Chlorhexidine-loaded functionalized mesoporous MCM-41 poly(methylmethacrylate) based composites with Candida antibiofilm activity

Abstract

The complete or partial dentures made of acrylic resins or composites constitute the support for Candida biofilm with consequent onset of stomatitis and candidiasis. In this paper, polymethylmethacrylate composites containing chlorhexidine are prepared. The drug was loaded inside the mesopores of the silicate MCM-41 which had been properly silanized with an acrylic coupling agent. Four different chlorhexidine loaded composites were prepared using MCM-41 silanized with four different amounts of 3-methacryloxypropyl-trimethoxisilane. The composites prepared showed a decreased resin polymerization degree in comparison to the pure resin. The composite obtained with MCM-41 with the lowest silanization degree, showed a good polymerization degree, higher than that obtained when free chlorhexidine was added to the resin. This composite was able to decrease Candida biofilm adhesion and formation and inhibited Candida biofilm proliferation for the duration of the test (120 days).

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Introduction

Polymethylmethacrylate resins and composites are widely used in restorative and prosthetic dentistry. In particular polymethylmethacrylate resins are the choice materials for bases of complete or partial dentures. They have desirable qualities such as good esthetics and good processability. Unfortunately, they have several drawbacks. In fact they offer a good surface for biofilm adhesion and proliferation.^1,2 Biofilms are colonies of bacteria or fungi that adhere to a surface by means of flagellar proteins and secretion of an extracellular polymeric substance composed of polysaccharides and extracellular DNA.³ Biofilms are ubiquitous in nature and nearly all species of bacteria and fungi are able to form biofilms. They resist traditional antibiotic treatment and adhere readily to a wide variety of clinical apparatus such as catheter tubing and surgical and dental implants.

The physical and chemical characteristics of the surface of dental resins support biofilm formation^2,4,5 through the proliferation of plaque on the surface of dentures with consequent onset of oral stomatitis, hardly to eradicate. Candidiasis is one of the most common human oral infections, is expressed in several clinical manifestations especially in geriatric patients with poor oral hygiene, such as patients with physical or mental disabilities and has been identified as the major causative microbial agent in denture stomatitis. Thus, with the aim of preventing biofilm adhesion and proliferation, several attempts have been made to introduce chlorhexidine (CHX) salts as antimicrobial agent into dental composites.^6–9 These materials show a delay in biofilm formation compared to conventional resins or composites but some of them show polymer leakage, reduced polymerization degree and loss of their effectiveness over time.^10–12

Recently, mesoporous ordered silicates, such as MCM-41 and SBA-15, have been proposed as components for dental polymer silica composites and their presence in moderate amount in the polymer matrix improved composite hardness.^13–15 Mesoporous ordered silicates are inorganic materials synthesized in the presence of surfactants which act as templates for poly-condensation of silicic species, a source of silica, a solvent and a catalyst.¹⁶

When a cationic surfactant, such as alkylammonium salt, is present as a template, the M41S materials are obtained.¹⁷ The most famous representative of this group is MCM-41 which has a honeycomb structure that is the result of hexagonal packing of unidimensional hexagonal mesopores with pore size and wall thickness not beyond 4.0 and 2.0 nm respectively. MCM-41 has been largely proposed as suitable carrier to modify drug release¹⁸ and to improve dissolution rate of poorly soluble drugs.^19–22

In this paper polymethylmethacrylate composites containing CHX was prepared with the aim of obtaining a material having prolonged antibiofilm activity. However, as CHX could interfere with the resin polymerization process, it was loaded inside the pores of the filler MCM-41, properly modified with an acrylic coupling agent. The composites were prepared and characterized for polymerization degree. Then CHX release from composites and their anti-biofilm activity were evaluated as well.

Experimental

Materials

Cetyltrimethylammonium chloride (25% wt water solution), sodium metasilicate and 3-methacryloxypropyl-trimethoxysilane (γ-MPS) were obtained from Sigma-Aldrich Chemical (Milano, Italy). CHX as diacetate salt was kindly gifted by Degussa AG, Hanau-wolfgang, Germany.

A polymerized heat-curing dental acrylic resin (Paladon® 65)²³ kindly furnished by Heraeus Kulzer (Wehrheim, Germany) was used. Paladon® is a powder/liquid system in which the powder main component is polymethylmethacrylate and the liquid components are methylmethacrylate and dimethylmethacrylate.

Deionized water was obtained by a reverse osmosis process with a MilliQ system (Millipore, Rome, Italy). Other chemicals and solvents were of reagent grade quality and were used without further purification.

Methods

XRPD patterns were taken using a computer-controlled PW 1710 Philips diffractometer (Lelyweg, the Netherlands), using the Ni filtered Cu Kα radiation.

Thermogravimetric analyses (TG) were carried out by a thermoanalyzer (TG-DTA Netzsch STA 490) at heating rate of 10 °C min⁻¹ with 30 mL min⁻¹ air flow.

DSC analyses were performed using an automatic thermal analyzer (Mettler Toledo DSC821e). Temperature calibrations were achieved by using an indium standard. Holed aluminum pans were employed for all samples and an empty pan, prepared in the same way, was used as reference. Samples of 3–6 mg were loaded directly into the aluminum pans and thermal analyses were conducted at a heating rate of 10 °C min⁻¹ from 25 to 300 °C.

Nitrogen adsorption–desorption isotherms were determined with a Micromeritics ASAP 2010 instrument at 77 K on samples previously outgassed at room temperature overnight. The specific surface area was calculated with the Brunauer, Emmett, and Teller (B.E.T.) method.²⁴ For the pore size characterization, the considerations concerning the nitrogen adsorption on MCM-41 solids were considered²⁵ and the BJH–KJS method was followed.

Synthesis and silanization of MCM-41

MCM-41 was synthesized pouring an aqueous solution (300 mL) of sodium metasilicate (30 g) into an aqueous solution (1000 mL) of cetyltrimethylammonium chloride (68.8 g, 25 wt%). Then ethyl acetate (30 mL) was quickly added under vigorous stirring. The mixture was allowed to stay at room temperature for 2 h and then was heated for 40 h at 80 °C. The resulting solid was recovered by filtration, abundantly washed with water and ethanol, dried at room temperature, and at last calcined at 600 °C for 20 h in order to eliminate the surfactant.²⁶

MCM-41 particles were silanized with 4 different amounts of γ-MPS (0.032, 0.062, 0.125, 0.250 mL relative to 1 g of MCM-41). Thus, MCM-41 was suspended in n-hexane (100 mL), then the proper amount of γ-MPS was added and the mixture was heated at 65 ± 5 °C for 5 hours. The solid was recovered by filtration, plenty washed with ethanol and dried at 70 °C for 12 hours.²⁷

Preparation of CHX loaded silanized MCM-41

CHX was loaded aiming at a theoretical drug content of 10 wt% in the final products (that is 10 g of CHX in 100 g of the final loaded compound). Thus CHX (170 mg) was dissolved in EtOH (5 mL) and then the proper silanized MCM-41 (1.5 g) was added and at this step, in order to remove air from matrix pore and to make easy the drug entrance, the vacuum was applied at the dispersion until bubble airs were removed from the sample. Afterwards, the mixture was kept under stirring for 30 min. Finally, the solvent was removed by using a rotary evaporator.

CHX loading was determined both by TG and by UV spectrophotometry (Spectrophotometer Agilent 8453), after suspension of a well known amount of CHX loaded silanized MCM-41 (10 mg) with absolute ethanol (100 mL) for 24 h (λ_max = 262 nm).

Dental composite preparation

Six groups of disk-shaped polymethylmethacrylate specimens (12 ± 1 mm in diameter, 2.5 mm in thickness) were prepared. The mixing ratio (10 g of powder mixed with 4 mL of liquid component) and conditions for processing and polymerization recommended by the manufacturer were followed strictly. The obtained specimen were maintained in deionized water for 12 hours and then were dried at 37 °C for 4/5 hours.

Degree of conversion (DC)

The DC of monomer-to-polymer was determined by FTIR. Spectra were recorded in air, at room temperature on a Jasco FT/IR-410, 420 Herschel series (Jasco Corporation Tokyo, Japan) in KBr dispersion using the EasiDiff™ Diffuse Reflectance Accessory. Samples were prepared by grounding the powders with KBr. Spectra were obtained with 100 scans at a resolution of cm⁻¹ from 4000 cm⁻¹.

The DC of monomer-to-polymer was calculated by comparison of the absorbance ratio, using a standard baseline technique, of the C=C peak from the methacrylate group at 1640 cm⁻¹ and to that of the unchanging C=O peak from the ester group at 1720 cm⁻¹, which was used as a reference peak, before and after polymerization.^28,29 The DC% was determined by the following formula:

where P₁ is peak area of C=C methacrylate peak and P₂ is the peak area relative to the C=O of the ester group.

CHX release

Release studies were performed on disks containing CHX (disks 2–5). The samples were accurately weighted and placed in a sealed vial containing 5 mL of deionized water, at 37 ± 1 °C under stirring (50 rpm). At appropriate intervals 0.5 mL of dissolution medium were removed from the vial and CHX concentration was determined by UV spectrophotometry at λ_max 255 nm (UV-vis spectrophotometer Agilent model 8453). After CHX determination, the removed sample was replaced in the vial.

All experiments were performed in triplicate and results were reported as an average.

Microorganisms and culture conditions

Candida albicans strain CAF 2-1 (SC5314, ATCC MYA-2876) was used. Stock cultures were maintained at 20 °C. After recovery, culture was maintained on Sabouraud dextrose agar (SDA) stored at 4–6 °C during the experimental period. To prepare the yeast inoculum, a loopful of the stock culture was streaked onto SDA and incubated at 37 °C for 24 h. Two loopfuls of this young culture were transferred to 7 mL of sterile Sabouraud broth medium and incubated at 37 °C for 24 h. Cells of the resultant culture were harvested, washed twice with sterile phosphate-buffered saline (PBS) (pH 7.2) at 2000 g for 10 min and re-suspended in Sabouraud broth. Candida cells were counted by hemocytometer and diluted to the desired concentration.

Adhesion and biofilm formation assays

The adhesion and biofilm formation assays were performed according to Wady A. F. et al.³⁰ with some modifications. Prior to the tests, the disks were sterilized to kill any microorganisms that may had contaminated the disks during preparation. All disks were pre-coated with pooled clarified human saliva for 30 minutes at 37 °C. Saliva was then removed and 100 μL of the C. albicans suspension (10⁶ cells per mL) were placed on each disk in a well of a 24-well microplate. The cells were incubated for 90 min (adhesion assay) and 72 h (biofilm formation) at 37 °C. After incubation, non-adherent cells were removed from the disks by gently washing twice with sterile 3 mL phosphate buffer solution. Each disk was placed in a tube containing 10 mL of sterile saline solution. In order to detach yeasts adherent to the biomaterial surfaces bringing them in suspension, all tubes were then placed in an ultrasonic bath cleaner operating at 47 kHz, 234 W and sonicated for 6 min. 50 μL of each suspension were diluted and plated on Sabouraud agar plates. Plates were incubated at 37 °C for 24 h and CFU were counted and expressed as CFU cm⁻². All experiments were performed in triplicate in two independent assays. In parallel experiments the biofilm mass was determined by Crystal violet assay as previously described.³¹ After biofilm formation, each disk was washed in a tube containing 10 mL of saline to remove unattached cells. Disks were then stained with 0.4% Crystal violet for 45 minutes, washed in 10 mL of saline and a picture was taken. In parallel experiments Crystal violet was detached by 95% ethanol and 200 μL of solution was read by microplate reader at 595 nm.

Kinetics of biofilm formation

For kinetics study, disks were sterilized and placed in a tube with 10 mL of sterile artificial saliva.³² Tubes were then placed at 37 °C for 120 days. Every seven days saliva was replaced. After 0, 7, 14, 28, 60 and 120 days 3 disks for each type were removed from saliva and used to determine the biofilm formation as described above.

Statistical analysis

The effect of incorporation of CHX on Candida albicans adhesion and biofilm formation were evaluated by t test. A value of P < 0.05 was considered significant.

Results and discussion

CHX loaded derivatized MCM-41 preparation and characterization

MCM-41 derivatization can be performed by two different approaches. The first is the co-condensation procedure, in which the functionalizing agent is appropriately mixed with the silica precursors and all the procedure is carried out in the same reaction vessel. The second is the post-synthesis grafting method, where the inorganic matrix already formed is reacted with the functionalizing agent usually in anhydrous conditions to yield the modified silica mesopores.³³

MCM-41 silanization was performed according to the post-synthesis procedure which assures that modifying agents are placed in the outer surface of the silica network, leading to a larger functionalization degree.³³ Silanized MCM-41 samples (Table 1) were characterized by TG and FT-IR analysis.

Table 1 Amount of silane attached on the MCM-41 surface determined by TG analysis and amount of loaded CHX determined by UV spectrophotometry and TG analysis

Compound	Silane chain attached on the silica (%)	Compound	CHX TG	(%) UV
MCM-41-acr-1	3.1	MCM-41-acr-1-CHX	10.2	9.9
MCM-41-acr-2	5.3	MCM-41-acr-2-CHX	10.3	9.6
MCM-41-acr-3	9.3	MCM-41-acr-3-CHX	10.8	9.7
MCM-41-acr-4	14.3	MCM-41-acr-4-CHX	9.8	10.1

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The amount of γ-MPS effectively attached to MCM-41 was quantitatively determined by TG analysis (not reported) and results are reported in Table 1.

FT-IR spectrum (Fig. 1) of MCM-41 showed the presence of a vibrational band at 3740 cm⁻¹, attributable to isolated terminal silanol groups, and a large band at 3550 cm⁻¹ due to germinal and terminal associate silanol groups.²⁰ The FT-IR spectra of silanized MCM-41 revealed many changes namely: (i) a remarkable decrease of the peak at 3740 cm⁻¹, due to the decrease of free silanols which condense with the hydrolyzed silane,^34,35 (ii) a band at 1636 cm⁻¹ due to the stretching vibration of the C=C bond in γ-MPS and (iii) the band at 1702 cm⁻¹ relative to the C=O stretching frequency.^34–37 These changes were the prove of the MCM-41 functionalization.

Fig. 1 FT-IR spectra of pristine and silanized MCM-41 (a.u. arbitrary units).

The CHX loading into the silanized MCM-41 samples was performed according to the solvent evaporation method as this presents many advantages in comparison to other loading procedures such as great reproducibility, short reaction time and employment of low solvent amount. Drug loading percentage of all samples is reported in Table 1.

In Fig. S-1,† the DSC thermal behaviors of CHX loaded silicates, CHX and the physical mixture MCM-41-acr-4/CHX are reported. CHX thermogram showed an endothermic peak at 154 °C due to its melting. CHX loaded MCM-41 derivatives did not show any peak attributable to drug melting, proving the lack of CHX in crystalline form, as previously observed.³⁸

The study of the change of the specific surface area, pore diameter and volume of MCM-41 before and after derivatization and CHX loading furnished important information and this aspect was evaluated by the nitrogen adsorption–desorption at 77 K measurements recorded for all MCM-41-silanized derivatives (MCM-41-acr-1, MCM-41-acr-2, MCM-41-acr-3 and MCM-41-acr-4) and the corresponding CHX loaded samples (MCM-41-acr-1-CHX, MCM-41-acr-2-CHX, MCM-41-acr-3-CHX, MCM-41-acr-4-CHX).

In Fig. S-2† the nitrogen adsorption and desorption at 77 K isotherms and the calculated mesopore size distribution for the pristine MCM-41 are showed. Typical adsorption and desorption isotherms were obtained. The calculated B.E.T. specific surface area is 740 m² g⁻¹ and the mesopore volume was 0.70 cm³ g⁻¹. A maximum in the pore size distribution around 3.1 nm of pore width was found.

In Fig. 2 the nitrogen adsorption and desorption at 77 K isotherms and the pore size distributions of MCM-41 with different percentage of derivatization are reported. The calculated B.E.T. specific surface area for these samples together with the pore volume and the pore size corresponding to the maximum in the pore size distribution curves are reported in Table 2.

Fig. 2 Nitrogen adsorption and desorption at 77 K isotherms (A) and the pore size distributions (B) of the silanized MCM-41 samples MCM-41-acr-1 (a), MCM-41-acr-2 (b), MCM-41-acr-3 (c) and MCM-41-acr-4 (d).

Table 2 B.E.T. surface area, mesopore volume and pore diameter for the precursor MCM-41 and silanized MCM-41 samples

	MCM-41	MCM-41-acr-1	MCM-41-acr-2	MCM-41-acr-3	MCM-41-acr-4
a a = pore diameter (nm) corresponding to the maximum in the pore size distribution curve.
B.E.T. surface area (m^2 g^−1)	740	744	734	745	654
Mesopore volume (cm^3 g^−1)	0.70	0.62	0.59	0.62	0.36
a^a	3.1	3.0	3.0	3.0	2.9

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The calculated B.E.T. surface area and pore volume values of the MCM-41-acr-1, MCM-41-acr-2 and MCM-41-acr-3 were similar. The isotherm curves, pore size distribution and B.E.T. surface area of these samples were also similar with those of the MCM-41 precursor while only a little decrease in mesopore volume was detected. This suggested that, for a percentage of derivatization at least until to 9.4% w/w, the silanols placed in the external surface were involved in the bond with the γ-MPS while those placed in the pore walls were less interested. When the γ-MPS content increased to 14.3% w/w (MCM-41-acr-4), a decrease of the adsorbed nitrogen in the isotherm curves, B.E.T. surface area and mesopore volume were found, while only a very little decrease in the pore size was detected. In this case, the higher γ-MPS loading caused the obstruction of a part of the pores of MCM-41.

As regards the CHX loaded MCM-41 derivatives, their isotherms and calculated mesopore size distribution are showed in Fig. 3.

Fig. 3 Nitrogen adsorption and desorption at 77 K isotherms (A) and the pore size distributions (B) of MCM-41-acr-1-CHX (a), MCM-41-acr-2-CHX (b), MCM-41-acr-3-CHX (c) and MCM-41-acr-4-CHX (d).

The calculated B.E.T. specific surface area, pore volume and pore size corresponding to the maximum in the pore size distribution curves of CHX loaded samples are reported in Table 3.

Table 3 B.E.T. surface area, mesopore volume and pore diameter for CHX loaded silanized MCM-41 samples

	MCM-41-acr-1-CHX	MCM-41-acr-2-CHX	MCM-41-acr-3-CHX	MCM-41-acr-4-CHX
a a = pore diameter corresponding to the maximum in the pore size distribution curve.
B.E.T. surface area (m^2 g^−1)	550	494	511	369
Mesopore volume (cm^3 g^−1)	0.37	0.35	0.30	0.19
a^a (nm)	2.88	2.74	2.47	—

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Consequently, to the CHX loading, reductions in the B.E.T. specific surface area and mesopore volume were found and thus sorption of CHX in mesopores could be assumed. Moreover, a decrease of the maximum position in the pore size distribution was also found in all the loaded samples. In the case of MCM-41-acr-4-CHX sample the pore sizes were less than 3.0 nm without evident maximum in the distribution curve. Considering that the density of the crystalline pure CHX is 1.39 g cm⁻³ (from http://www.guidechem.com/cas-55/55-56-1.htm) the specific volume of the adsorbed CHX could be evaluated assuming the same packing for both crystalline and adsorbed CHX. Taking into account the adsorption of the CHX into MCM-41-acr-1, it results that 1.000 g of loaded sample contains 0.095 g of adsorbed CHX and 1.000 − 0.095 = 0.905 g of the original MCM-41-acr-1 corresponding to 0.62 × 0.905 = 0.56 cm³ g⁻¹ of accessible mesopore volume. The measured mesopore volume reported in Table 3 (0.37 cm³ g⁻¹) indicated that the reduction in mesopore volume was higher than that corresponding to the adsorbed pure CHX (i.e. 0.095/1.39 = 0.068 cm³). Similar results were obtained applying these calculations to the other samples. This seemed to indicate that the CHX molecules were adsorbed in mesopores, which were partially occluded.

Composite characterization

First, the preparation of composites with 10% of CHX loaded silanized MCM-41 was attempted but the mixture obtained before polymerization was too hard and not homogeneous. Thus, the 10% silicate concentration was not investigated and disks containing 5% of CHX loaded silanized MCM-41 were prepared. Then, a group of disks containing CHX was prepared for comparison. All disks, with the exception of disk 1, have a CHX content of 0.5%. For comparison a disk containing only silanized MCM-41 was prepared. Composition of the specimen is reported in Table 4.

Table 4 Composition of the disks

Sample	CHX (0.02 g) or CHX loaded silanized MCM-41 (0.18 g, 5%)	Polymer powder (g)	Paladon liquid (mL)
Disk 1	—	2.5	1
Disk 2	CHX	2.48	1
Disk 3	MCM-41-acr-1-CHX	2.32	1
Disk 4	MCM-41-acr-2-CHX	2.32	1
Disk 5	MCM-41-acr-3-CHX	2.32	1
Disk 6	MCM-41-acr-4-CHX	2.32	1
Disk 7	MCM-41-acr-4	2.32	1

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FT-IT spectra showing the peaks detected of DC determination are reported in Fig. 4, whereas DC values are shown in Table 5.

Fig. 4 FT-IR spectra of the unpolimerized mixtures and the corresponding composites.

Table 5 Conversion degree (DC) values of the prepared composites

Resin/composites	DC (%)
Disk 1	99
Disk 2	80
Disk 3	94
Disk 4	75
Disk 5	70
Disk 6	56

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The highest DC value (99%) was obtained for disk 1, made of Paladon® 65, whereas disk 2, which contained crystalline CHX, presented a lower DC value (80%), confirming, as expected, that the presence of the drug caused a negative effect on the polymerization process.³⁹ Also disks containing CHX masked inside the pore of silanized MCM-41 (disks 4–6) showed a decrease of the DC in comparison to Paladon®, and this negative effect was dramatic for disks containing high derivatization. Disk 3 containing CHX loaded into MCM-41 with the lowest percentage of silanization showed a good DC. Results here described show that the best percentage derivatization, as concerns the conversion degree, is 3.1%.

CHX release from different samples

The results of CHX release from disks 2–6 are reported in Fig. 5. Analyzing the release profiles, it can be highlighted that CHX was very slowly released from all disks and reached almost 6% for disk 2 after 78 days, versus less than 4% for other disks. Thus CHX release was faster from disk 2 containing free CHX than from the other disks.

Fig. 5 CHX release from disks.

The CHX release from the disk 2 presented a burst effect and then a slower release. This behavior is in agreement with other studies that reported a similar burst effect of polymer-containing drugs⁴⁰ due to the drug molecules present on the outer surface of the disks. Then slow release can be explained with water diffusion into the matrix and dissolution of drug particles upon contact. Once dissolved, drug molecules diffuse out through the interconnecting pores. While drug solubilization and diffusion take place, the released drug molecules leave empty pores that can favor drug diffusion.

Disks 3 and 4 too presented a burst effect followed by a slower release in comparison to disk 2. CHX release from disk 5 and disk 6 showed a lag time of 1 and 2 days respectively, then the drug was gradually released. In the case of disks 3–6 containing the siliceous filler, the drug release is due to two steps: the first step is the water diffusion inside the disks, as described for disk 2, the second step is the water diffusion inside the narrow mesopores of MCM-41 in which the drug molecules are located. Finally, drug diffusion across the interconnecting pores can take place.

As concerns the silanized composites (disks 3–6), disks 3 and 4 showed a burst effect whereas disks 5 and 6 showed a lag time before CHX release and a slower drug release. This different behavior can be explained with the increasing percentage of silanization from disks 3 to disk 6. In fact, disks containing MCM-41 with higher silanization, notwithstanding the lower DC, could have a closer network surrounding the silicate and the presence of narrower interconnecting pores. This could slow down the drug diffusion through the composite.

Candida adhesion assay and biofilm formation on disks 1–6

Disk 1, constituted by Paladon® resin was easily colonized by C. albicans. Colonization of tooth surface is initiated by microbial adhesion to the salivary pellicle, after this step, adherent microbes produced a complex matrix that consists in polysaccharide and extracellular nucleic acid.⁴¹ In a first series of experiments, the ability of Candida albicans yeast cells to adhere to composites after pre-treatment with pooled human saliva was evaluated after 90 min.

The adhesion of Candida biofilm on the disk is mainly related to the presence of CHX on the disk surface. Results reported in Fig. 6, after 90 min of treatment, showed that disk 2 resulted the most active in inhibiting Candida adhesion. This was an evidence of the presence of CHX molecules on the disk surface. Disks 3 and 5 only partially inhibited the Candida adhesion and no activity was detected for disks 4 and 6. It has to be highlighted that CHX in disks containing the filler is mainly present inside the mesopores of the silicates and not on the disk surface and this can explain the less antibiofilm adherence inhibition for these disks in comparison to disk 2.

Fig. 6 Candida albicans adhesion to acrylic resin disks containing CHX after 90 min of treatment. Histograms represent means and standard deviation of three different determinations. *P < 0.05; **P < 0.01.

A second series of experiments were performed to evaluate the formation of Candida biofilm after 72 h of incubation. As shown in Fig. 7 disks 3, 4 and 5 showed a significant reduction (P < 0.01) of live cells in biofilm attached to the resin. This effect is due to the slow diffusion of CHX molecules towards the disk surface following the prolonged contact with the water.

Fig. 7 Candida albicans biofilm on acrylic resin disks containing CHX. Histograms represent means and standard deviation of nine different determinations. *P < 0.05; **P < 0.01 (disks containing CHX versus disk 1 without CHX). Picture represents disks stained by Crystal violet assay.

Disk 2 had the best antibiofilm activity. Disk 6 did not show a significant reduction of the biofilm formation after 72 h and this is in agreement with the drug in vitro release test, which showed the highest lag time in CHX release. An anti-Candida effect due to the presence of uncured monomers and dimers leached in the culture medium was excluded as the disk with the lowest conversion degree (disk 6), and thus with the highest amount of uncured monomers and dimers resulted the less active disk in inhibiting Candida biofilm formation.

The biofilm mass was determined by Crystal violet assay and results obtained by this test overlapped with data obtained by CFU determination. One representative picture for each sample is reported in Fig. 7.

The effect of the time on the antibiofilm effect after immersion of disks in artificial saliva is summarized in Fig. 8. All disks were sterilized and submerged in artificial saliva for different time periods. Artificial saliva was replaced weekly. Disk 2 was able to reduce the biofilm formation on resin starting from the beginning of the test until day 14. After 14 days it did not show any effect, and this maybe because CHX concentration on disk surface decreased following to the higher CHX release. As concerns the other disks, even if at the beginning they showed a weaker antibiofilm activity, disks 3, 4 and 5 were still able to reduce the biofilm formation after 60 days. In particular disks 3 showed a significant reduced number of CFU cm⁻² until 120 days, allowing a inhibition of Candida for a longer time in comparison to disk 2 containing free CHX.

Fig. 8 Candida albicans biofilm on acrylic composite disks containing CHX after immersion in artificial saliva. Data represent means of three different determinations. *P < 0.05; **P < 0.01 (disks containing CHX versus disk 1 without CHX).

Conclusions

Because of the large number of oral candidiasis associated with the biofilm formation due to the use of acrylic resin and composite dental prostheses, it is desirable to develop materials able to reduce the adherence of biofilms and the microorganism proliferation. Thus, an acrylic composite loaded with CHX was successfully prepared by using MCM-41 properly functionalized as a filler. Among the prepared composites interesting results were obtained with the composite containing MCM-41 with the lowest silane derivatization. This composite (disk 3) showed good polymerization degree (ca. 94%), not far from the polymerization degree of the reference resin. Moreover, in in vitro Candida adhesion and biofilm formation assays disk 3 resulted effective and it showed anti-biofilm activity from the beginning of the test until 120 days. On the other hand, the reference resin loaded with CHX (disk 2) showed a lower polymerization degree. Its biofilm adherence and formation inhibition was the best at the beginning of the experience but decreased during the test. In conclusion the good results obtained make CHX loaded silanized MCM-41 a promising filler for dental materials and worthy of further studies.

References

1. C. Koch, R. Bürgers and S. Hahnel, Gerodontology, 2013, 30, 309–313.
2. A. S. Kagermeier-Callaway, B. Willershausen, T. Frank and E. Stender, Int. Dent. J., 2000, 50, 79–85.
3. S. Neethirajan, M. A. Clond and A. Vogt, J. Biomed. Nanotechnol., 2014, 10, 2806–2827.
4. A. Cochis, L. Fracchia, M. G. Martinotti and L. Rimondini, Oral Surg., Oral Med., Oral Pathol., 2012, 113, 755–761.
5. P. Curtois, Biomaterials – Physica and Chemistry, R. Pignatello ED, IBNS 978-953-307-418-4, http://www.intechopen.com/books/biomaterials-physics-and-chemistry/candida-biofilms-on-oral-biomaterials.
6. K. D. Jandt and B. W. Sigusch, Dent. Mater., 2009, 25, 1001–1006.
7. S. Ryalat, R. Darwish and W. Amin, Ther. Clin. Risk Manage., 2011, 7, 219–225.
8. P. D. Riggs, M. Braden and M. Patel, Biomaterials, 2000, 21, 345–351.
9. D. Leung, D. A. Spratt, J. Pratten, K. Gulabilava, N. J. Mordan and A. M. Young, Biomaterials, 2005, 26, 7145–7153.
10. K. Kuroki, T. Hayashi, K. Sato, T. Asai, M. Okano and Y. Kominami, et al., Dent. Mater. J., 2010, 29, 277–285.
11. N. Salim, J. Satterthwaite, R. Rautemaa and N. Silikas, Dent. Mater. J., 2012, 31, 1008–1013.
12. S. Imazato, Y. Kinomoto, H. Tarumi, S. Ebisu and F. R. Tay, Dent. Mater., 2003, 19, 313–319.
13. L. Z. Bing, L. Bois, B. Grosgogeat, F. Chassagneux, F. Toche, R. Chiriac, N. Pradelle-Plasse, B. Gardiola, P. Colon and A. Brioude, Microporous Mesoporous Mater., 2013, 175, 1–7.
14. S. P. Samuel, S. Li, I. Mukherjee, Y. Guo, A. C. Patel, G. Baran and Y. Wei, Dent. Mater., 2009, 25, 296–301.
15. S. Praveen, Z. F. Sun, J. G. Xu, A. Patel, Y. Wei, R. Ranade and G. Baran, Mol. Cryst. Liq. Cryst., 2006, 448, 223–231.
16. G. Øye, J. Sjöblom and M. Stöcker, Adv. Colloid Interface Sci., 2001, 89–90, 439–466.
17. J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T. W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Higgins and J. L. Schlenker, J. Am. Chem. Soc., 1992, 114, 10834–10843.
18. M. Vallet-Regi, A. Rámila, R. O. del Real and J. Pérez-Pariente, J. Mater. Chem., 2001, 13, 308–311.
19. V. Ambrogi, Properties and Development, in Comprehensive Guide for Mesoporous Materials, Nova Science Publishers, Hauppauge NY (USA), 2015, vol. 3, ch. 1, pp. 1–18.
20. V. Ambrogi, L. Perioli, F. Marmottini, O. Accorsi, C. Pagano, M. Ricci and C. Rossi, Microporous Mesoporous Mater., 2008, 113, 445–452.
21. V. Ambrogi, L. Perioli, F. Marmottini, S. Giovagnoli, M. Esposito and C. Rossi, Eur. J. Pharm. Sci., 2007, 32, 216–222.
22. V. Ambrogi, L. Perioli, C. Pagano, L. Latterini, F. Marmottini, M. Ricci and C. Rossi, Microporous Mesoporous Mater., 2012, 147, 343–349.
23. http://heraeus-kulzer-us.com/media/webmedia_local/media/msds___dfu/dfu/Paladon_65_DFU_0112–2.pdf.
24. S. Brunauer, P. H. Emmet and E. Teller, J. Am. Chem. Soc., 1938, 60, 309–319.
25. J. Choma, M. Jaroniec, W. Burakiewicz-Mortka and M. Kloske, Appl. Surf. Sci., 2002, 196, 216–223.
26. G. Schulz-Ekloff, J. Rathousky and A. Zukal, Microporous Mesoporous Mater., 1999, 27, 273–285.
27. N. Wang, Q. Fang, J. Zhang, E. Chen and X. Zhang, Mater. Sci. Eng., A, 2011, 528, 3321–3325.
28. V. Migliorini Urban, A. L. Machado, C. E. Vergani, É. Gouveia Jorge, L. P. Serejo dos Santos, E. R. Leite and S. V. Canevarolo, Mater. Res., 2007, 10, 191–197.
29. E. K. Viljanen, L. V. J. Lassila, M. Skrifvars and P. K. Vallittu, Dent. Mater., 2005, 21, 172–177.
30. A. F. Wady, A. L. Machado, V. Zucolotto, C. A. Zamperini, E. Berni and C. E. Vergani, J. Appl. Microbiol., 2012, 112, 1163–1172.
31. L. Peng, J. DeSousa, Z. Su, B. M. Novak, A. A. Nevzorov, E. R. Garland and C. Melander, Chem. Commun., 2011, 47, 4896–4898.
32. M. R. C. Marques, R. Loebenberg and M. Almukainzi, Dissolution Technol., 2011, 18, 15–28,  DOI:10.14227/DT180311P15.
33. M. Vallet-Regi, F. Balas, M. Colilla and M. Manzano, Solid State Sci., 2007, 9, 768–776.
34. I. D. Sideridou and M. M. Karabela, Dent. Mater., 2009, 25, 1315–1324.
35. K. J. Söderholm and S. W. Shang, J. Dent. Res., 1993, 72, 1050–1054.
36. F. Bauer, H. Ernst, U. Decker, M. Findeisen, H.-J. Glasel, H. Langguth, E. Hartmann, R. Mehnert and C. Peuker, Macromol. Chem. Phys., 2000, 201, 2654–2659.
37. H.-J. Glasel, F. Bauer, H. Ernst, M. Findeisen, E. Hartmann, H. Langguth, R. Mehnert and R. Schuber, Macromol. Chem. Phys., 2000, 201, 2765–2770.
38. V. Ambrogi, L. Perioli, F. Marmottini, M. Moretti, E. Lollini and C. Rossi, J. Pharm. Sci. Innovation, 2009, 4, 156–164.
39. S. Pallan, M. V. Furtado Araujo, R. Cilli and A. Prakki, Dent. Mater., 2012, 28, 687–694.
40. A. Karmaker, A. Prasad and N. K. Sarkar, J. Mater. Sci.: Mater. Med., 2007, 18, 1157–1162.
41. N. S. Jakubovics and P. E. Kolenbrander, Oral Dis., 2010, 16, 729–739.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra11876j

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