polymethylmethacrylate dermal fillers: evaluation of the systemic toxicity in rats

YIJOM-2718; No of Pages 6

Research Paper

Cosmetic Surgery

Int. J. Oral Maxillofac. Surg. 2013; xxx: xxx–xxxhttp://dx.doi.org/10.1016/j.ijom.2013.06.009, available online at http://www.sciencedirect.com

Polymethylmethacrylate dermalfillers: evaluation of thesystemic toxicity in ratsC. C. G. Medeiros, R. L. Borghetti, N. Nicoletti, V. D. da Silva, K. Cherubini, F. G.Salum, M. A. Z. de Figueiredo: Polymethylmethacrylate dermal fillers: evaluation ofthe systemic toxicity in rats. Int. J. Oral Maxillofac. Surg. 2013; xxx: xxx–xxx.# 2013 International Association of Oral and Maxillofacial Surgeons. Published byElsevier Ltd. All rights reserved.

Abstract. This study evaluated local and systemic reactions after an intravascularinjection of polymethylmethacrylate (PMMA) at two concentrations in a murinemodel. Thirty rats were divided equally into three groups: 2% PMMA, 30% PMMA,and a control group (normal saline only injection). The filler was injected into theranine vein. The rats were sedated at 7 and 90 days and a clinical evaluationperformed. After euthanasia, the right lung, liver, and right kidney were removed,weighed, and microscopically analyzed. The submandibular lymph nodes andtongue were removed and examined microscopically. Serum was subjected to liverand kidney function tests. No groups showed clinical alterations. Microspheres werenot observed at any distant organ. Two samples from the 2% PMMA group showeda local inflammatory response at day 7 and another two samples from the 30%PMMA group at day 90. The group injected with 30% PMMA presented higherlevels of alanine aminotransferase (P = 0.047) after 90 days when compared withthe other groups. The data obtained in this study demonstrate that intravascularinjections of PMMA fillers show potential health risks such as chronic inflammationat the implantation site.

Please cite this article in press as: Medeiros CCG, et al. Polymethylmethacrylate dermal fil

rats, Int J Oral Maxillofac Surg (2013), http://dx.doi.org/10.1016/j.ijom.2013.06.009

0901-5027/000001+06 $36.00/0 # 2013 International Association of Oral and Maxillofacial Surge

C. C. G. Medeiros1, R. L. Borghetti1,N. Nicoletti2, V. D. da Silva3,K. Cherubini1, F. G. Salum1,M. A. Z. de Figueiredo1

1Division of Oral Medicine, PUCRS, PontificalCatholic University of Rio Grande do Sul,Porto Alegre, RS, Brazil; 2PostgraduateProgram of Cellular and Molecular Biology,PUCRS, Pontifical Catholic University of RioGrande do Sul, Porto Alegre, RS, Brazil;3Division of Pathology, PUCRS, PontificalCatholic University of Rio Grande do Sul,Porto Alegre, RS, Brazil

Key words: oral medicine; polymethylmetha-crylate; adverse effects; toxicology.

Accepted for publication 17 June 2013

Introduction

The use of facial fillers is one of theoptions to minimize the signs of facialageing and to correct skin imperfections.They are increasingly preferred over con-ventional plastic surgery due to their lowfinancial cost and painless and noninva-sive application.1–5

Each type of soft-tissue filler has its owntechnique of implantation, period of per-manence in the tissues, and adverse effectsthat are inherent to its composition.1,2

Several chemical compounds can triggertoxic reactions, and there are differentways to evaluate the possible outcomes.6

Facial fillers containing polymethyl-methacrylate (PMMA) can be classifiedas non-biodegradable or permanent, sincethey consist of non-absorbable micro-spheres that are suspended in an aqueouscarrier or bovine collagen.7 Permanentfillers must be carefully monitored dueto the severity of possible complicationsthat can occur late and can present adifficult or impossible resolution.8–10

The term ‘migration’ is found in theliterature to define the displacement ofPMMA microspheres in two differentways: when they are injected into bloodvessels,11,12 or when they are transportedthrough phagocytosis.13

PMMA has a specific implantation tech-nique and must not be injected into bloodvessels, to avoid particle migration and toachieve a satisfactory result. However,there are several published case reportsof disastrous local complications due toinjections being intravascular or very

lers: evaluation of the systemic toxicity in

ons. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ijom.2013.06.009



2 Medeiros et al.


close to a blood vessel.14–17 A study on thesystemic manifestations caused by intra-venous injection of PMMA fillers inhumans or animals has not been publishedpreviously.

The aim of this study was to identify andanalyze fast-occurring as well as latentsystemic and local tissue reactions andthe occurrence of particle migration todistant organs after intravascular injectionof PMMA at different concentrations in amurine model. It is necessary to betterunderstand the mechanism of a possibletoxicity induced by this material in orderto improve the safety of this treatment.The ventral surface of the rat tongue wasselected as the site of injection owing tothe fact that it is less vulnerable to trau-matic factors18 and also as it is highlyvascularized, comprising a plexus com-posed of a superficial vascular networkand the ranine veins.19

Materials and methods

This research was initiated after approvalfrom the scientific and ethics committee

Please cite this article in press as: Medeiros

rats, Int J Oral Maxillofac Surg (2013), htt

Fig. 1. Phase contrast microscopy showing PMM100�, (C) and (D) 400�.

and the ethics committee for animal use;the procedures were carried out in accor-dance with institutional guidelines for ani-mal care and use.

The material used for this experimentwas PMMA microspheres suspended inhydroxyethylcellulose gel. Two extremesof concentration (2% and 30%) were cho-sen to compare the possible systemic toxiceffects when the product is injected into ablood vessel. The materials were injectedat the beginning of the experiment. Clin-ical and histological evaluations were per-formed at 7 and 90 days (Fig. 1).

Animal model

The sample comprised 30 female Wistarrats (Rattus norvegicus) from the samebreeder. Each animal weighed about200 g and had an average age of 2 monthsat the beginning of the experiment. Theywere obtained from the animal facility ofthe State Foundation of Production andHealth Research (FEPPS), Porto Alegre,Brazil. Animals were individually num-bered on the tail and housed in plastic

CCG, et al. Polymethylmethacrylate dermal fil

p://dx.doi.org/10.1016/j.ijom.2013.06.009

A microspheres in the tongue with different man

cages placed in ventilated racks at a tem-perature of 22 8C with a 12-h light–darkcycle. Animals were fed a standard diet ofrat chow (Nuvilab-CR1) and given waterad libitum. The animals were randomlyallocated to one of three groups, accordingto the treatment received: group 1(n = 10): 2% PMMA; group 2 (n = 10):30% PMMA; and group 3 (n = 10): con-trol, 0.9% NaCl; each group was dividedequally into experimental periods of 7days (A) and 90 days (B).

Anaesthesia

Initially, each rat was weighed on a digitalscale (Urano model UDI 2500/0.5; Urano,Canoas, RS, Brazil) to calculate the dose ofanaesthetic. This procedure was performedwith an intraperitoneal injection of a mix-ture of 20 mg/ml xylazine hydrochloride(0.05 ml/100 g), a sedative, analgesic, andmuscle relaxant, with 50 mg/ml ketaminehydrochloride (0.1 ml/100 g), an anaes-thetic for veterinary use. Animals fromthe respective groups, chosen randomly,were successively anesthetized.4,18


ipulations and magnifications: (A) 200�, (B)


Evaluation of systemic toxicity of PMMA 3


Injection of the material

When sedation was evident, the animalwas placed on a surgical table in supineposition and its paws tied with the use ofelastic strips. The tongue was pulled outwith tweezers to expose the ventral tongueregion. Using a disposable insulin syringe(1/2 in. 26G; 13 mm � 0.45 mm), 0.05 mlof each material (group 1: 2% PMMA;group 2: 30% PMMA; and group 3: 0.9%NaCl) was injected into the right raninevein (lingual vein), which is located lateralto the lingual frenulum.19 The needle wasinclined as parallel as possible to themucosa, with the bevel facing up.

Clinical evaluation

Before euthanasia, the animals wereweighed and sedated in such a way thatthe tongues could be clinically evaluated.The clinical examination sought to iden-tify possible tissue alterations, such asswelling, nodules, ulceration, necrosis,and/or suppuration.

Euthanasia

After injection of the material, animalswere sacrificed and terminally bled bycardiac puncture at the respective moni-toring periods.

Sample processing

Immediately before euthanasia, the ani-mals were anesthetized via isofluraneinhalation. After anaesthesia, a thoracot-omy was performed and blood samplescollected without haemolysis by cardiacpuncture. Samples were centrifuged at8000 rpm at 4 8C to separate serum. Theanimals underwent necropsy; their rightkidney, right lung, and liver wereremoved, weighed, and microscopicallyanalyzed. The submandibular lymphnodes and the tongue were removed andexamined microscopically. Sample fixa-tion was carried out with the use of 10%neutral-buffered formalin for a minimumof 24 h. Samples of the tongue, lymphnodes, right lung, right kidney, and liverwere sectioned longitudinally into twofragments. The inclusion was performedso that the edge of the sample had its longaxis parallel to the paraffin block section.For each specimen, there was one histo-logical section of 6 mm per slide, whichwas stained with haematoxylin and eosin(H&E).

Histological evaluation

The analysis of the slides was carried outin a blinded fashion (using masks for all



the evaluated slides) by an examinerwho had previously been calibrated.The analysis of histological sectionswas conducted in the pathology unitusing a biological microscope (ZeissAxioskop 40, Carl Zeiss, Jena, Ger-many) coupled to a camera (CoolSNAP-Pro cf, Media Cybernetics,Bethesda, MD, USA) connected to aDell Optiplex GX620 computer (Dell,Round Rock, TX, USA), at 100�,200�, and 400� magnifications. Imagesof the tongue samples were transferredto Image-Pro Plus, version 4.5.1 (MediaCybernetics, Inc.; 2005). Some slideswere analyzed with a fixed stage micro-scope for electrophysiological research(ECLIPSE FN1, Nikon, Tokyo, Japan)for phase contrast microscopy.

Inflammatory reaction of the tongue

Histological evaluation of the tongue wasperformed with an analysis of the presenceor absence of lymphocytes, plasma cells,macrophages, giant cells, neutrophils,eosinophils, oedema, and hyperemia, at200� magnification.

Migration

The microscopic evaluation of migrationwas based on the presence or absence ofmicrospheres in the submandibular lymphnode, right kidney, right lung, and liver ofeach animal.

Histological scoring of liver injury

Liver tissue was scored for histologicalnecrosis and inflammation according tothe modified activity index (HAI) grad-ing.20,21

Histological scoring of kidney and lung

injury

Kidney and lung were evaluated based onthe presence or absence of an inflamma-tory reaction.

Serum analysis

Serum was subjected to liver and kidneyfunction tests. Aspartate aminotransfer-ase (AST) and alanine aminotransferase(ALT) activity in serum was quantita-tively measured using a VITROS DT60Chemistry System (Ortho Clinical Diag-nostics, Rochester, NY, USA) by akinetic reaction with multiple measuringpoints. The serum creatinine level wasdetermined by two-point kinetic method(Labtest Diagnostics, Lagoa Santa, MG,


p://dx.doi.org/10.1016/j.ijom.2013.06.009

Brazil). Samples were processed in anautomatic biochemical system (VitrosFusion 5.1; Ortho Clinical Diagnostics,Rochester, NY, USA), using specificreagents for each test. The tests wereperformed in the clinical laboratory.

Statistical analysis

All data were tabulated and analyzedusing SPSS 17 software (SPSS Inc., Chi-cago, IL, USA), with a two-way analysisof variance (ANOVA) parametric test,complemented by the Bonferroni correc-tion test, at a significance level of 5%.

Results

Clinical alterations

No group showed any clinical alteration at7 and 90 days.

Migration

Microspheres were not observed in anydistant organs (lung, liver, and kidney) orlymph nodes.

Inflammatory reaction of the tongue

Two samples from the 2% PMMA groupshowed moderate inflammation at 7 days,while another two samples from the 30%PMMA group also showed inflammationat 90 days—one being moderate with aninfiltrate of mononuclear cells and sparseneutrophils and eosinophils, and the otherbeing severe with a lymphoplasmacyticinfiltrate and granuloma formation. Mas-tocytosis was observed during the histo-logical evaluation of several samples ofthe tongue, lymph nodes, and submandib-ular gland at all times of the study (Fig. 2).

Liver function tests

At 90 days, there was a significant differ-ence in ALT levels (P = 0.047) betweenthe group injected with 30% PMMA andthe other two groups (2% PMMA andcontrol) (Fig. 3). The levels of AST werenot significantly different between thegroups at any time of the study.

Creatinine

At 90 days, all groups showed a significantincrease in creatinine levels (P = 0.001),but there was no difference between thePMMA groups and the control group(Fig. 3).



4 Medeiros et al.


Fig. 2. Mastocytosis was evident after staining with toluidine blue.

Organ weight evaluation

Changes in liver weight did not differsignificantly between the groups at anyobservation time. Lung weight was statis-tically different between the PMMAgroups (P = 0.048) only at 7 days, whilethe kidney weight increase was associatedwith the animals’ gain in body weightduring the study period (Fig. 4).



Fig. 4. The lung weight of samples from the 2%days (A). There was a proportional increase in

Fig. 3. Serum levels of alanine aminotransferasecontrol group over time, while for creatinine th

Discussion

The lack of published studies on the pos-sible toxic effects that could be caused bythe systemic distribution of PMMA micro-spheres prompted this study. Since it hasbeen reported widely that needle mispla-cement due to professional inexperiencemay result in an intravascular injectionand because it has been suggested by some


p://dx.doi.org/10.1016/j.ijom.2013.06.009

and 30% PMMA groups was statistically higher

kidney weight in all groups at 90 days (B).

(ALT) (A) and creatinine (B). There was a differene differences were only due to time.

authors that PMMA particles couldmigrate to distant organs, this study eval-uated the aspects involved in a possiblesystemic reaction to this material using amurine model. The experiments were con-ducted through the methodology used inmost toxicity studies.

Kidney and liver are considered targetorgans for drug metabolism. The kidneyplays an important role in the filtration ofplasma and metabolic homeostasis, andtherefore medications can induce somelevel of toxicity by interacting directlywith the organ’s structure or indirectlyinducing some damage to the electrolytebalance or blood circulation.22,23 Serumcreatinine levels should be measured toidentify possible toxic effects on the kid-ney that are influenced by the followingfactors: drug pharmacokinetics, dose tol-erance, drug interactions, physiologicalvariations, pathological factors, andgenetic factors.24

Due to the major role of liver in themetabolism and excretion of drugs andxenobiotics, it will frequently manifestsigns of toxicity. In the USA, the principalcause of death due to acute liver failure isdrug-induced liver injury.23,25 Serumlevels of AST and ALT are consideredan important sign of hepatocellular


(P = 0.048) than that of the control group at 7

ce in ALT between the PMMA groups and the


Evaluation of systemic toxicity of PMMA 5


damage.26 ALT is present basically in theliver, while AST is also involved in otherorgans.

Although histological signs of liver andkidney damage were not found in thisstudy, the level of ALT activity in serumwas higher in the group injected with 2%PMMA than in the other two groups at 7days, while at 90 days the 30% PMMAgroup showed higher levels of ALT. Thisindicates that for each group, the effect ofPMMA differed from the control groupover time. Previous studies have demon-strated that ALT levels higher than 51 U/lcould be considered a sign of hepatotoxi-city.27 However, no difference was foundwhen the AST levels were assessed, and incomparing creatinine levels, only timeseemed to account for increases. This factcould be related to the ageing process inthe animals.

In toxicology experiments, a compari-son of the organ weights of the treatedgroup and control group is conventionallyused to evaluate the toxic effect of the testproduct, and changes may represent asensitive indicator of chemically inducedtoxicity. However, organ weight data mustbe interpreted with caution and consideredin combination with clinical and histo-pathology findings, since the changesmay not be related to the treatment.28,29

Our evaluation of weight changes was notable to determine whether they were theresult of toxicity induced by PMMA sincea correlation between clinical and histo-pathology findings was not established,despite the statistically different lungweight data between the PMMA groupsand the control group.

The systemic distribution of the injectedmaterial was evaluated microscopically inall experimental groups on the basis of thepresence or absence of an inflammatoryresponse and/or PMMA particles in theright submandibular lymph node, lung,kidney, and liver of each animal. Micro-spheres were not observed in this study atany distant organ and inflammation wasalso not present at any distant organ. Thesubmandibular glands of some animalswere removed by accident and surpris-ingly microspheres were found in twospecimens of the 2% PMMA group after7 days. Our findings are in line with mostprevious studies, indicating that PMMAmicrospheres are not able to migrate todistant organs, in contradiction to the find-ings of Rosa and de Macedo,30 whoobserved hepatic and renal inflammationafter PMMA injection in mouse ears.However, the microspheres were observedin the submandibular gland, so the resultscould indicate a different situation if the



organs were entirely examined, or if otherorgans were also assessed, such as theheart and spleen. Surprisingly throm-boembolic complications were notdetected at any observation time, and alsounexpected is the fact that inflammation inthe tongue was observed in only fouranimals, two in the 2% PMMA group at7 days and two animals in the 30% PMMAgroup at 90 days, with just one case beinga granuloma formation. These findingsdiffer from the results obtained by Moureet al.4; the authors performed an injectionat the ventral surface of the tongue of agreater amount of PMMA (0.07 ml) andfound an intense inflammatory reactionwith polymorphonuclear neutrophil infil-tration after 7 days and chronic inflamma-tion with moderate intensity at 60 and 90days, represented by the presence of alymphoplasmacytic infiltrate and giantcells next to the PMMA particles at allobservation times.

Mastocytosis was observed during thehistological evaluation of several samplesof the tongue and lymph nodes at all timesof the study. In an attempt to establish acorrelation between their presence and apossible foreign-body reaction that couldbe mediated by this type of cell, mast cellswere scored in the tongue samples. How-ever, the statistical analysis demonstratedthat mast cells decreased in number in thetongue samples after 90 days and thatthere was no difference between PMMAgroups and the control group. It can beassumed that since mast cells are found inlarger quantities in acute wounds, partici-pating in the inflammatory reaction,angiogenesis, and extracellular matrixreabsorption and remodeling,31,32 eventhe trauma caused by needle insertioncould result in an increased presence ofmast cells at the injection site. It shouldalso be highlighted that recent studieshave demonstrated that plasma estradiollevels increase in the presence of mastcells in several tissues in a dose-dependentway,33 and since female rats wereemployed in our study, we have to con-sider the possibility that our findings maybe related to changes in the animals’estrous cycle.

Considering our methods and the dataobtained in this study, intravascular injec-tion of PMMA fillers shows potentialhealth risks, such as chronic inflammationat the implantation site. Furthermore, thegroup injected with 30% PMMA had ele-vated ALT levels at 90 days and bothPMMA groups showed increased lungweight at 7 days, indicating a non-specificsystemic reaction. Considering our presentresults and the lack of previous studies


p://dx.doi.org/10.1016/j.ijom.2013.06.009

examining the possible reactions of sys-temic toxicity and genotoxicity caused bycosmetic fillers containing PMMA, furtherresearch on this issue is warranted.

Funding

None.

Competing interests

None declared.

Ethical approval

This study was carried out with theapproval of the Scientific and Ethics Com-mittee (protocol 0060/11) and the EthicsCommittee for Animal Use (protocol 11/00261) of the Pontifical Catholic Univer-sity of Rio Grande do Sul, PUCRS, Brazil.

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Address:Clarissa Castro Galvao MedeirosServico de Estomatologia do Hospital SaoLucasPUCRSPontifıcia Universidade Catolica do RioGrande do SulAv. Ipiranga6690 – 28 andar/sala 231Porto AlegreRSCEP 90610-000BrazilTel: +55 51 33203254;Fax: +55 51 33203254E-mails: [email protected],[email protected]


































































































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mailto:[email protected]


polymethylmethacrylate dermal fillers: evaluation of the systemic toxicity in rats

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