Introduction
Breast implant mammoplasty is one of the most performed plastic surgeries worldwide1. Thus, breast implants have been the target of research, mainly focusing on their
coating's surface, which can be divided into smooth and textured, the latter being
subdivided into micro and textured macro2-5.
Thus, in recent decades, there has been a great evolution in breast implants' lining
surface, resulting in the prevention of mammoplasties complications, such as capsular
contracture5,6, infection7, and, more recently, large cell anaplastic lymphoma (BIA-ALCL)8.
Bia-ALCL has been the subject of studies in recent years, as there has been an increase
in its incidence, especially in patients undergoing mammoplasties with implants3,4,6,8. In the pathophysiology of this disease, it is known that neoplasia is directly related
to the host's inflammatory response to the implant6,8.
One way to evaluate the inflammatory response is the blood count, which is divided
into red and white sets, and platelets. Its advantages are to be a quick, inexpensive
and high availability exam9.
Through this examination, the biological response to the implant can be evaluated
by toxicity and rejection (number of red blood cells) and by the response to cellular
stress (neutrophil/lymphocyte/NLR ratio)10, correlated with mortality in response to device implantation or infections11.
As an example, we can mention a rare complication, toxic shock syndrome, after breast
implant surgery. This disease has as characteristic the lack of local signs such as
edema or erythema but presents fever and a marked increase in white blood cell count,
suggesting severe infection. Therefore, early diagnosis is usually difficult, and
appropriate treatment initiation can be delayed without the knowledge of this characteristic12.
In the inflammatory response, neutrophils are one of the first immunological cells
to be recruited to go to the injured site and secrete various inflammatory cytokines
that contribute to the beginning of this response. However, the biological response
of neutrophils to implanted devices remains uncertain13.
OBJECTIVES
The present study aims to evaluate at the laboratory through blood biomarkers the
rats submitted to the placement of mini-implants of silicone nanotextured and mini-implants
coated with polyurethane foam.
METHODS
The research was carried out in the Universidade Estadual de Ponta Grossa (UEPG) experimental
surgery vivarium after being approved by the Ethics Committee on the Use of Animals
(CEUA) of UEPG. CEUA Case - 041/2018. EUPG Protocol: 16450/2018. All procedures strictly
followed existing animal research regulations.
The study design was a primary study (randomized clinical trial), interventional,
experimental in animals (rats), prospective, analytical, controlled, randomized, double-blind
and unicentric.
A total of 60 albino rats (Rattus norvegicus albinus, Rodentia Mammalia) weighing between 190 to 250 grams and 30 to 60 days of life had
free access to water and diet specific to the species, with room temperature and circadian
cycles of 12 hours.
They were randomly divided into two groups of 30 animals for each type of mini-silicone
implant (nanotextured and polyurethane foam) and subdivided into three subgroups,
according to the euthanasia time of the animals (30, 60 and 90 days).
In the nanotextured group, n = 30, mini-implants with a nanotextured surface (Silimed®, Rio de Janeiro, Brazil) were placed, and in the polyurethane group, n = 30, mini-implants
with polyurethane foam coating were placed (Silimed®.
The implanted materials had the same layers of a human breast silicone implant, discoid
shape, with 22 +/- 1 mm (mm) in diameter and 9 +/- 1mm in height in mini-implants
with a nanotextured surface, and with 24 +/- 1 mm in diameter and 11 +/- 1mm high
in polyurethane foam-coated mini-implants. Height was defined as the point of greatest
implant projection on the vertical axis (Figure 1).
Figure 1 - Mini silicone implants nanotextured and mini implants coated with polyurethane foam.
Figure 1 - Mini silicone implants nanotextured and mini implants coated with polyurethane foam.
Concerning pores on the surface of mini-implants, those with nanotextured surface
had the following dimensions: diameter 0.3 to 8.7 micrometers (300 to 8700 nanometers);
average roughness (Ar) 4.12 micrometers (4120 nanometers); and depth 3.08 to 10.74
micrometers. The mini-implants coated by polyurethane foam had the following dimensions:
diameter 120 to 320 micrometers; average roughness (Ar) 1500 micrometers; and pore
depth 480 to 1200 micrometers.
After group distribution, the rats were randomly removed from the cages and anesthetized
by intraperitoneal injection, composed of an association of ketamine hydrochloride
1% (Dopalen®, Hertape, Belo Horizonte, Brazil) at a dose of 40mg/kg and xylazine hydrochloride
2% (Dopasen®, Hertape) at a dose of 8mg/kg according to the anesthesia and analgesia guide of
laboratory animals - UNIFESP/CEUA (2017)14.
The effectiveness of anesthesia was evaluated by the absence of movement, corneal-eyelid
reflex, and motor reaction after gripping with tweezers of one of the hind legs' adipose
cushions, in addition to a good ventilatory pattern.
With the rats positioned in ventral decubitus, trichotomy was performed in the dorsal
region, with subsequent antisepsis and sterile surgical field placement.
The incision's delimitation was performed regarding a subcostal horizontal line, following
the posteroinferior costal edge, which met with the middle sagittal line. With a scalpel
cable no. 3, coupled with a blade no. 15, a horizontal incision was made, with an
extension of 20 mm at the intersection of these reference lines.
The store was made for the mini-implants in a retromuscular plane (below the Panniculus
carnosus), and, later, the mini-implant was introduced vertically and positioned horizontally
according to the group (nanotextured or polyurethane). The suture of the skin was
intradermal with mononylon 5-0 (Ethico® with buried knots. There was no removal of the stitches in the postoperative period,
and the surgical wound was kept exposed (Figure 2).
Figure 2 - Immediate post-op.
Figure 2 - Immediate post-op.
Postoperative analgesia was with a single intramuscular application of sodium dipyrone
(20mg/kg) in the posterior limb's lateral region. No postoperative dressings or stitches
were performed.
Euthanasia occurred according to the subgroups of 30, 60 and 90 days by applying four
times the therapeutic dose of Dopalen® and Dopasen® subsequent cervical dislocation. There was no death, infection of the surgical site,
and no implants' extrusion, so no rats were excluded.
Evaluation methodology
Blood samples with 4mL were obtained on the day of euthanasia, according to each subgroup,
by intracardiac puncture with a 10mL syringe performed by the veterinarian (Video1), and were arranged in tubes with anticoagulant Ethylenediamine tetraacetic acid
(EDTA) and homogenized for 1 minute.
After that, the tubes were immediately placed on the veterinary hematological analyzer
Max Cell 200 - KT 6200 VET® (Green Medical Instrument, Tokyo, Japan) previously calibrated for blood count reading,
in which blood separation was performed in red and white series, and platelets.
The use of this equipment is a way of not committing cell counting errors that frequently
occur through a blood smear. After that, the results were analyzed 15,16.
Statistical evaluation
The results were described by median, minimum and maximum values. For comparing the
groups (nanotextured and polyurethane), the Mann-Whitney non-parametric test was used
in each subgroup (30, 60 and 90 days). The differences between the subgroups for each
group were made using the Kruskal-Wallis non-parametric test. P<0.05 values indicated
statistical significance. The data were analyzed with the computer program Stata/SE
v.14.1. StataCorpLP, USA.
RESULTS
Red series
Hypochromic anemia was found in the animals evaluated on the changes in the red series
in the postoperative period.
Hemoglobin and hematocrit
In all groups and subgroups evaluated, the results were very similar and without statistical
significance (Tables 1 and 2, Figures 3 and 4).
Table 1 - Hemoglobin analysis in the nanotextured and polyurethane groups over time
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
13 (3.9-13.7) |
13 (12-13.6) |
0.762 |
| 60d |
12.5 (7.2-13.9) |
12.2 (11.5-37.1) |
0.720 |
| 90d |
13 (12.3-14.7) |
13.4 (10.5-13.9) |
0.631 |
| p** (30 x 60 x 90d)
|
0.210 |
0.165 |
|
Table 1 - Hemoglobin analysis in the nanotextured and polyurethane groups over time
Table 2 - Analysis of hematocrit in the nanotextured and polyurethane groups over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
48.4 (16.3-50) |
48.3 (44-50.6) |
0.829 |
| 60d |
46.7 (0.7-51.2) |
47.5 (45.2-51.1) |
0.780 |
| 90d |
46.3 (44.2-51.3) |
48.5 (37.2-49.9) |
0.631 |
| p** (30 x 60 x 90d)
|
0.594 |
0.632 |
|
Table 2 - Analysis of hematocrit in the nanotextured and polyurethane groups over time.
Figure 3 - Hemoglobin analysis in the nanotextured and polyurethane groups over time.
Figure 3 - Hemoglobin analysis in the nanotextured and polyurethane groups over time.
Figure 4 - Analysis of hematocrit in the nanotextured and polyurethane groups over time.
Figure 4 - Analysis of hematocrit in the nanotextured and polyurethane groups over time.
Mean corpuscular hemoglobin
When the different subgroups were compared among themselves, statistically significant
results were obtained (Table 3 and Figure 5).
Table 3 - Analysis of mean corpuscular hemoglobin in the nanotextured and polyurethane groups
over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
15.5 (13.6-16.3) |
15.8 (15.4-16.5) |
0.515 |
| 60d |
15.7 (14.7-16.5) |
15.2 (14.9-16) |
0.190 |
| 90d |
16.5 (14.8-16.8) |
16.4 (14.5-16.9) |
0.631 |
| p** (30 x 60 x 90d)
|
0.032*** |
0.007*** |
|
Table 3 - Analysis of mean corpuscular hemoglobin in the nanotextured and polyurethane groups
over time.
Figure 5 - Analysis of mean corpuscular hemoglobin in the nanotextured and polyurethane groups
over time.
Figure 5 - Analysis of mean corpuscular hemoglobin in the nanotextured and polyurethane groups
over time.
White series
Normal leukocytes count at the expense of neutrophilia and lymphopenia was evidenced
in the changes in the white series postoperatively.
Leukocytes
When comparing the different subgroups among each other, within each group, results
with statistical significance were obtained (Table 4 and Figure 6).
Table 4 - Analysis of leukocytes in the nanotextured and polyurethane groups over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
8.1 (3.3-12.6) |
10.5 (3.8-17.6) |
0.122 |
| 60d |
8.3 (2-11.9) |
7.9 (1.1-11.7) |
0.905 |
| 90d |
11.9 (4.4-16.3) |
12 (6-30.6) |
0.631 |
| p** (30 x 60 x 90d)
|
0.038*** |
0.034*** |
|
Table 4 - Analysis of leukocytes in the nanotextured and polyurethane groups over time.
Figure 6 - Analysis of leukocytes in the nanotextured and polyurethane groups over time.
Figure 6 - Analysis of leukocytes in the nanotextured and polyurethane groups over time.
Neutrophils and lymphocytes
In all groups and subgroups evaluated, the results were similar and without statistical
significance (Tables 5 and 6, Figures 7 and 8).
Table 5 - Analysis of neutrophils in % in the nanotextured and polyurethane groups over time
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
35.1 (25.5-44.1) |
37.8 (28.4-46.3) |
0.408 |
| 60d |
39 (25.9-43.8) |
35.1 (22.2-43.9) |
0.211 |
| 90d |
40.4 (28.7-49.1) |
41 (23.4-60.2) |
0.631 |
| p** (30 x 60 x 90d)
|
0.589 |
0.148 |
|
Table 5 - Analysis of neutrophils in % in the nanotextured and polyurethane groups over time
Table 6 - Analysis of lymphocytes in % in the nanotextured and polyurethane groups over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
53.6 (44.5-70.8) |
51.6 (43.6-62.5) |
0.360 |
| 60d |
49.6 (44-68.1) |
55.9 (45.7-72.6) |
0.079 |
| 90d |
48 (41.4-60.2) |
47.2 (23.5-65.8) |
0.481 |
| p** (30 x 60 x 90d)
|
0.372 |
0.075 |
|
Table 6 - Analysis of lymphocytes in % in the nanotextured and polyurethane groups over time.
Figura 7 - Análise dos neutrófilos em % nos grupos nanotexturizado e poliuretano ao longo do
tempo.
Figura 7 - Análise dos neutrófilos em % nos grupos nanotexturizado e poliuretano ao longo do
tempo.
Figure 8 - Analysis of lymphocytes in % in the nanotextured and polyurethane groups over time.
Figure 8 - Analysis of lymphocytes in % in the nanotextured and polyurethane groups over time.
Platelets
Platelets presented with thrombocytopenia, and the results were very similar between
the two groups, in the various subgroups evaluated, and without statistical significance
(Table 7 and Figure 9).
Table 7 - Analysis of platelets in the nanotextured and polyurethane groups over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
polyurethane median (min-max) |
| 30d |
114 (37-667) |
211 (42-739) |
0.573 |
| 60d |
146 (110-759) |
292 (134-645) |
0.489 |
| 90d |
301.5 (137-662) |
203.5 (98-839) |
0.247 |
| p** (30 x 60 x 90d)
|
0.265 |
0.535 |
|
Table 7 - Analysis of platelets in the nanotextured and polyurethane groups over time.
Figure 9 - Analysis of platelets in the nanotextured and polyurethane groups over time.
Figure 9 - Analysis of platelets in the nanotextured and polyurethane groups over time.
DISCUSSION
Choice of animal model
Based on Zarini et al. (2004) 15, it was considered that the life expectancy for a laboratory rat is approximately
2.5 years. Correlating with human life, three months of this animal correspond to
90 months (approximately eight years) in a human being, based on an average life expectancy
of 75 years. Due to this, we chose to divide into subgroups of 30, 60 and 90 days
to give greater veracity to a late postoperative period.
The rat (Rattus novergicus Albinus, Rodentia Mammalia) chosen by the authors is the animal most used in healing studies
around silicone implants due to the easy reproducibility of results, resistance to
surgical interventions and high availability in bioteries17,18.
Red series
According to reference values in Wistar rats (19,) hemoglobin, hematocrit and mean corpuscular hemoglobin were analyzed. Concerning
hemoglobin, there was a total decrease and, on the other hand, hematocrit increased,
regardless of the type of mini-implant.
This configured a possible postoperative hypochromic anemia and may suggest iron deficiency,
blood loss, inflammation, and postoperative infection. As a consequence, anemia can
increase postoperative morbidity and mortality20.
Partially agreeing with Ersoy et al. (2015) 21, who implanted intracardiac device in humans, demonstrated that hemoglobin and hematocrit
decreased significantly in the postoperative period. They attributed this gastrointestinal
bleeding and hemolysis of red cells secondary to high shear tension, which the implanted
device could cause.
Disagreeing with Zarini et al. (2004) 15, who placed hydrogel implants in female mice, the authors mentioned above found no
alteration in the red series, indicating no postoperative systemic involvement in
the red series due to the implant.
Disagrees with Csendes et al. (2014) 9, who underwent gastrectomy in humans, did not show a change in hemoglobin's reference
values before and after surgery. However, concerning hematocrit, they obtained a decrease
in this parameter, which contradicts our study. We had increased hematocrit values,
even if not absolutely, in all subgroups, regardless of the type of mini-implant.
White series
When analyzing leukocytes in the present study, a large amplitude was evidenced between
the minimum and maximum values; however, the median remained within the reference
value. When the subgroups were compared longitudinally, there was a gradual increase
in leukocytes, indicating a higher production of these cells.
Neutrophilia accompanied by lymphopenia was also evidenced. In this case, lymphopenia
is considered relative because there is an increase in neutrophils, unbalancing the
leukocyte equation, which is a marker of inflammation11.
Neutrophilia is immediate in the postoperative period because interleukin-1 is formed
by stimulation of the inflamed and traumatized areas, resulting in the neutrophil
reserve's release to the periphery13.
In the present study, neutrophilia was persistent, probably due to the perpetuation
of the implant's inflammatory reaction, especially in the polyurethane group, which
showed relatively higher values.
It is partially agreed with Csendes et al. (2014) 9, which in their study found an increase in leukocytes at the expense of neutrophilia
in the postoperative period; however, according to these authors, the values returned
to the reference on the fifth day, suggesting a physiological inflammatory process
due to surgical stress, unlike the present study in which the values remained high
until 90 days9.
We disagree with Zarini et al. (2004) 15, who described a mild leukocytosis accompanied by neutrophilia attributed to the
foreign body reaction15; on the contrary, in the present study, normal leukocyte values were found due to
a balance between neutrophilia and lymphopenia.
Regarding the leukocyte equation, Hashimoto et al., in 201811), reported that the neutrophil/lymphocyte ratio (NLR) might be a method of stratification
of mortality risk, with the characteristics of being noninvasive, inexpensive and
readily available for patients with implants.
The advantage of using both neutrophils and lymphocytes can be explained because NLR
combines two different immunological pathways. The first pathway, composed of neutrophils,
is involved in rapid response, while the second pathway contemplates lymphocytes in
the long-term adaptive response of the immune system, which is a synonym for physiological
stress11.
Platelets
Regarding platelet changes, thrombocytopenia was observed, suggesting splenic sequestration
or decreased production, destruction or accelerated consumption of platelets20.
It corroborates with Zarini et al. (2004) 15, who in their study demonstrated early and transient thrombocytopenia as a consequence
of hemostatic activation due to microhemorrhage at the implant site.
It partially disagrees with Zhang et al. (2015)22, who evaluated the alteration in platelets after stent implantation in humans in
their study. These authors found patients with decreased platelet count (mild, moderate
and large amounts) and reported patients with increased platelet count, but these
values returned to normality after six months.
The same authors attributed the alteration in platelets due to the minimization of
surgical stress and the accelerated consumption of these cells to stent-related hypercoagulability.
This study also demonstrated that the change in platelet count was positively correlated
with the change of leukocytes and not with the number of stents22.
CONCLUSION
After placement of mini silicone implants, the rats of both groups evolved with hypochromic
anemia, normal leukocyte count at the expense of neutrophilia and lymphopenia, and
thrombocytopenia.
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1. Universidade Federal de São Paulo, Graduate Program in Translational Surgery, São
Paulo, SP, Brazil.
2. Universidade Estadual do Rio de Janeiro, Graduate Program in Pathophysiology and
Surgical Sciences, Rio de Janeiro, RJ, Brazil.
3. Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil.
Corresponding author: Eduardo Nascimento Silva, Avenida Doutor Francisco Burzio, 991, Centro, Ponta Grossa, PR, Brazil. Zip Code:
84010-200. E-mail: dr_eduardosilva@yahoo.com.br
Article received: October 09, 2020.
Article accepted: November 17, 2020.
Conflicts of interest: none
