Effect of propranolol on capsular reaction around silicone implants in guinea pigs 1

PURPOSE: To evaluate the effect of propranolol on capsular architecture around silicone implants by measuring the inflammation, capsular thickness, and collagen fiber density, using a guinea pig experimental model. METHODS: Thirty six adult male guinea pigs randomly divided into two groups (n=18) were used. Each one received a silicone implant with textured-surface. The capsular tissue around implants from untreated or treated animals with the beta-adrenoceptor antagonist propranolol (10 mg/kg, dissolved in daily water) were analyzed for inflammation by histological scoring, capsular thickness by computerized histometry, and collagen fibers type I and Type III density by picrosirius polarization at different time points (7, 14 or 21 days after silicone implantation). RESULTS: Propranolol treatment reduced inflammation and impaired capsular thickness and delayed collagen maturation around the textured implant. CONCLUSION: Propranolol reduces the risk of developing capsular contracture around silicone implants with textured surface.


Introduction
Concepts about beauty have increased the demand for breast implants.According to the International Society for Aesthetic Plastic Surgery (ISAPS), over 1.2 million procedures were performed worldwide exclusively for breast augmentation in 2011 1 .In Brazil, according to the Brazilian Society of Plastic Surgery (SBCP), this number is over 150.000 each year 2 .
Foreign body reaction often interferes with the longterm functionality and performance of implanted biomedical devices through fibrous capsule formation 3,4 .Both the wound healing and immune system have very complex interconnections and implanting biomaterials are likely to affect many of these pathways [3][4][5] .Despite silicone has being considered relatively inert, as any biomaterial implanted in the human body, its elicits this protective immune-inflammatory reaction from the organism, starting a rejection process that interferes with wound healing process and finish by encapsulating this foreign body too large to be eliminated by phagocytosis [6][7][8][9][10][11] .Ordinarily beneficial, in some cases such capsules become pathologically active, and undergo an adverse capsular contracture (ACC), leading to swelling, spherical constrictive fibrosis, distortion, hardness and pain in the breast, with physical and psychological distress [10][11][12][13][14] .
Histologically, breast implant capsules have been well characterized.Long time ago it was hypothesized that collagen production and deposition might be increased by inflammation in contracted capsules since there is histological evidence for increased lymphocytes, macrophages and multinuclear giant cells [6][7][8][12][13][14] . Also, he ratio of collagen type I:type III increases in rigid tissue compared with more distensible ones 15 .The management of ACC is a wellknown problem for plastic surgeons.ACC occurs in 0-74% of breast implant operations depending on the series, follow-up, implant shell and content, surface texture, presence of subclinical infection or accumulation of tissue fluids in the implant pocket, patient age, surgical technique and anatomical location of implants and others [9][10][11][12][13][14][15][16] .
Just as there is no universally accepted etiology, no single unified hypotheses that explain pathogenesis of ACC have been identified.Nowadays, capsular contracture etiology and its development seem to be multi-factorial 13 .Also, no effective treatments for adverse capsular contractures have been identified.Revision surgery for capsulotomy or capsulectomy with implant exchange are the standard treatments 14 .Unfortunately, these procedures are associated with a high risk of complications, recurrences included [11][12][13][14] .
A non-surgical therapy for ACC would be desirable; however, so far, no study has clearly demonstrated their effectiveness.
Researchers and surgeons have used antimicrobials agents, steroids, non-steroidal anti-inflammatory drugs, and more recently, leukotriene antagonists to skirmish contracture, with little success and often with collateral effects [16][17][18] .Propranolol is a non-selective betaadrenoceptor antagonist that have a well recognized antihypertensive and antiarrythmic action and has been widely used in cardiovascular disease, migraine and post-traumatic stress disorder 19,20 .Propranolol now is used as a first-line treatment in infantile hemangiomas when intervention is required [19][20][21] .Since propranolol's invention by Sir James Black and co-workers in 1960s 19 , it has been demonstrated that the peripheral nervous system has important participation in wound healing and that beta-blockers modulates stress-related injury, both wound healing and inflammation, facts that attracted the interest of numerous scholars and led to various hypotheses [19][20][21][22] .
Souza et al. 22 demonstrated that sympathetic denervation accelerates wound contraction but delays reepithelialization in rats.
Recently was shown that early propranolol administration to severely injured patients can improve bone marrow dysfunction 23 .There are also interactive effects between polymorphisms in the beta-adrenergic receptors and longitudinal changes in obesity, an inflammatory-stateprone disease 24 .The potential use of propranololol to curb fibrosis conditions, as like ACC, was well demonstrated by another study where beta-adrenoceptor blockade has antifibrotic effects in a murine model of nonsinusoidal liver fibrosis 25 .
Fibrogenesis is a mechanism of wound healing and repair.However, prolonged injury promotes deregulation of normal processes and results in extensive deposition of extracellular matrix (ECM) proteins and fibrosis 26 .Based in these facts and evidences, we hypothesized that propranolol could impairs capsular fibrosis surrounding silicone implants.To test this hypothesis, we conducted an experimental study to evaluate the effect of propranolol on capsular formation by measuring the capsule thickness, scoring inflammation and analyzing deposition of collagen type I and type III using an animal model.Thirty-six mature male guinea pigs weighing 520 to 750g were used.Animals were maintained in adequate standard conditions at the LABEX.Each animal was kept in a standard polypropylene box labeled with information on each animal.These animals were randomly divided in two groups (n=18).In the dorsum of each animal a textured silicone gel-filled mini-prosthesis, with pore size of 0.05-0.25 mm, shell-shaped and round-based, 2.2 cm in diameter, and volume of two mL was implanted subcutaneously.

Methods
All mini-prosthesis were produced and donated by SILIMED™ Company,São Paulo, Brazil) (Figure 1).A group of 18 animals was administered 10 mg/kg of propranolol hydrochloride (PROP) orally in 150 ml of daily water.The control group (CTRL)was administered daily the same volume of tap water.Each group of animals was split into three subgroups of six animals Results were evaluated at 7, 14 and 21 days after implantation surgery.Following samples collection the animals were killed by an overdose of anesthetics.

Surgical procedure
All surgical procedures were performed under general anesthesia by intraperitoneal injection of 100 mg/kg of ketamine hydrochloride (Vetanarcol™, 50 mg/ml, König S/A, Sao Paulo, Brazil) associated to 10 mg/kg of xylazine (Kensol™, 20 mg/ml, König S/A, Sao Paulo, Brazil).Under anesthesia, each animal was placed in the prone position.After epilation of the dorsum from tail up to cervical region, the skin was prepared and draped in a sterile fashion.A surgical transverse incision of 2 cm, symmetrically crossing the midline above lumbar region was done 3-cm above the hind limbs.Following, a tunnel was built above the panniculus carnosus, up to the shoulder blades.The implant (Figure 1) was inserted through the incision and placed on a pocket between scapulas, approximately 3 cm apart from the incision (Figure 2).Incision was closed using 4-0 polyamide interrupted sutures.

Follow-up and macroscopic assessment
After surgical procedures, animals were placed on a warm environment at 25ºC until complete recovering from anesthesia, and afterwards re-allocated to their respective boxes.Animals were daily observed for their level of activity, respiratory distress, diarrhea, starvation or weight gain, and refusal of food of food or water (indirect signals of infection or sepsis).The macroscopic aspects of wound and pocket implant were carefully inspected for dislocation or extrusion of the prostheses, as well for abscess, hematoma, seroma or wound dehiscence.If any sign of suffering was seen, the veterinarian was authorized to interrupt the research and to euthanize the animal.

Microscopic assessment
The involving skin, subjacent tissues, capsules and implants were removed en bloc and preserved for later qualitativequantitative analysis of histologic samples sectioned 5 to 7µm and stained with hematoxylin-eosin (H&E) and histochemical Sirius Red dye (SR) saturated with picric acid as described elsewhere [27][28][29][30] .SR-stained slides were used to analysis of collagen morphology and deposition under polarization.

Qualitative histologic analysis
H&E stained slides were used to establish the type and intensity of inflammation by scoring and to measure capsule thickness.Grading was done by an independent pathologist.Briefly, acute inflammation was characterized by the findings of neutrophyls, polymorphonuclear cells, vascular congestion and diapedesis; as to the chronic inflammatory infiltrate, the presence of mononuclear cells (lymphocytes, plasmocytes and monocytes).
As described elsewhere 31  Quantitative analysis of capsule thickness H&E-stained sections were examined by light microscopy.Microscopic digital images were obtained as previously described 28 .The points of greatest thickness were located by scanning the sections at a magnification of x100, with the selection of three regions of interest (ROIs): a middle point correspondent to maximal thickness and two lateral adjacent points 50 micra apart from the middle point.Each digitized image (640 x 480 pixels) corresponded to a microscope field of 0.29 mm 2 (one pixel =0, 53 micrometer).Digital images were processed and analyzed for capsule thickness using the calibrated free hands measurement tools of the public domain NIH ImageJ program (developed at the U.S. National Institute of Health, Baltimore-MD, and available at htttp://rsb.info.nih.gov/nih-image/).Three slides were examined per animal and three ROIs in each section were measured by digital planimetry (total = 54 observations by subgroup).The averages mean thickness/ capsule/group/ that were measured at that ROIs of capsules were expressed as mean ± standard deviation (SD).

Quantitative collagen analysis
New histological sections of 7 micrometers thickness were cut from the paraffin blocks that were utilized for the histopathological study.These sections were taken from the point of greatest capsular thickness from inner layer to outer lamina of tissue.Three slides of three SR-stained samples of the capsular layer of each animal were used to assess morphometry and density of collagen type III -Col 3 (fibrilar, non-polymerized) and type I -Col 1(thick, polymerized) under an optic polarizedlight microscope.
Polarized images (100X, each field area = 0.29 mm 2 ) were captured with a high-resolution digital camera (Sony™ DSC H-10016.1 MP, Japan) coupled to a polarized microscope.
Digitized images were transferred to a personal computer and saved to disc as bmp files (640 x 480 pixels) based on RGB (red, green, blue) color mode, forming an image bank.The computer was equipped with the SAMM, an image analysis software developed and validated by the Federal University of Ceará, Brazil [32][33] .Due to their anisotropic properties, under polarization, collagen type I (thicker and bundled mature collagen fibers) turns red (reddish birrefringence) and collagen type III (fibrilar, thinner and dispersed immature collagen) turns green.Based on the color spectrum, the "separate colors" tool was used to split images into color channels (red, green, blue) 28,32 . in order to analyze by differential colorimetric method the deposition of the collagen fibers.The results were expressed as percentages of areas of collagen type I (Col 1) or collagen type III (Col 3) positive pixels per recorded field (the total area of the histologic cut).Subsequently, Col 1/Col 3 ratios were calculated for each group.The average mean percentage for each capsule thin section was determined by calculating the average of means obtained from reading the three ROIs.
In our pilot-study, these assessments were performed in two occasions separated by a lapse time of three months by two observers without knowledge of the clinic pathological stage and the results from the histological study using H.E. staining.
No significant inter-observer and intra-observer variability has been noticed.

Statistical analysis
In the descriptive analysis, data were summarized as means, medians, standard deviations, standard errors, 1 st and

Macroscopic analysis
No infection or mortality was observed during the experiments, and no local complications were clinically detectable.Also, no evidence of inactivity, diarrhea, starvation or weight gain was observed.A thin, well vascularized, and loosely adherent capsule was found surrounding the implant in all animals, independently of the time of harvesting samples (Figure 2).Although difficult to analyze, apparently the capsules from control group were more opaque and adhered to implant than capsules from propranolol-treated group.

Capsule morphology and inflammation scores
A tri-laminar structure of the capsule was found in all specimens both in H&E and picrosirius stained samples.There was an inner cellular layer adjacent to the implant, an intervening lamina comprising loose connective tissue including an internal vascular supply and a rich cellular presence, and an outer layer collagenous layer of densely packed collagen fibres aligned parallel to the prosthese surface.The inner layer exhibited an epitheliallike single layer composed from fibrocytes and histiocytes, the so called synovial-type metaplasia (STM).The STM was less pronounced in propranolol group at 21 st post-operative day, although without statistical significance.Also, in some capsules villous hyperplasia was found (Figure 3).Except for the apparent lesser vascularization (not quantified) in the middle layer from propranolol-treated group as compared to controls we did not observed gross differences between capsules.All capsules tended to become less cellular with time.
Propranolol-treated capsules demonstrated less inflammatory acute response moderate/intense in about 70% of samples at 7 th post-operative (PO) day; absente/discrete in about 84% at 14 th PO day, but persistent at 21 st day (33%), and no giant cells anytime (Table 1).Also, chronic inflammation was absent    2).

Capsule thickness
Propranolol was highly effective in reducing capsule thickness at 7 th and 21 st days periods when compared to control group (Figure 4 and Table 3).
There was a trend to increasing collagen type I/type III ratios along the time in control group while in propranololtreated group these ratios exhibited lower and almost constant values along the time.was higher in control group.Proportionally, with the evolution of the post-operatory period, type I collagen increased, and was present in both groups (Figure 7).The highest proportion of type III collagen in propranolol-treated group was of statistical significance at all time-points when both groups were compared; also it was significant into each group comparison.

FIGURE 4 -Thickness measures of the capsular reaction
Bars represent control and propranalol groups at 7 th , 14 th and 21 st timepoints.Significant differences were encountered on 7 th (p<0.001) and 21 st timepoints (p<0.05), when comparing control and propranolol groups.ANOVA test.ns = non significant.

Collagen density
Propranolol was effective in impairing deposition of collagen type I fibers into capsule tissue surrounding silicone implants (p-value < 0.0001, one-way ANOVA, Kruskal-Wallis test) when compared to control group (Figure 5).In the initial phase of the capsular formation (7 th day) the presence of collagen type I (reddish) was almost the same for all groups.Density of capsular collagen type III is illustrated in Figure 6.

Discussion
Adverse capsular contracture (ACC) should be regarded as an exaggeration of normal wound healing 6 .In patient populations, this condition is accompanied by overt symptoms and loss of esthetic results of breast augmentation 13 Since the first reports on capsular contracture in the literature, a number of preclinical studies have been conducted in an attempt to prevent its development, using different drugs (such as leukotriene receptor antagonists, antibiotics, anti-neoplastic and antifibroticagents, steroids and non-steroidal anti-inflammatory drugs) administered locally or systemically, but with disappointing results [3][4][5][6][7][8][9][13][14][15][16] . Thus, here is currently no proven pharmacological therapy for ACC 13 .
Special attention should be paid to the possibility of identifying an oral drug for the treatment of a complication that has serious physical, psychological and financial consequences and for which the only effective treatment is surgery.Propranolol is commonly used in the treatment of cardiovascular diseases 19 .
Epinephrine or norepinephrine adrenoceptors, stress-released catecholamines,have been implicated in the pathophysiology of delayed wound healing of cutaneous lesions, acting by delaying the migration and activation of macrophages and neutrophils, inhibiting keratinocyte migration and re-epithelialization in vitro and in vivo wound models [22][23] .Besides alteration of cellular activity related to inflammation, catecholamines stimulate vasoconstriction that causes hypoxia at the wound area and the increase of endothelial cell proliferation, vascular endothelial growth factor (VEGF) expression and angiogenesis.Also, catecholamines promote inhibition of eosinophil influx [23][24] .
High concentrations of catecholamines may delay fibroblasts proliferation, myofibroblastic differentiation and collagen deposition [24][25][26] .Strack et al. 25  As for the method of measuring the thickness of the capsule and the collagen type I and type III deposition, the picrosirius polarization microscopy method plus digital measurement, previously described in another context was utilized [28][29][30][31] .The use of conventional histology method to assess inflammation was necessary because it remains the most utilized method to assess capsular inflammation [32][33][34][35][36][37] .Baker's classification and applanation tonometry did not seem very appropriate to investigate contractures or fibrosis in rodent models [16][17] .Nowadays, the use of implants with smooth surfaces is no longer acceptable due to the fact that they behave differently regarding the occurrence of capsular contracture 11 Therefore, it was decided to use one type of textured prostheses so that responses to the use of propranolol could be verified efficiently without excessive number of variables.In this study, all samples from both groups exhibited a tri-layered capsule as described elsewhere by several scholars in human capsules and animal capsules surrounding textured implants 11,[33][34][35][36][37] .
Various authors mention that synovial-like metaplasia present in the interface capsule-textured implant is due to mechanic friction between implanted materiel and the peri-prosthetic tissue [37][38][39] .
The tissue invasion of STM directed to the coating texture (aka, villous hyperplasia) was found in all capsules examined, although without significant difference between groups.Despite these fact, propranolol was capable of reduce the thickness of capsular reaction tissue at all timepoints, suggesting an anti-proliferative or cytotoxic effect of the drug.
Lyras 40 , analyzing the tissue reaction to rough and smooth silicone implants in a rodent model detected intense acute inflammatory reaction to textured implants.Here, in nonpropranolol-treated group, this kind of reaction was absent or discrete in 1/3 of capsules at 7 th post-operative (PO) day, 2/3 at 14 th PO day and 5/6 at 21 st PO day, confirming the findings already described for textured surface [41][42][43] .In our study we was found that in propranolol-treated capsules the acute inflammatory reaction was absent or discrete in 1/3, 5/6 and 2/3 at 7 th , 14 th and 21 st post-operative day, whereas in control group this reaction was moderate or intense in 2/3, 1/3 and 1/6 at the same periods.As to chronic inflammatory reaction, in control group this reaction was moderate or intense in 1/3 at 7 th PO day, 1/6 at 14 th PO day and 100% at 21 st PO day, while in propranolol group an absent or discrete chronic inflammatory reaction was found in 5/6, 2/3 and 5/6 of the capsules at 7 th , 14 th and 21 st PO day respectively.Clearly, propranolol exerted an anti-inflammatory effect that persisted through all the experiment.
The effect of propranolol in reducing capsular inflammation and thickness, and in modulating collagen deposition on textured implants observed in this experiment is consistent with findings from previous studies of others fibrosis conditions 19 , which showed that the application of nonselective beta-adrenoceptors antagonists inhibits maturation of collagen, reduces the number of vessels, collagen density, number of mast cells, eosinophils and myofibroblasts compared with control 22,23,25 .
These concerted actions of propranolol provide a new opportunity to support pharmacotherapeutic approaches to human adverse capsular contracture.

Conclusion
Propranolol reduces the risk of developing capsular fibrosis around silicone implants with textured surface in this guinea pig model.
the two kinds of inflammation were classified by scores (0=absent; 1=discrete; 2=moderate, and 3=intense), three slides / animal.Collected data were used to calculate the average score from each group by timepoint.FIGURE 1 -Textured silicone implant.
rd quartiles, minimum and maximum values.Scores from the histological assessment of inflammation were analyzed with Kruskal-Wallis test with Dunn's multiple comparison tests when necessary.Capsule thickness data were analyzed by one factor analysis of variance (ANOVA).Collagen type I and type III deposition percentage were analyzed with Kruskal-Wallis test with Dunn's multiple comparison tests when necessary.Col 1/Col 3 ratios were analyzed with Sudent's t test.The used statistical programs were: Statistical Package for Social Sciences (SPSS) version 18.0 and GraphPad Prism 5.0.For evaluating normality conditions of the variables the Shapiro-Wilks test was used.Statistical significance was set for a confidence interval of 95% (p<0.05).

FIGURE 3 -
FIGURE 3 -Microscopic images of capsules.(A) Tri-layered structure of capsule.(B) Negative image the three layers with bright intervening lamina.(C) Zooming of tri-layered capsule.(D) Synovial-type metaplasia with villous hyperplasia of inner capsular layer (x100).

FIGURE 5 -
FIGURE5 -Density of capsular collagen type I Boxes represent control and propranalol groups at 7 th , 14 th and 21 st timepoints.Significant differences were encountered on 14 th (p<0.001) and 21 st timepoints (p<0.0001), when comparing control and propranalol groups.ANOVA test.

FIGURE 6 -
FIGURE 6 -Density of capsular collagen type III Boxes represent control and propranalol groups at 7 th , 14 th and 21 st timepoints.No significant differences were encountered when comparing timepoints.

FIGURE 7 -
FIGURE 7 -Collagen type I/Type III ratio Lines represent control and propranolol groups at 7 th , 14 th and 21 st timepoints.

Figure 8
Figure 8 depicts significant picrosirius red-stained images with and without polarization at 7 th , 14 th and 21 st timepoints.

FIGURE 8 -
FIGURE 8 -Photomicrography of capsules obtained from propranolol-treated animals (right) or untreated (left) and stained with Sirius Red under polarization.Collagen type I fibers stain reddish (orange or red).Collagen type III stain greenish (x100).
demonstrated that propranolol was effective in reducing inflammation and liver fibrosis progression in a model of nonsinusoidal fibrosis.The transcript level of profibrogenic cytokines TNF-alfa, TGF-beta and CTGF was reduced >50% in fibrotic portal areas.Also, propranolol treatment results in levels of proinflammatory mediators (angiotensinogen and endothelin-1) of vasoconstriction under sympathetic nervous system control.In this study, the option for silicone instead of saline prostheses was due to the greater incidence of capsular contracture found in the use of the former27 .PubMed database was extensively searched for reviewing models of capsular contracture.Capsular contracture is a refractory human-specific condition, such as keloids are.The proposed models found have provided valuable information about pathogenesis and treatment possibilities of ACC.The length of the observation time (three weeks) used in this study was based on other studies dealing with wound healing.

TABLE 1 -
Intensity of acute inflammatory reaction.

TABLE 2 -
Intensity of chronic inflammatory reaction.
in 100% of propranolol-treated capsules at 21 st PO day.Acute inflammation was practically resolved by 21 st PO day in the control group.However, 100% of tissue samples from this group harvested at 21 st day exhibited a moderate to intense chronic inflammatory infiltrate, and some sparsely giant cells were found at that moment (Table