In vivo biocompatibility of silicon dioxide nanofilm used as antimicrobial agent on acrylic surface

Abstract

the focus ofthis study was to testthe hypothesisthatthere would be no difference betweenthe biocompatibility of silicon dioxide nanofilms used as antimicrobial agents. Sixty male Wistar rats were divided into 4 groups (n=15): Group C (Control,Polyethylene), Group AR (Acrylic Resin), Group NP (Acrylic Resin coated with NP-Liquid), Group BG (Acrylic Resin coated with Bacterlon).the animals were sacrificed with 7,15 and 30 days and tissues analyzed as regardsthe events of inflammatory infiltrate, edema, necrosis, granulation tissue, mutinucleated giant cells, fibroblasts and collagen. Kruskal-Wallis and Dunn tests was used (P<0.05). Intense inflammatory infiltrate was shown mainly in Groups BG and AR, with significant difference from Control Group inthe time interval of 7days (P=0.004). Necrosis demonstrated significant difference between Group BG and Control Group (P<0.05) inthe time intervals of 7 days. For collagen fibers,there was significant difference betweenthe Control Group and Groups AR and BG inthe time interval of 7 days (P=0.006), and between BG and Control Groups inthe time intervals of 15 days (P=0.010).the hypothesis was rejected. Bacterlon demonstratedthe lowest level, and NP-Liquid Glassthe highest level of tissue compatibility, and best cell repair.the coating with NP-Liquid Glass was demonstrated to be highly promising for clinical use.

Key words
biomaterials; biocompatibility; dentistry; inflammation

INTRODUCTION

The entire surface ofthe dental environment is subject to colonization by microorganisms (Mutters et al. 2014). In addition tothis medium being subject tothe proliferation of microorganisms, other surfaces could also serve as a medium for biofilm formation, such as acrylic resin dental prostheses and removable orthodontic appliances (Olsen & Birkeland 1977). Studies (Compagnoni et al. 2014, Sesma et al. 2005) were conducted forthe purpose of obtaining an inhibition of initial biofilm formation onthese surfaces, either with antimicrobial agents incorporated intothe methacrylate (Compagnoni et al. 2014), or by applying coatings or glazing substances (Sesma et al. 2005). However, some aspects ofthe results were not satisfactory, such as rapid depletion ofthe antimicrobial agent, or bythe formation of cracks and or failures inthe coatings (Compagnoni et al. 2014, Sesma et al. 2005).

The silicon dioxide based nanofilm (SiO₂) has been used as a bioprotective substance for surfaces susceptible to colonization by microorganisms, by means of an invisible <250nmthick layer of coating,that could be associated withthe inclusion of antimicrobial agents (Jurgens & Schwindt 2007).the SiO₂ nanofilm has characteristics such as hydrophobia, oleophobia, a cationic nature, resistance to acids, excellent flexibility, antibacterial and antifungal properties, resistance to abrasion and corrosion, which inhibitthe adhesion and proliferation of microorganisms (Jurgens & Schwindt 2007, Wolinsky 2006). Inthis context,the SiO₂ nanofilm has been suggested as an antimicrobial bioprotective coating for dental devices made of acrylic resin (Vilar 2014).

However,the silicon nanoparticles have great power of penetration intothe systemic circulation (Napierska et al. 2010), and authors (Montanaro et al. 2005) have suggested studies to be conducted withthe purpose of verifying possible biological damage, by means of cytotoxicity and biocompatibility tests at cellular level (Montanaro et al. 2005).

Consideringthe lack of in vivo biocompatibility studies ofthe SiO₂ nanofilm (Napierska et al. 2010),the focus ofthis double-blind randomized study was to testthe hypothesisthatthere would be no difference histological betweenthe biocompatibility of conventional SiO₂ (Montanaro et al. 2005) nanofilm-NP Liquid glass, andthe enriched with antibacterial substances-Bacterlon, used as inhibitors of cellular growth onthe acrylic surface.

MATERIALS AND METHODS

Animal model and experimental groups

Forthis study 60 adult male Wistar rats were used, weighing between 250-350g, belonging tothe Vivarium ofthe Federal University of Campina Grande, UFCG.the animals were divided into 4 experimental groups (n=15, per group): Group C (Control, Polyethylene), Group AR (Acrylic Resin), Group NP (Acrylic Resin coated with NP-Liquid Glass), Group BG (Acrylic Resin coated with Bacterlon Glass) (Table I).the animal experiment was approved bythe Ethics Committee on Animal Research, CSTR/UFCG/No.102016.

Table I
Silicon dioxide nanofilms tested inthis study.

Groups	Material	Composition	Manufacturer	Lot
AR	Acrylic Resin	Powder: Poly(methyl methacrylate), benzoyl- peroxide, biocompatible pigmentsLiquid: Methyl methacrylate Monomer, Inhibitor	OrtoCril, VIPI, Pirassununga, SP,Brazil	1253
NP	NP-Liquid Glass	Silicon Dioxide	Nanopool GmbH (Schwalbach, Germany)	A-LGPL/ 141009
BG	Bacterlon Glass	Silicon Dioxide, Chitosan, Trichloro-2’-hydroxydiphenyl Ether (Triclosan) and quaternary ammonium salts	Nanopool GmbH (Schwalbach, Germany)	A-BLPL/ 140603

The acrylic resin samples were manipulated bythe mass technique, in accordance withthe manufacturer’s instructions (Dos Santos et al. 2013), withthe powder and liquid manipulated inthe ratio of 3:1.the samples were fabricated by using a condensation silicon mold (Zhermack, Badia Polesine, RO, Italy), with an internal diameter of 6mm by x 2mm height.

Polymerization occurred within a resin polymerizer M-1000 (EDG, São Carlos, SP, Brazil), at a temperature of 20°C, pressure of 25psi (1.75 kg/cm²), for a period of 15 minutes, in accordance withthe manufacturer’s instructions. When excess material was present, it was progressively removed manually using abrasive papers with 150, 400, 600 and 800 grits. To obtainthe desired dimensions,the specimens were measured with a precision pachymeter (123m-150; Starrett, SP, Brazil). Allthe samples were fabricated and polished bythe same operator and stored in deionized water at 37ºC (Millipore, Bedford, MA, USA) for 24 hours to allowthe superficial residual monomers to be released (Rocha Filho et al. 2007). Afterthis, both sides ofthe acrylic samples were previously sterilized with ultraviolet light (Labconco, Kansas City, MO, USA) for 30 minutes (Dos Santos et al. 2012).

Groups NP and BG were coated with SiO₂ nanofilm NP-Liquid Glass and Bacterlon respectively. To ensurethat allthe sample walls would come into contact withthe coating, each sample was placed in contact with 3 mL ofthe respective nanofilm. After 30 seconds, each sample was carefully removed and stored at ambient temperature for 24 hours to guaranteethatthe nanofilm had been correctly formed and dried.the samples were kept in a laminar flow chamber withthe purpose of avoiding any type of contamination (Vilar 2014).

Inthis study, polyethylene discs withthe same dimensions asthose ofthe acrylic resin discs were used as controls ofthe trauma induced, andthese were washed with deionized water and autoclaved at a temperature of 120⁰C for 20 minutes. After fabricating allthe samples,the rats were anesthetized with intraperitoneal injection of sodiumthiopental (50mg/kg, Cristália, SP, Brazil). Afterthis, trichotomy was performed with razor blades inthe dorsal region of each animal (4x4cm).

For antisepsis ofthe operative field 4% chlorhexidine gluconate was used. Onthe midline, equidistant fromthe insertion ofthe animal’s tale and head, two incisions approximately 8mm long was made using a No.15 scalpel blade.

Withthe aid of a blunt tipped scissors,the subcutaneous tissue was laterally parted to promote a tunnel inthe lateral direction, forming two surgical recesses, each approximately 18mm deep. Each rat received two implants ofthe samples.

Afterthe materials were implanted,the surgical recesses were sutured with a 4.0 suturethread (Ethicon, Jonhson&Jonhson, SP, Brazil) and afterthe procedure,the animals received an injection of sodium dipyrone (0.3ml/100g, Sanofi-Aventis, Suzano, SP, Brazil).

Allthe procedures inthis study were performed in compliance withthe guidelines ofthe Canadian Council on Animal Care (1981).the animals were kept in individual cages and under adequate conditions with balanced rations and water ad libitum. After time intervals of 7, 15 and 30 days,the animals were anesthetized to obtain excisional biopsies ofthe implant area, including sufficient normal surrounding tissue, afterwardsthe rats were sacrificed bythe cervical dislocation technique.

Biocompatibility

After fixation in 4% formaldehyde (Milony solution) for 24 hours,the samples were embedded in paraffin to obtain serial histologic cuts 6 µmthick, and stained with hematoxylin and eosin.the inflammatory reaction induced bythe samples was evaluated by using a light microscope (BX40; Olympus, Hamburg, Germany) at 100, 200 and 400x magnifications. Double blind examination was performed by two calibrated examiners (kappa=0.85).

The histological sections were made transversal-perpendicular direction tothe operated area. For each sample ofthe study, five sections representative ofthe histological condition ofthe tissue adjacent tothe implanted materials were analyzed.the cellular events with regard tothe presence of inflammatory infiltrate, edema, necrosis, granulation tissue, mutinucleated giant cells, young fibroblasts and collagen, were awarded points according tothe following scores: 1–absent (when absent inthe tissue); 2–scarce (when scarcely present,or in very small groups), 3–moderate (when densely present,or in some groups) and 4–intense (when found inthe entire field,or present in large numbers).the histological sections were randomly assessed at 5 different points ofthe tissue, adjacent tothe specimen, when all five sections ofthe tissue showedthe same histological condition. Scores: 1, absent (5.00); 2, scarce (10.00); 3, moderate (15.00); and 4, intense (20.00).these values representthe mean of scores ofthe sum of five representative histological sections ofthe tissue evaluated (n=5, per group).

Statistical analysis

The data were tabulated and analyzed inthe statistical program GraphPad Prism version 5.0 (San Diego, CA, USA).the statistical method was chosen based onthe model of distribution and variance of data evaluated bythe Kolmogorov- Smirnov and Levene tests, respectively.the results ofthe cellular events did not present normal distribution,therefore,they were submitted tothe Kruskal-Wallis non parametric test, followed bythe Dunn test to determinethe differences amongthe groups(P<.05).

RESULTS

Inthe initial period, intense inflammatory infiltrate was shown mainly in Groups BG and AR, with significant difference from Control Group inthe time interval of 7days (P=0.004). Intense inflammatory infiltrate was also demonstrated in Group BG inthe time interval of 15 days, with significant difference fromthe Control Group (P=0.003). No significant difference was demonstrated betweenthe Groups evaluated inthe time interval of 30 days (P=0.454) (Table II) (Figure 1a-i).

Table II
Mean ofthe scores attributed tothe materials, afterthe time intervals of 7, 15 and 30 days, forthe seven conditions evaluated.

Condition	Time, Days	Groups				P*
		AR	NP	BG	C
Inflammatory infiltrate	7	18.75A	12.50AB	20.00A	10.00B	.004
	15	15.00AB	10.00AB	16.25A	8.75B	.003
	30	7.50	6.25	8.75	6.25	.454
Edema	7	8.75AB	7.50AB	10.00A	5.00B	.039
	15	6.25	5.00	6.25	5.00	.543
	30	5.00	5.00	5.00	5.00	1.000
Necrosis	7	7.50AB	5.00B	10.00A	5.00B	.011
	15	5.00	5.00	7.50	5.00	.092
	30	5.00	5.00	5.00	5.00	1.000
Granulation tissue	7	15.00A	10.00AB	15.00A	8.75B	.002
	15	10.00	8.75	10.00	7.50	.237
	30	5.00	5.00	5.00	5.00	1.000
Mutinucleated giant cells	7	12.50AB	7.50AB	15.00A	6.25B	.010
	15	12.50A	6.25AB	12.50A	5.00B	.008
	30	7.50	5.00	8.75	5.00	.061
Young fibroblasts	7	7.50	10.00	7.50	12.50	.071
	15	11.25A	15.00AB	11.25A	18.75B	.012
	30	15.00	13.75	16.25	12.50	.237
Collagen	7	5.00A	8.75AB	5.00A	10.00B	.006
	15	12.50AB	16.25AB	10.00A	18.75B	.010
	30	16.25	18.75	16.25	20.00	.092

• a For each sample ofthe study, five representative sections ofthe histological condition ofthe tissue were analyzed, when all five sections ofthe tissue showedthe same histological condition. Scores: 1, absent (5.00); 2, scarce (10.00); 3, moderate (15.00); and 4, intense (20.00). *P indicates nonparametric Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. A or B Means followed bythe same single letter do not express statistically significant difference (P>.05). AB Means followed by different letters express statistically significant difference (P<.05).

Figure 1
Photomicrographs of histological samples. a) 7 days after implantation, Group AR: intense inflammatory infiltrate (III), granulation tissue (GT) and congested blood vessels (CV) (HE, 100X magnification, scale:100µm). b) 7 days after implantation, Group BG: intense inflammatory infiltrate (III), congested blood vessels (CV) and presence of extracellular fluid (ECF) (HE, 100X magnification, scale:100µm). c) 7 days after implantation, Group C: cavity surrounded by moderate inflammatory infiltrate (MII) and granulation tissue reaction (GT) (HE, 100X magnification, scale:100µm). d) 15 days after implantation, Group AR: presence of moderate inflammatory infiltrate (MII) adjacent tothe cavity, congested vessels (CV) and presence of multinucleated giant cells (MGC) (HE, 400X magnification, scale:25µm). e) 15 days after implantation, Group BG; presence of moderate inflammatory infiltrate (MII), small areas of necrosis (AN) adjacent tothe cavity, and presence of multinucleated giant cells (MGC) (HE, 400X magnification, scale:25µm). f) 15 days after implantation, Group C; slight mononunclear inflammatory infiltrate, presence of young ovoid and fusiform fibroblasts (YF), congested blood vessels (CV) and deposition of collagen fibers (DCF), (HE, 400X magnification, scale:25µm). g) 30 days after implantation, Group AR; cavity surrounded bythick band with deposition of collagen fibers (DCF), young ovoid and fusiform fibroblasts (YF), presence of congested blood vessels (CV) and slight chronic inflammatory infiltrate (HE, 200X magnification, scale: 50µm). h) 30 days after implantation, Group BG; cavity surrounded by collagenization band with deposition of collagen fibers (DCF) sometimes disposed in parallel bands and sometimes in varied bands, young ovoid and fusiform fibroblasts (YF), presence of slight chronic inflammatory infiltrate, congested blood vessels (CV) and multinucleated giant cells (MGC) (HE, 200X magnification, scale:50µm). i) 30 days after implantation, Group C; deposition of collagen fibers (DCF) disposed in parallel bands involvingthe area ofthe cavity, young fibroblasts (YF) and presence of blood vessels (CV), (HE, 200X magnification, scale:50µm). Area of polyethylene tube implant (PT).

Circulatory changes (edema) and tissue degeneration (necrosis) were significant, only inthe time interval of 7 days, with significant difference between Group BG and Control Group (P<0.05); and between Groups BG and NP forthe presence of necrosis (P=0.011). However, some necrotic events of little significance were still observed in Group BG inthe time intervals of 15 days (P>0.05) (Figure 1e). Granulation tissue was shown to be densely present in Groups AR and BG inthe time interval of 7 days, with significant difference fromthe Control Group (P=0.002), and subsequentlythere was a reduction inthis cellular event, without statistical differences betweenthe Groups inthe time intervals of 15 (P=0.237) and 30 days (P=1.000).

There were more mutinucleated giant cells present in Group BG inthe time intervals of 7 days (P=0.010). Groups AR and BG demonstrated a similar condition forthe presence ofthese cells inthe time intervals of 15 days, with significant difference fromthe Control Group (P=0.008) (Figure 1d-e).

Inthe tissue repair events, Groups AR and BG demonstratedthe smallest quantity of young fibroblasts amongthe experimental Groups inthe time intervals of 7 and 15 days, with significant difference only betweenthe Control Group and Groups AR and BG (P=0.012) inthe time intervals of 15 days.the quantity of collagen fibers increased overthe course ofthe experimental time intervals evaluated (Figure 1g-i), andthere was significant difference betweenthe Control Group and Groups AR and BG inthe time interval of 7 days (P=0.006), and between BG and Control Groups inthe time intervals of 15 days (P=0.010) (Table II).

DISCUSSION

Microorganisms, such asthe acidogenic (Streptococcus mutans, Streptococcus gordinii) and proteolytic bacteria (Porphyronomas gingivalis, Prevotella intermedia) and fungi, such as Candida albicans, are frequently found inthe oral cavity (Piovano 1999).thus it is common to find large quantities of biofilm on dental and orthodontic appliances, leading to inflammation, stomatitis and erythema inthe mucosa (Lacerda-Santos et al. 2014, Mesquita et al. 2017, Uzunoglu et al. 2014). To preventthese sources of harm, it is important to implement control and biosafety measures whose efficiency has been proved.

Inthis context, SiO₂-based nanofilm has appeared as an alternative for providing complementary biosafety care (Vilar 2014). Silica nanoparticles,the main component of nanofilm, have demonstrated good results both in vitro, and in vivo (Fruijtier-Polloth 2012), and more serious inflammations have been observed only on exposure to high levels of inhalation or injection of nanoparticles (Johnston et al. 2000, Sayes et al. 2010).

Inthe present study,the inflammatory infiltrate found was shown to be significantly greater inthe Acrylic Resin (AR) and Bacterlon Glass (BG) Groups inthe time interval of 7 days. Inthe period of 15 days, significant infiltrate continued to persist in Group BG.these results suggested a greater capacity for aggression againstthe Bacterlon tissues, due tothe presence/or concentration ofthe antimicrobial substances such as chitosan, triclosan and quaternary ammonia salts present inthis nanofilm. Studies (Thanou et al. 2001, Wedmore et al. 2006) have reportedthat chitosan is considered compatible withthe tissues, however, changes made inthis drug to adjust formulations may have a direct influence on its capacity to cause tissue damage and influence inflammatory events (Kean &thanou 2010). In addition tothis, authors (Lyman & Furia 1969) have demonstratedthe toxicity of triclosan and its influence onthe cellular events in epithelial cells of human gingival cells (Zuckerbraun et al. 1998). In conjunction, quaternary ammonia has been shown to be cytotoxic tothe mitochondria of epithelial cells (Inacio et al. 2013) and to havethe potential to increase cell damage.

There was significant presence of edema and necrosis only in Group BG inthe time interval of 7 days, which demonstrated a capacity for initial aggression, butthat was not persistent inthe subsequent time intervals. Although irreversible cell damage and subsequent cell death inthe short term have been found,the histological evaluations suggested a low capacity of Bacterlon to lead to significant damage inthe long term. Onthe other hand, granulation tissue was shown to be densely present in Groups AR and BG inthe time interval of 7 days, a conditionthat did not persist significantly inthe following time intervals.

Multinucleated giant cells were shown to be more present in Group BG inthe time interval of 7 days;their significant presence persisted in Group BG, and was also demonstrated in Group AR inthe time interval of 15 days, which corresponded tothe organism’s response to phagocytingthe foreign bodythroughthese cells (Lacerda-Santos et al. 2016). In Group BG,the presence/or concentration of antimicrobial substances present inthis material could be related tothe increase inthese cells; for Group AR,the presence of multinucleated giant cells suggestedthatthis could be linked tothe toxicity ofthe acrylic resin due tothe presence of residual monomer released after its polymerization, withthe degradation of its components overthe course of time (Ivković et al. 2013).

In tissue repair,there was growing presence of young fibroblaststhat was not significant amongthe materials, except for Groups AR and BGthat demonstrated a lower number of young fibroblasts inthe time interval of 15 days.the presence of collagen was shown to be lower in Groups AR and BG inthe time interval of 7 days;this suggestedthatthe tissue toxicity of Groups AR and BG hadthe capacity to interfere inthe production of collagen and non-collagen protein, as has been seen in other substances (Ivković et al. 2013).

The tissue toxicity in Group AR was directly related tothe release of monomer residues (Dos Santos et al. 2013, Ivković et al. 2013), and Bacterlon withthe presence of its antimicrobial agents, which corroboratedthe findings of a study (Vilar 2014)that demonstrated its cellular toxicity in vitro associated with its potential to inhibitthe growth of bacteria ofthe S.mutans and S.aureus types, and fungi ofthe C. albicans type (Vilar 2014).

Studies (Thanou et al. 2001, Wedmore et al. 2006) have reportedthatthe changes made inthe density ofthe molecular load of chitosan and its route of administration are directly related to its toxicity (Thanou et al. 2001, Wedmore et al. 2006) andthe type of cell affected (Kean &thanou 2010). Triclosan (Zuckerbraun et al. 1998), in tests for verifying cellular apoptosis, has also presented considerable cellular damage, particularly whenthe time of exposure to it and its concentration were increased (Jirasripongpun et al. 2008). Added tothese factorsthe quaternary ammonia salts have demonstrated cytotoxicity in epithelial cells (Inacio et al. 2013), although other authors (Grabińska-Sota 2011) have found no strong evidence of risks to human health. In conjunction,these agents have been suggested to have significant potential for increasing initial cellular damage and to slow downthe time of response for tissue repair.

The SiO₂ nanofilm withoutthe presence of antimicrobial agent, NP-Liquid Glass, demonstrated significantly promising results in comparison with Bacterlon, with a higher level of tissue biocompatibility for allthe cellular events evaluated, which corroboratedthe finding of studies (Vilar 2014)that evaluatedthe in vitro cellular cytotoxicity ofthese nanofilms in L929 fibroblasts; moreover, NP-Liquid Glass demonstratedthe potential to inhibitthe growth of bacteria ofthe S. aureus type (Vilar 2014). Authors (DeLoid et al. 2017, Landgraf et al. 2017) have demonstrated a low cytotoxicity profile of SiO₂ nanopartícles, however,they have pointed outthat variations inthe preparation/ formulation ofthe nanopartícles may have a significant influence onthe results of cellular tests (Landgraf et al. 2017), sothat further analyses and standardizations are necessary (DeLoid et al. 2017).

The biocompatibility presented by NP-Liquid Glass demonstrated clinical applicability promising as a bioprotective coating with low deleterious risk tothe individual. However, it is suggestedthe use of Bacterlon only in small acrylic devices for individuals who are not allergic tothe antimicrobial substances present inthis nanofilm.the elevated cytotoxicity of Bacterlon has been suggested to be caused bythe high antibacterial power of its components, however, whenthis toxicity has ceased,the material could continue to be an excellent option for coating surfacesthat come into contact with live beings (Vilar 2014). Forthe purpose of confirmingthis hypothesis, long-term studies about cytotoxicity/biocompatibility are necessary to evaluate until whenthe material generates cellular/tissue damage, and up to what point its antimicrobial capacity remains active.

CONCLUSIONS

The hypothesis was rejected. Bacterlon demonstratedthe lowest level, and NP-Liquid Glassthe highest level of tissue compatibility, and best cell repair.the coating with NP-Liquid Glass was demonstrated to be highly promising for use in clinical practice.

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Publication Dates

• Publication in this collection
  17 Apr 2020
• Date of issue
  2020

History

• Received
  23 Oct 2018
• Accepted
  11 Oct 2019