Abstract
This study evaluated local and systemic reactions after an intravascular injection of polymethylmethacrylate (PMMA) at two concentrations in a murine model. 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 the ranine vein. The rats were sedated at 7 and 90 days and a clinical evaluation performed. After euthanasia, the right lung, liver, and right kidney were removed, weighed, and microscopically analyzed. The submandibular lymph nodes and tongue were removed and examined microscopically. Serum was subjected to liver and kidney function tests. No groups showed clinical alterations. Microspheres were not observed at any distant organ. Two samples from the 2% PMMA group showed a 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 higher levels of alanine aminotransferase ( P = 0.047) after 90 days when compared with the other groups. The data obtained in this study demonstrate that intravascular injections of PMMA fillers show potential health risks such as chronic inflammation at the implantation site.
Introduction
The use of facial fillers is one of the options to minimize the signs of facial ageing and to correct skin imperfections. They are increasingly preferred over conventional plastic surgery due to their low financial cost and painless and noninvasive application.
Each type of soft-tissue filler has its own technique of implantation, period of permanence in the tissues, and adverse effects that are inherent to its composition. Several chemical compounds can trigger toxic reactions, and there are different ways to evaluate the possible outcomes.
Facial fillers containing polymethylmethacrylate (PMMA) can be classified as non-biodegradable or permanent, since they consist of non-absorbable microspheres that are suspended in an aqueous carrier or bovine collagen. Permanent fillers must be carefully monitored due to the severity of possible complications that can occur late and can present a difficult or impossible resolution.
The term ‘migration’ is found in the literature to define the displacement of PMMA microspheres in two different ways: when they are injected into blood vessels, or when they are transported through phagocytosis.
PMMA has a specific implantation technique and must not be injected into blood vessels, to avoid particle migration and to achieve a satisfactory result. However, there are several published case reports of disastrous local complications due to injections being intravascular or very close to a blood vessel. A study on the systemic manifestations caused by intravenous injection of PMMA fillers in humans or animals has not been published previously.
The aim of this study was to identify and analyze fast-occurring as well as latent systemic and local tissue reactions and the occurrence of particle migration to distant organs after intravascular injection of PMMA at different concentrations in a murine model. It is necessary to better understand the mechanism of a possible toxicity induced by this material in order to improve the safety of this treatment. The ventral surface of the rat tongue was selected as the site of injection owing to the fact that it is less vulnerable to traumatic factors and also as it is highly vascularized, comprising a plexus composed of a superficial vascular network and the ranine veins.
Materials and methods
This research was initiated after approval from the scientific and ethics committee and the ethics committee for animal use; the procedures were carried out in accordance with institutional guidelines for animal care and use.
The material used for this experiment was PMMA microspheres suspended in hydroxyethylcellulose gel. Two extremes of concentration (2% and 30%) were chosen to compare the possible systemic toxic effects when the product is injected into a blood vessel. The materials were injected at the beginning of the experiment. Clinical and histological evaluations were performed at 7 and 90 days ( Fig. 1 ).
Animal model
The sample comprised 30 female Wistar rats ( Rattus norvegicus ) from the same breeder. Each animal weighed about 200 g and had an average age of 2 months at the beginning of the experiment. They were obtained from the animal facility of the State Foundation of Production and Health Research (FEPPS), Porto Alegre, Brazil. Animals were individually numbered on the tail and housed in plastic cages placed in ventilated racks at a temperature of 22 °C with a 12-h light–dark cycle. Animals were fed a standard diet of rat chow (Nuvilab-CR1) and given water ad libitum. The animals were randomly allocated to one of three groups, according to the treatment received: group 1 ( n = 10): 2% PMMA; group 2 ( n = 10): 30% PMMA; and group 3 ( n = 10): control, 0.9% NaCl; each group was divided equally into experimental periods of 7 days (A) and 90 days (B).
Anaesthesia
Initially, each rat was weighed on a digital scale (Urano model UDI 2500/0.5; Urano, Canoas, RS, Brazil) to calculate the dose of anaesthetic. This procedure was performed with an intraperitoneal injection of a mixture of 20 mg/ml xylazine hydrochloride (0.05 ml/100 g), a sedative, analgesic, and muscle relaxant, with 50 mg/ml ketamine hydrochloride (0.1 ml/100 g), an anaesthetic for veterinary use. Animals from the respective groups, chosen randomly, were successively anesthetized.
Injection of the material
When sedation was evident, the animal was placed on a surgical table in supine position and its paws tied with the use of elastic strips. The tongue was pulled out with tweezers to expose the ventral tongue region. Using a disposable insulin syringe (1/2 in. 26G; 13 mm × 0.45 mm), 0.05 ml of each material (group 1: 2% PMMA; group 2: 30% PMMA; and group 3: 0.9% NaCl) was injected into the right ranine vein (lingual vein), which is located lateral to the lingual frenulum. The needle was inclined as parallel as possible to the mucosa, with the bevel facing up.
Clinical evaluation
Before euthanasia, the animals were weighed and sedated in such a way that the tongues could be clinically evaluated. The clinical examination sought to identify possible tissue alterations, such as swelling, nodules, ulceration, necrosis, and/or suppuration.
Euthanasia
After injection of the material, animals were sacrificed and terminally bled by cardiac puncture at the respective monitoring periods.
Sample processing
Immediately before euthanasia, the animals were anesthetized via isoflurane inhalation. After anaesthesia, a thoracotomy was performed and blood samples collected without haemolysis by cardiac puncture. Samples were centrifuged at 8000 rpm at 4 °C to separate serum. The animals underwent necropsy; their right kidney, right lung, and liver were removed, weighed, and microscopically analyzed. The submandibular lymph nodes and the tongue were removed and examined microscopically. Sample fixation was carried out with the use of 10% neutral-buffered formalin for a minimum of 24 h. Samples of the tongue, lymph nodes, right lung, right kidney, and liver were sectioned longitudinally into two fragments. The inclusion was performed so that the edge of the sample had its long axis parallel to the paraffin block section. For each specimen, there was one histological section of 6 μm per slide, which was stained with haematoxylin and eosin (H&E).
Histological evaluation
The analysis of the slides was carried out in a blinded fashion (using masks for all the evaluated slides) by an examiner who had previously been calibrated. The analysis of histological sections was conducted in the pathology unit using a biological microscope (Zeiss Axioskop 40, Carl Zeiss, Jena, Germany) coupled to a camera (Cool SNAP-Pro cf, Media Cybernetics, Bethesda, MD, USA) connected to a Dell Optiplex GX620 computer (Dell, Round Rock, TX, USA), at 100×, 200×, and 400× magnifications. Images of the tongue samples were transferred to Image-Pro Plus, version 4.5.1 (Media Cybernetics, Inc.; 2005). Some slides were analyzed with a fixed stage microscope for electrophysiological research (ECLIPSE FN1, Nikon, Tokyo, Japan) for phase contrast microscopy.
Inflammatory reaction of the tongue
Histological evaluation of the tongue was performed with an analysis of the presence or absence of lymphocytes, plasma cells, macrophages, giant cells, neutrophils, eosinophils, oedema, and hyperemia, at 200× magnification.
Migration
The microscopic evaluation of migration was based on the presence or absence of microspheres in the submandibular lymph node, right kidney, right lung, and liver of each animal.
Histological scoring of liver injury
Liver tissue was scored for histological necrosis and inflammation according to the modified activity index (HAI) grading.
Histological scoring of kidney and lung injury
Kidney and lung were evaluated based on the presence or absence of an inflammatory reaction.
Serum analysis
Serum was subjected to liver and kidney function tests. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in serum was quantitatively measured using a VITROS DT60 Chemistry System (Ortho Clinical Diagnostics, Rochester, NY, USA) by a kinetic reaction with multiple measuring points. The serum creatinine level was determined by two-point kinetic method (Labtest Diagnostics, Lagoa Santa, MG, Brazil). Samples were processed in an automatic biochemical system (Vitros Fusion 5.1; Ortho Clinical Diagnostics, Rochester, NY, USA), using specific reagents for each test. The tests were performed in the clinical laboratory.
Statistical analysis
All data were tabulated and analyzed using SPSS 17 software (SPSS Inc., Chicago, IL, USA), with a two-way analysis of variance (ANOVA) parametric test, complemented by the Bonferroni correction test, at a significance level of 5%.
Materials and methods
This research was initiated after approval from the scientific and ethics committee and the ethics committee for animal use; the procedures were carried out in accordance with institutional guidelines for animal care and use.
The material used for this experiment was PMMA microspheres suspended in hydroxyethylcellulose gel. Two extremes of concentration (2% and 30%) were chosen to compare the possible systemic toxic effects when the product is injected into a blood vessel. The materials were injected at the beginning of the experiment. Clinical and histological evaluations were performed at 7 and 90 days ( Fig. 1 ).
Animal model
The sample comprised 30 female Wistar rats ( Rattus norvegicus ) from the same breeder. Each animal weighed about 200 g and had an average age of 2 months at the beginning of the experiment. They were obtained from the animal facility of the State Foundation of Production and Health Research (FEPPS), Porto Alegre, Brazil. Animals were individually numbered on the tail and housed in plastic cages placed in ventilated racks at a temperature of 22 °C with a 12-h light–dark cycle. Animals were fed a standard diet of rat chow (Nuvilab-CR1) and given water ad libitum. The animals were randomly allocated to one of three groups, according to the treatment received: group 1 ( n = 10): 2% PMMA; group 2 ( n = 10): 30% PMMA; and group 3 ( n = 10): control, 0.9% NaCl; each group was divided equally into experimental periods of 7 days (A) and 90 days (B).
Anaesthesia
Initially, each rat was weighed on a digital scale (Urano model UDI 2500/0.5; Urano, Canoas, RS, Brazil) to calculate the dose of anaesthetic. This procedure was performed with an intraperitoneal injection of a mixture of 20 mg/ml xylazine hydrochloride (0.05 ml/100 g), a sedative, analgesic, and muscle relaxant, with 50 mg/ml ketamine hydrochloride (0.1 ml/100 g), an anaesthetic for veterinary use. Animals from the respective groups, chosen randomly, were successively anesthetized.
Injection of the material
When sedation was evident, the animal was placed on a surgical table in supine position and its paws tied with the use of elastic strips. The tongue was pulled out with tweezers to expose the ventral tongue region. Using a disposable insulin syringe (1/2 in. 26G; 13 mm × 0.45 mm), 0.05 ml of each material (group 1: 2% PMMA; group 2: 30% PMMA; and group 3: 0.9% NaCl) was injected into the right ranine vein (lingual vein), which is located lateral to the lingual frenulum. The needle was inclined as parallel as possible to the mucosa, with the bevel facing up.
Clinical evaluation
Before euthanasia, the animals were weighed and sedated in such a way that the tongues could be clinically evaluated. The clinical examination sought to identify possible tissue alterations, such as swelling, nodules, ulceration, necrosis, and/or suppuration.
Euthanasia
After injection of the material, animals were sacrificed and terminally bled by cardiac puncture at the respective monitoring periods.
Sample processing
Immediately before euthanasia, the animals were anesthetized via isoflurane inhalation. After anaesthesia, a thoracotomy was performed and blood samples collected without haemolysis by cardiac puncture. Samples were centrifuged at 8000 rpm at 4 °C to separate serum. The animals underwent necropsy; their right kidney, right lung, and liver were removed, weighed, and microscopically analyzed. The submandibular lymph nodes and the tongue were removed and examined microscopically. Sample fixation was carried out with the use of 10% neutral-buffered formalin for a minimum of 24 h. Samples of the tongue, lymph nodes, right lung, right kidney, and liver were sectioned longitudinally into two fragments. The inclusion was performed so that the edge of the sample had its long axis parallel to the paraffin block section. For each specimen, there was one histological section of 6 μm per slide, which was stained with haematoxylin and eosin (H&E).
Histological evaluation
The analysis of the slides was carried out in a blinded fashion (using masks for all the evaluated slides) by an examiner who had previously been calibrated. The analysis of histological sections was conducted in the pathology unit using a biological microscope (Zeiss Axioskop 40, Carl Zeiss, Jena, Germany) coupled to a camera (Cool SNAP-Pro cf, Media Cybernetics, Bethesda, MD, USA) connected to a Dell Optiplex GX620 computer (Dell, Round Rock, TX, USA), at 100×, 200×, and 400× magnifications. Images of the tongue samples were transferred to Image-Pro Plus, version 4.5.1 (Media Cybernetics, Inc.; 2005). Some slides were analyzed with a fixed stage microscope for electrophysiological research (ECLIPSE FN1, Nikon, Tokyo, Japan) for phase contrast microscopy.
Inflammatory reaction of the tongue
Histological evaluation of the tongue was performed with an analysis of the presence or absence of lymphocytes, plasma cells, macrophages, giant cells, neutrophils, eosinophils, oedema, and hyperemia, at 200× magnification.
Migration
The microscopic evaluation of migration was based on the presence or absence of microspheres in the submandibular lymph node, right kidney, right lung, and liver of each animal.
Histological scoring of liver injury
Liver tissue was scored for histological necrosis and inflammation according to the modified activity index (HAI) grading.
Histological scoring of kidney and lung injury
Kidney and lung were evaluated based on the presence or absence of an inflammatory reaction.
Serum analysis
Serum was subjected to liver and kidney function tests. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in serum was quantitatively measured using a VITROS DT60 Chemistry System (Ortho Clinical Diagnostics, Rochester, NY, USA) by a kinetic reaction with multiple measuring points. The serum creatinine level was determined by two-point kinetic method (Labtest Diagnostics, Lagoa Santa, MG, Brazil). Samples were processed in an automatic biochemical system (Vitros Fusion 5.1; Ortho Clinical Diagnostics, Rochester, NY, USA), using specific reagents for each test. The tests were performed in the clinical laboratory.
Statistical analysis
All data were tabulated and analyzed using SPSS 17 software (SPSS Inc., Chicago, IL, USA), with a two-way analysis of variance (ANOVA) parametric test, complemented by the Bonferroni correction test, at a significance level of 5%.
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