible to identify that some AgNPs synthesised during the hydrogel fabrication reaction presented a triangular shape, but most of them had rounded contour in a nanometre scale (Figure 2D). Cryo-SEM and EDS analysis were used to evaluate the presence of AgNPs in hydrogel (Figure 2E, 2F). The energy-dispersive element mapping (Figure 2F) and the x-ray spectrum from the same area (Figure 2G) confirm the presence of Ag with Lα and Lβ Ag x-ray peaks being visible at 2.98 keV and 3.15 keV, respectively. Platinum peaks from the sample coating are also visible in the x-ray spectrum. Confocal Raman spectroscopy confirmed the presence of CHX within CHX-AgNP hydrogel samples (Supplementary Material Figure 1).

The degradation of CHX-AgNP hydrogels was determined based on the weight loss of samples, which were incubated in water at 37 °C. On day 0 (immediately after synthesis), dry hydrogel discs weighed 202 mg on average. The hydrogels’ weight was decreasing steadily over time, and at the end of the assessment period the average weight was 121 mg (Figure 3A). In 52 days, the hydrogel samples lost 40% of their dry weight, representing a significant degradation rate (P < .0001).

CHX-AgNP hydrogels leached a significant amount of silver ions (approximately 30.7% of silver present in the initial formulation) during the first 7 days of incubation in water. Figure 3B shows a 4-day period of rapid leaching with the rate of leaching declining rapidly to almost zero by day 7. From day 10 onward, the silver leaching continues at a low rate until reaching zero at day 43.

A semi-quantitative analysis based on OD readings was used to assess the antimicrobial effect of hydrogels in a broth culture system. All hydrogels with antimicrobial agents significantly inhibited the growth of P. gingivalis at 24, 36, and 48 hours, especially the CHX and CHX-AgNP hydrogels (P < .001; Figure 4A). The control hydrogel showed no antimicrobial effect (Figure 4B), and its OD readings were higher than the test hydrogels for all readings taken. The percentage of bacterial growth inhibition (Table) was lower for AgNP hydrogel compared to CHX and CHX-AgNP in all time intervals. The antimicrobial effect of CHX and CHX-AgNP hydrogels were very similar, although CHX-AgNP hydrogel presented a slightly higher percentage of inhibition. Control, CHX, and CHX-AgNP hydrogels were further investigated in a disc diffusion assay, which confirmed that the control hydrogel did not have antimicrobial effects. CHX and CHX-AgNP hydrogels had similar antimicrobial effects based on the inhibition zone around each sample (Figure 4C).

A previous study from our group developed porous pHEMA sponges with antimicrobial capacity using a single-step methodology and light activation. Based on this previous protocol, we have formulated a novel methodology to synthesise dual-cured polymeric hydrogels loaded with 2 important antimicrobial agents: CHX and AgNPs. The former is one of the most potent chemotherapeutic agent used in dentistry to control oral biofilms, and the latter has been used in the medical field as an antimicrobial agent that promotes and accelerates the healing process. In terms of physical characteristics, our hydrogels containing AgNPs had a firmer consistency compared to hydrogels containing only CHX. It has been shown that the addition of AgNPs not only changes the colour of hydrogels but also accelerates polymerisation and affects the morphology of the resulting polymer. In our pilot studies, higher concentrations of potassium persulfate displayed rapid polymerisation of hydrogel samples, but compromised synthesis of AgNPs within hydrogels. Therefore, the concentration of potassium persulfate was adjusted to allow sufficient time of UV light exposure to form AgNPs before completion of hydrogel polymerisation. The CHX-AgNP hydrogel was found to be an efficient drug-delivery system, as it contained CHX in the form of chlorhexidine gluconate and sustained the release of AgNPs for more than a month. In contrast, previous experimental hydrogels containing AgNPs lost most of their silver content in the first 3 days postsynthesis. The efficient and sustainable delivery of CHX and AgNPs promoted significant antimicrobial effect against P. gingivalis, an important periodontopathogen in the periodontal and peri-implant diseases. In our study, the antimicrobial tests demonstrated that both antimicrobial agents were responsible for the anti–P. gingivalis effect of the hydrogels. However, further studies on the duration of antimicrobial activity would need to be conducted to determine whether this sustained release of antimicrobials in the form of CHX and AgNPs would correspond to a similar length of antimicrobial activity. Even though the addition of AgNPs in the hydrogel formulation did not significantly increase the short-term antimicrobial effect of CHX-AgNP hydrogels compared to CHX hydrogels, the use of AgNP is believed to be beneficial for long-term antimicrobial effect due to the sustained release of AgNPs. In contrast to existing drug delivery systems, this new hydrogel formulation allows the synthesis of an antimicrobial scaffold within the periodontal defect, using a curing light source and body temperature to activate and accelerate AgNP formation and hydrogel polymerisation. Therefore, this drug delivery system could be individually synthesised for any periodontal or peri-implant defect to achieve intimate contact with tissues and increase its retention within the defect. Additionally, cryo-SEM analysis showed CHX-AgNP hydrogels with a monolithic PHEMA structure exhibiting porosity, with pores having dimensions 10 to 30 μm, and mass-loss studies showed that the hydrogels degrade slowly over about 50 days in aqueous media at 37 °C. These traits suggest that the hydrogels have potential to act as a scaffold to support cell adhesion and growth. Previous studies found that the size of pores in scaffolds for tissue engineering influences the cell behaviour, such as cell proliferation, gene expression, and cartilage-like matrix deposition. As our hydrogels presented relatively small pores, they are likely to provide more adhesion support for tissue cells, despite the reduced space for cell growth. Cross-linkers were not used in our reaction; therefore, the hydrogel did not have a polymer droplet morphology like in previous study. Biodegradability and biocompatibility are essential properties in scaffolds used in tissue regeneration. Our polymer hydrogels, which had no addition of cross-linkers, presented a favourable degradation rate in water at body temperature, thus indicating that it could be biodegradable in the oral environment. On the contrary, the previous study testing pHEMA sponges doped with AgNPs as antibacterial agents used the cross-linking agent EDGMA (ethylene glycol dimethacrylate) in the hydrogel formulation and, therefore, degradability was not evidenced. In the same study, hydrogels formulated with HEMA and AgNO3 were shown to be nontoxic to human corneal epithelial cells. Our new formulation would require further cytotoxicity tests before its application in animal and clinical studies, as we have incorporated additional components to obtain dual (photo and thermal) activation of polymerisation.

A novel methodology was devised and successfully produced dual-cured pHEMA hydrogels loaded with antimicrobial agents. These hydrogels showed to be an efficient and effective drug-delivery system against P. gingivalis. The hydrogel has microporosities, is biodegradable, and exhibits promising potential as a scaffold for tissue regeneration when treating periodontal or peri-implant diseases.

None disclosed.