Introduction
The advancement of materials' functions and performance is the main objective of surface
engineering, aiming to produce implants that trigger increasingly controlled biological
responses1. When an alloplastic material is placed in contact with the living organism, the
proteins present in the blood interact with the surface identified as foreign. Protein
absorption in these biomaterials is one of the main points during the development
of technologies in bio-devices2.
This is because, when a material is implanted, the absorption of proteins is one of
the first steps in the biological process, as well as the coagulation cascade, which
triggers immune and inflammatory reactions2,3. Therefore, the protein absorption phenomenon's control is necessary to define biomaterials'
properties and their specific uses3. Blood plasma contains several different proteins, including fibrinogen, which is
a signaling molecule with a wide spectrum of functions, which can lead from a balance
between coagulation and protection against infections to processes of fibrosis and
intense inflammation4. Thus, fibrinogen shows an important role in cell adhesion and, consequently, in
the biocompatibility results of implants3.
During the evolution of breast implants, several coating surfaces were proposed to
minimize tissue reactions5, of which nanotextured implants, through nanotechnology, proved to be safer concerning
anaplastic large cell lymphoma6 and polyurethane foam if proved effective in reducing the rate of capsular contracture7.
Segmented polyurethane is a thermoplastic elastomer that has been used extensively
in surgical procedures due to its excellent physical and mechanical properties, thermoplasticity
and biocompatibility8-10.
Currently, with the advancement of nanotechnology, materials with surfaces of smaller
and smaller sizes and more similar to biological structures have been proposed, thus
allowing increasingly natural tissue reactions between the implant and adjacent tissues,
which should decrease the intensity of the inflammatory response and influence the
absorption of proteins 1,11.
OBJECTIVES
The present study aims to evaluate in the laboratory the rats submitted to the placement
of nanotextured silicone implants and coated with polyurethane foam, with the following
parameters:
- measurement of fibrinogen;
- measurement of plasma protein.
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) UEPG. CEUA process - 041/2018. UEPG Protocol: 16450/2018. All procedures strictly
followed the existing regulations for animal research.
The study design was a primary study (randomized clinical trial), interventional,
experimental in animals (rats), prospective, analytical, controlled, randomized, double-blind
and single-centered.
A total of 60 albino rats (Rattus norvegicus albinus, Rodentia mammalia) weighing between 190 to 250 grams and 30 to 60 days old had free access to water
and a species-specific diet, with room temperature and 12-hour circadian cycles.
They were randomly divided into two groups of 30 animals for each type of silicone
mini-implant (nanotextured and polyurethane foam), and subdivided into 3 subgroups,
according to the animals' euthanasia time (30, 60 and 90 days).
In the nanotextured group, n = 30, mini-implants with nanotextured surfaces (Silimed®), Rio de Janeiro, Brazil) were placed. In the polyurethane group, n = 30, mini-implants
with polyurethane foam coating (Silimed®) were placed.
The implanted materials had the same layers as a human breast implant, discoid in
shape, with 22 +/- 1 mm (mm) in diameter and 9 +/- 1 mm in height in mini-implants
with a nanotextured surface, and with 24 + / - 1 mm in diameter and 11 +/- 1 mm high
in mini-implants coated with polyurethane foam. The height was defined as the point
of greatest implant projection on the vertical axis (Figure 1). Concerning the pores on the surface of the mini-implants, those with a 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 with polyurethane foam had the
following dimensions: diameter 120 to 320 micrometers; average roughness (Ar) 1500
micrometers; and pore depth 480 to 1200 micrometers. After distribution to groups,
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 hydrochloride of xylazine
2% (Dopasen®, Hertape) at a dose of 8mg/kg according to the guide for anesthesia and analgesia
of laboratory animals - UNIFESP/CEUA (2017) 12. The effectiveness of anesthesia was assessed by the absence of movement, corneal-eyelid
reflex, and motor reaction after grasping the fat pad of one of the hind legs, in
addition to a good ventilatory pattern. With the rats positioned in the prone position,
trichotomy was performed on the dorsal region, with subsequent antisepsis and sterile
surgical field placement.
Figure 1 - Mini-implants of nanotextured silicone and coated by polyurethane foam.
Figure 1 - Mini-implants of nanotextured silicone and coated by polyurethane foam.
The incision's delimitation was performed regarding a subcostal horizontal line, following
the posteroinferior costal edge, which was 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 pocket was made for the mini-implants in a retromuscular plane (below the Panniculus carnosus), and, later, the mini-implant was introduced vertically, being positioned horizontally
according to the group (nanotextured or polyurethane). The skin's suture was intradermal
with mononylon 5-0(Ethicon ®) 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 subgroups of 30, 60 and 90 days by applying four
times the therapeutic dose of Dopalen® and Dopasen® and subsequent cervical dislocation. There was no death, infection of the surgical
site or extrusion of the implants, so no rats were excluded.
Evaluation methodology
Blood samples were obtained on the day of euthanasia of the animals, according to
each subgroup, by intracardiac puncture performed by the veterinarian (Video 1), and were placed in tubes without and with anticoagulant ethylenediaminetetraacetic
acid (EDTA) 13. The triplicate thermal precipitation technique was used for each animal, consisting
of filling six capillary tubes with blood up to 3/4 of the capacity, properly closed
at one end. After that, they were centrifuged at 8.0rpm in a microhematocrit centrifuge
to separate the plasma for 5 minutes. After being centrifuged, three of the capillaries
were randomly chosen and broken to obtain a drop, which was later placed on the Goldberg
refractometer to measure total plasma protein (TPP) 13,14. The remaining three capillary tubes were taken to the water bath (temperature of
56-58 ° C, for three minutes) and afterward, again centrifuged, as previously described,
obtaining this time the serum, which was measured in the refractometer, resulting
in serum protein (SP) 13.
The fibrinogen value was obtained by the difference between the total plasma protein
and the serum protein. The result was multiplied by 1,000, as fibrinogen is evaluated
in mg.dL-¹13.
Statistical evaluation
The results were described by median, minimum and maximum values. For the comparison
of groups (nanotextured and polyurethane), in each subgroup (30, 60 and 90 days),
the Mann-Whitney non-parametric test was used. The comparisons between the subgroups
for each group were made using the Kruskal-Wallis non-parametric test. Values of
p <0.05 indicated statistical significance. The data were analyzed with the computer
program Stata/SE v.14.1. StataCorpLP, USA.
RESULTS
The groups (nanotextured and polyurethane) were compared for the variables fibrinogen
and plasma protein in the subgroups of 30, 60 and 90 days.
When the groups were compared to each other, it was observed that the nanotextured
group had a greater amount of fibrinogen and plasma protein in the 90-day subgroup,
with statistical significance (p = 0.004) (Tables 1 and 2 and Figures 3 and 4).
Table 1 - Comparison of fibrinogen in the nanotextured and polyurethane groups over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
Polyurethane median (min-max) |
| 30d |
5.6 (5-5.6) |
5.5 (4.8-6) |
0.962 |
| 60d |
9.2 (8.8-9.8) |
9.2 (8.8-10) |
0.673 |
| 90d |
7.2 (6.4-7.8) |
6.3 (5-7.4) |
0.004 |
| p** (30 x 60 x 90d)
|
<0.001*** |
<0.001*** |
|
Table 1 - Comparison of fibrinogen in the nanotextured and polyurethane groups over time.
Table 2 - Comparison of plasma protein in the nanotextured and polyurethane groups over time.
| Subgroups |
Groups |
p*
|
| Nanotextured median (min-max) |
Polyurethane median (min-max) |
| 30d |
5.6 (5 - 6) |
5.6 (4.8 - 6) |
0.813 |
| 60d |
9.4 (9 - 10) |
9.4 (8.2 - 10) |
0.393 |
| 90d |
7.9 (6.4 - 8) |
7 (5.2 - 7.4) |
0.002 |
| p** (30 x 60 x 90d)
|
<0.001*** |
<0.001*** |
|
Table 2 - Comparison of plasma protein in the nanotextured and polyurethane groups over time.
Figure 3 - Comparison of fibrinogen in the nanotextured and polyurethane groups over time.
Figure 3 - Comparison of fibrinogen in the nanotextured and polyurethane groups over time.
Figure 4 - Comparison of plasma protein in the nanotextured and polyurethane groups over time.
Figure 4 - Comparison of plasma protein in the nanotextured and polyurethane groups over time.
When comparing the subgroups, a significant difference was observed (p <0.001) (Tables 1 and 2 and Figures 3 and 4).
DISCUSSION
The foreign body reaction is the inflammatory sequence triggered by the implantation
of biomaterials15, corresponding to the absorption of proteins on the implant surface, infiltration
of inflammatory cells, fusion of macrophages and giant cells, activation of fibroblasts
and, finally, formation of a fibrous capsule16.
The acute phase of healing is closely related to the activation of macrophages, which
produce a variety of growth factors (IGF-1, VEGF-α, TGF-b and Wnt) that are proteins
that regulate the proliferation of endothelial and epithelial cells, activate myofibroblasts,
and can differentiate into progenitor cells and neovessel formation. Macrophages,
therefore, recover tissue homeostasis by activating anti-inflammatory cells and regulating
the deposition of collagen and fibrin17.
When there is inflammation, the presence of fibrinogen and fibrin is frequent. Like
the macrophage, fibrinogen exerts its effects depending on the context in which it
is, both in inflammation of the tissue and in its repair, in wound healing or in the
development of fibrosis18.
Thus, the mechanisms that regulate these different activations of macrophages and
fibrinogen have become active areas of research18. It is known today that these responses vary according to the characteristics of
the implanted material, such as its size, biological behavior, pore size, surface
topography and sterilization techniques17.
Biomaterial's engineering has focused on creating implants that more accurately simulate
human tissues, both physically and chemically15. With the annual growth in the number of mammoplasty surgeries, the ideal implant's
choice remains a challenge, aiming for natural results and safety procedures19.
Currently, breast implants can be classified according to their filling (silicone
or saline), shape (round or anatomical) and surface texture (smooth, micro and macrotextured)
20. The surface texture is determined from the material's roughness: smooth (less than
10µm), microtextured (10-50µm) and macrotextured (greater than 50µm)5.
The silicone implant coated with polyurethane foam is considered a macrotextured implant.
Its use began in 1970, motivated by a supposed reduction in capsular contracture21. The polyurethane foam forms a foamy mass in situ containing pores; this porosity
allows for cell growth inside, leading to the incorporation of the implant lining
into the adjacent tissue. After forming the capsule around the implant, the polyurethane
coating degrades and merges with the capsule22.
More recently, with the advent of nanotechnology, greater tissue mimicry has become
reality23, making it possible to build surfaces with specific nano protrusions, depending on
the need, both for promotion and to prevent the absorption of proteins2.
Nanotextured surfaces demonstrated the ability to more efficiently control the interactions
between the recipient tissue and the implant surface, decreasing the foreign body
reaction, inflammation and scar tissue formation, in addition to greater control of
colonization by pathogens24.
CONCLUSION
The nanotextured implants showed a lower protein absorption in relation to polyurethane
foam-coated implants in the 90-day subgroup.
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1. State University of Ponta Grossa, Ponta Grossa, PR, Brazil.
2. Federal University of São Paulo, Graduate Program in Translational Surgery, São
Paulo, SP, Brazil.
3. State University of Rio de Janeiro, Graduate Program in Pathophysiology and Surgical
Sciences, Rio de Janeiro, RJ, 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: December 01, 2020.
Article accepted: January 10, 2021.
Conflicts of interest: none
