ORIGINAL ARTICLE
Wound healing modulation by a latex protein-containing
polyvinyl alcohol biomembrane
Márcio V. Ramos1&Nylane Maria N. de Alencar2&Raquel S. B. de Oliveira3&
Lyara B. N. Freitas2&Karoline S. Aragão2&Thiago Antônio M. de Andrade4&
Marco Andrey C. Frade4&Gerly Anne C. Brito5&Ingrid Samantha T. de Figueiredo3
Received: 17 January 2016 / Accepted: 23 March 2016 / Published online: 1 April 2016 #Springer-Verlag Berlin Heidelberg 2016
Abstract In a previous study, we performed the chemical characterization of a polyvinyl alcohol (PVA) membrane sup-plemented with latex proteins (LP) displaying wound healing activity, and its efficacy as a delivery system was demonstrat-ed. Here, we report on aspects of the mechanism underlying the performance of the PVA-latex protein biomembrane on wound healing. LP-PVA, but not PVA, induced more intense leukocyte (neutrophil) migration and mast cell degranulation during the inflammatory phase of the cicatricial process. Likewise, LP-PVA induced an increase in key markers and mediators of the inflammatory response (myeloperoxidase ac-tivity, nitric oxide, TNF, and IL-1β). These results demon-strated that LP-PVA significantly accelerates the early phase of the inflammatory process by upregulating cytokine release. This remarkable effect improves the subsequent phases of the healing process. The polyvinyl alcohol membrane was fully absorbed as an inert support while LP was shown to be active. It is therefore concluded that the LP-PVA is a suitable bioresource for biomedical engineering.
Keywords Biomembrane .Calotropis procera. Polyvinyl alcohol . Wound
Introduction
Wounds are characterized by damage to the epidermis caus-ing, with wide-ranging intensities, the exposure of internal tissue layers. The most common wounds result from small accidents and represent superficial tissue injury, which promptly heal. However, surgical wounds and lesions from physiological disorders such as psoriasis or chronic infectious diseases such as leprosy deserve appropriate care. Accordingly, medical supports capable of accelerating the cic-atricial process as well protecting lesions against opportunistic infections are needed.
Calotropis procera(Ait.)R. Br. (Asclepiadaceae), a wide-spread plant found in the tropics, is commonly known as a medicinal plant. The latex from this plant has been found to display toxic properties; nevertheless, when appropriately fractionated, various biological effects, such as anticancer, an-algesic, antipyretic, inflammatory, and anti-inflammatory properties, have been described (Chaudhary et al. 2015; Oliveira et al.2010). Hence, the pharmacological potential of latex proteins has been investigated (Kumar et al. 2014; Ramos et al.2012). Our studies on the soluble latex protein (LP) fraction have revealed dual activity in such a way that the release or downregulation of pro- and anti-inflammatory me-diators occurs, depending on the protocol (Freitas et al.2012; Oliveira et al.2012).
In a previous study, we successfully elaborated a biomem-brane composed of LP soaked in polyvinyl alcohol (PVA) and tested its ability to improve the healing process of incisional and excisional wounds. The macroscopic analysis of the wounds revealed that the use of the LP-PVA membrane
* Márcio V. Ramos [email protected]
1 Departamento de Bioquímica e Biologia Molecular/UFC Campus do Pici 6033, Fortaleza, Ceará, Brazil
2
Departamento de Fisiologia e Farmacologia/UFC, Fortaleza, Ceará, Brazil
3 Centro Universitário Estácio do Ceará, Via Corpvs, Fortaleza, Ceará, Brazil
4
Divisão de Dermatologia, Faculdade de Medicina de Ribeirão Preto, São Paulo, Brazil
5
improved the cicatrical process, stimulating fibroplasia and collagen deposition (Figueiredo et al.2014). It was necessary to perform further characterization of the healing effect of LP-PVA membrane to gain new insights into its usefulness as a wound healing promoter by determining its interference in the release of or downregulation of inflammatory mediators in-volved in the process. This study describes the mechanisms underlying the beneficial use of LP-PVA as a promoter of incisional and excisional wound healing and therefore con-firms LP-PVA as a suitable tool for investigating wound healing.
Materials and methods
Plant material
The latex ofC. procerawas obtained from young specimens distributed along the coastline of Fortaleza City, State of Ceará (Brazil). Following collection, the latex was processed in the laboratory, as previously reported (Alencar et al.2006). The soluble latex protein fraction obtained at the end of the process was freeze-dried and stored at 25 ± 4 °C until use. The proteins obtained as a result of collecting different specimens were pooled to provide a unique sample to be used in all experi-ments; this was called latex proteins (LP). Polyvinyl alcohol (PVA; Mw 9000–10,000, 80 % hydrolyzed) was purchased from Sigma-Aldrich (São Paulo, SP., Brazil).
Biomembrane preparation
The biomembranes composed of LP/PVA or PVA were pre-pared as previously reported (Figueiredo et al. 2014). PVA (1 %,w/v) and LP (0.2 or 1 %w/v) dissolved in 10 mL of distilled water were prepared. These solutions were mixed to obtain a final concentration of 1 % PVA + 0.2 % LP (v/v) or 1 % PVA + 1 % LP (v/v). After stirring at 50 °C for 2 h, the mixtures were filtered and decanted (20 mL) into Petri plates to dryness in an air circulation device (40 °C). The membranes containing 1 % PVA or PVA associated with LP were desig-nated PVA and BioMem PVA/LP 0.2 % (v/v), respectively (Fig.1). The membranes were carefully detached from the Petri plates and cut into 1.2-cm2slices. Both faces of the slices were kept in a laminar flow hood and exposed to ultraviolet light (UV) irradiation for 20 min to sterilize.
Animals
Incisional and excisional wounds were created in Swiss mice, 12 weeks of age (25 g ± 3.0 g), provided by the Universidade Federal do Ceará (Brazil). The animals were maintained indi-vidually in plastic cages with access to water and food ad libitum (commercial sterile diet). The environmental
conditions of maintenance were set at 25 ± 3.0 °C and humid-ity 55 ± 10 %. The light cycle was 12/12 h. Before handling the animals, the experimental procedures were evaluated by the institutional committee for animal use and care following approval under the number 39/2014.
Incisional and excisional wounds
Before handling, animals were anesthetized with 10 % keta-mine chloridrate (115 mg/kg) and 2 % xylazine chloridrate (10 mg/kg) (Hall and Clark 1991). The dorsal surface skin was shaved and further prepared for aseptic surgery using 1 % iodopovidone followed by 70 % ethanol. One wound, either incisional or excisional, was introduced. Linear incisional wounds were created using a surgical scalpel blade no. 15 and were 5 mm long (Chang et al.1980). Circular excisional wounds of approximately 1 cm2were obtained by using a punch biopsy instrument (Cardoso et al.2007).
Biomembrane implantation
The skin edges of the incisional wounds were minimally spaced to introduce blunt tweezers, aiming for complete sep-aration of the subcutaneous tissue and muscle fascia. Next, the membrane was accommodated into the wound. The wounds were sutured with mononylon 5–0 (PolySuture, Brazil) so that the healing occurred by primary intention (Fig. 2a–d). The excisional wounds were covered by membrane 1.2 cm2size. The edges remained fully accommodated under the intact skin. Excisional wounds were not sutured so that the healing progress occurred by secondary intention (Fig.2e-h). The an-imals were randomly divided into groups, representing five animals per group (n= 5) in the time point of experiment (days 2, 7, and 14), as follows: (i) animals with a 1 % PVA mem-brane implanted into the incisional or (ii) excisional wound; (iii) animals with BioMem PVA/LP 0.2 % (v/v) used in incisional or (iv) excisional wounds; (v) animals with BioMem PVA/LP 1 % (v/v) used in incisional wounds or (vi) animals subjected only to surgical procedures that did not receive any membrane (SHAM).
PVA BioMem PVA/LP 0.2% (v/v)
Histology
Samples of neoformed tissue were removed from wounds and were fixed for 24 h in 10 % formaldehyde dissolved in PBS 0.01 M at pH 7.2 and later submitted to histopathology pro-cedures. The sections (5μm) were stained with hematoxylin-eosin, and edema and leukocyte infiltrates were scored. The number of degranulated mast cells was quantified by staining the slides with toluidine blue. Edema and leukocyte infiltrates were evaluated in sections obtained on the days 2, 7, and 14 after surgery according the scores: (0) absent, (1) mild, (2) moderate, and (3) intense. For this, 15 random fields per slice (n = 5/group/day) were analyzed at ×400 magnification in granulation tissue for edema, also including the crust region for leukocytes. Results are expressed as the median and range of scores (Akkol et al.2009). Photomicrographs of sections were obtained on days 2 and 7 after surgery with a Leica DM microscope equipped with a Leica DFC 280 camera. Differential mast cell counting (degranulated and non-degranulated cells) was performed in 15 fields/slice at ×400, around the edges of normal skin (adjacent to the wound) and the wound area in sections obtained on days 2, 7, and 14 after surgery. The percentage of degranulated mast cells was esti-mated using the following formula: percentage of degranulated mast cells (%) = (number of degranulated mast cells/number of total mast cells) × 100 (Matsumura2007).
Markers and mediators of inflammation
Neutrophil infiltration in wounds was estimated indirectly by myeloperoxidase (MPO) activity, as previously described (Bradley et al.1982). Biopsies were homogenized individual-ly in 50 mM potassium phosphate buffer, pH 6.0, containing 0.5 % hexadecyl-trimethyl-ammonium bromide (Sigma). Samples of 50 mg of tissue were homogenized in 1 mL using a crusher Polytron® PT 3100 (5000g). The resulting samples were centrifuged for 15 min at 1500×gand 4 °C. The super-natant was subjected to three cycles of freeze-thawing at
−20 °C for 10 min and centrifuged again as before. A volume of 7μL was obtained and added to a 96-well microplate, in triplicate. MPO activity in the supernatant was detected using 200 μL of a solution containing 2.78 mg of O-dianisidine dihydrochloride (Sigma), bidistilled water (27 mL), potassium phosphate buffer (3 mL), and 1 % H2O2(15 mL). Enzyme activity was determined by measuring the absorbance (460 nm) at 0 and 1 min. The results were reported as the total number of PMN cells × 103/mg tissue using a standard curve of neutrophils obtained from rat peritoneal exudate and interpreted as myeloperoxidase activity. All analyses were performed in triplicate with samples obtained from three in-dependent experiments and were reproduced without signifi-cant differences.
Nitrite levels in biopsy lysates were determined indirectly as the total content of nitrite and nitrate (NO3−/NO2−) by spec-trophotometry using a method reported for the Griess reaction (Green et al.1982). Aliquots of each sample (80 μL) were incubated with 100μL of Griess reagent (1 % sulfanilamide in 1 % phosphoric acid and 0.1 % naphthalene diamine dihydrochloride in water) and left at 25 °C for 10 min. Optical densities were measured at 540 nm in a microplate reader. Nitrite concentrations in the samples were determined according to a standard curve generated by different concen-trations of sodium nitrite (0.1–100 mM). Data are expressed as micromoles of nitrite. All analyses were performed in tripli-cate with samples obtained from three independent experi-ments and were reproduced without significant differences.
Biopsies were homogenized individually in phosphate-buffered saline (PBS), pH 7.4 and processed (Safieh-Garabedian et al. 1997). The levels of TNF and IL-1β in biopsy lysates were determined using a sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, microtiter plates (96 wells) were coated with 100 μL of anti-TNF or IL-1βprimary antibodies (0.8 or 4μg/mL, kits from R&D Systems) and incubated overnight at 4 °C. The coating solu-tion was removed, and the plates were washed by filling the wells with 290μL of diluted wash buffer (R&D Systems).
Surgical excision and biomembrane implantation Surgical incision and biomembrane implantation
a
b
c
d
e
f
g
h
Fig. 2 Surgical procedures to induce incisional or excisional wounds and biomembrane implantation.aInduction of linear incisional wound.bAppearance of incisional wound.cSeparation of skin edges followed
Then, the remaining protein-binding sites were blocked in the coated wells by adding 290μL of 1 % bovine serum albumin in PBS, pH 7.4. The plates were washed again. Aliquots of samples (100μL) were added in duplicate to wells and incu-bated at 4 °C for 2 h. The plates were washed, and 100μL of anti-TNF or anti-IL-1βsecondary antibodies (R&D Systems) were added to the wells. After further incubation at room temperature for 2 h, the plates were washed and incubated with 100μL of HRP-conjugated streptavidin. The plates were washed, and 100μL of substrate solution (1:1 mixture of H2O2and tetramethylbenzidine) was added in the absence of light. The enzyme reaction was stopped by adding 50μL of H2SO4(2 N), and the absorbance was measured at 450 nm. The results are expressed as picograms/milliliter (pg/mL) and reported as mean ± standard error mean (S.E.M.). All analyses were performed in triplicate with samples obtained from three independent experiments and were reproduced without signif-icant differences.
In vitro analysis of macrophages activated by LP
Peritoneal macrophages of mice were harvested with RPMI (pH 7.4) 4 days after i.p. injection of 3 % thioglycolate (3 mL/ cavity) and cultured in plastic tissue culture dishes (24 wells) at 37 °C (5 % CO2). After 2 h, non-adherent cells were re-moved by three washes with 1 mL of RPMI medium and the
adherent population (95 % macrophages) was maintained at 37 °C (5 % CO2) (Cunha and Ferreira1986). After 24 h, the cells were incubated in fresh medium or in medium containing LP (500 μg/mL) for 1 h. The supernatants were then discarded, and after three further washes, the cells were incu-bated with medium (1.5 mL) or LPS (1μg/mL) for 5 h. The supernatants were used for the cytokine assay. The cell viabil-ity of each macrophage culture was analyzed by the trypan blue exclusion method (Renzi et al.1993).
Statistical analysis
All results are expressed as mean values ± S.E.M.. Statistical significance was assessed by ANOVA follow-ed by Bonferroni’s test for multiple comparisons of the mean or Kruskal-Wallis for the median. The level of significance was determined as P < 0.05 using GraphPad Prism, version 3.02.
Results
Leukocyte recruitment was stimulated in wounds im-planted with BioMem PVA/LP 0.2 % (v/v) (Figs. 3 and 4). This effect was not seen in the controls. The intense edema observed by histology was accompanied
CT
a
b
c
d
e
f
C C C
C
C
C
CT CT
CT
CT CT
PC
PC
SHAM PVA
BioMem PVA/LP 0.2% (v/v)
Excision
al woun
ds
Fig. 3 Histology of excisional wounds on day 2 after surgery.a SHAM,bPVA,cBioMem PVA/ LP 0.2 % (v/v):arrowshows edema in the connective tissue (×100 magnifications).dSHAM, ePVA,fBioMem PVA/LP 0.2 %
(v/v):arrowshows crust with
inflammatory infiltrate (×400 magnifications).Ccrust,CT
connective tissue,PC
with leukocyte infiltrate on day 2 (P < 0.05) (Table 1). Accordingly, the activity of MPO (Fig. 5a, b) was in-creased in both wounding processes, mediated by BioMem PVA/LP 0.2 % (v/v) (day 2), and declined on the following days similar to the SHAM group. This result suggested that BioMem PVA/LP 0.2 % (v/v) pro-moted a more intense inflammatory phase of the healing process.
The subsequent progress of healing was characterized by the displacement of polymorphonuclear cells with mononu-clear cells observed at day 7, which progressed to a lesser extent at day 14 (Tables1and2). It is worth noting that the infiltration of leukocytes persisted even at day 14 in both wound models treated with BioMem PVA/LP 0.2 % (v/v). It should be mentioned that the residual inflammation did not alter the main cellular events observed in the proliferating and
remodeling phases, i.e., fibroplasia and collagen deposition. Meanwhile, the residual inflammation was characterized by mononuclear cells.
Since activated leukocytes are involved in the release of pro-inflammatory cytokines, the levels of TNF and IL-1β were measured in the wounds. There was no significant dif-ference between the SHAM and PVA control groups in both wound models (Fig.6). However, the TNF level was higher in wounds stimulated by BioMem PVA/LP 0.2 % (v/v). Curiously, IL-1βwas augmented in incisional wounds but not in excisional wounds. Among other cytokines, TNF and IL-1βare released by activated macrophages and recruit neu-trophils. Indeed, the in vitro activation of macrophages by LP led to TNF and IL-1βrelease (Fig.7).
The levels of nitric oxide (NO) increased on day 2 in wounds treated with BioMem PVA/LP 0.2 % (v/v) and
Day
2
PC
a
d
g
Day 7
E
PC
Incision
al
woun
ds
SHAM
CT
CT
CT
CT
M
E
PC
b
e
h
EPVA
CT
CT
CT
i
PCc
Ef
CCT
BioM PVA/LP 0
CT
E
CT
*
T
Mem .2% (v/v)
E
T
E Fig. 4 Histology of incisional
wounds on days 2 and 7 after surgery.aSHAM,bPVA, andc BioMem PVA/LP 0.2 %:arrow
shows crust with inflammatory infiltrate on the second day.d SHAM,ePVA, andfBioMem PVA/LP 0.2 %:asteriskshows leukocyte infiltrate in the connective tissue on the seventh day (×100 magnifications).g SHAM,hPVA, andiBioMem PVA/LP 0.2 %: predominance of mononuclear leukocyte in the connective tissue on the seventh day (×400 magnifications).E
epidermis,CTconnective tissue,
subsequently declined, similar to what was observed in the control groups (Fig.8). Curiously, BioMem PVA/LP 1 % (v/ v) had no effect on NO production. It seems that in vivo NO release by activated macrophages is LP dose dependent.
Activated mast cells in wounds were characterized as degranulated by histology (Fig.9a, b). This effect was more intense in both wounding models treated with BioMem PVA/ LP 0.2 % (v/v) (day 2) and was similar on the following days to the control groups. Chemical mediators released by degranulated mast cells enhance the inflammatory process and fibroplasia. Accordingly, the inflammatory and prolifera-tive phases of the healing process were probably stimulated by LP via mast cell activation.
Discussion
The skin plays a pivotal role in maintaining health, and there-fore, the loss of its integrity deserves attention. The skin pro-tects the body against excessive perspiration and serves as the primary protective barrier against pathogens and microorgan-isms (Madison2003, Proksch et al.2008). Skin wounds result from tissue damage caused by a multitude of factors, such as hypoxia, trauma, burns, pressure, or underlying medical con-ditions such as diabetes or surgery (Gulcan et al. 2012; Lokmic et al.2012). Regardless of the starting point, tissue injury to the skin must to be repaired quickly. Biomaterials have been developed in order to promote specific chemical and biological responses along with healing (Brandl et al. 2007). These supports for tissue growth can be produced in the form of foam, hydrogels, or membranes, such as artificial skin (Weigel et al.2006; Weinand et al.2006).
Table 1 Semi-quantitative evaluation of microscopic flogistic signs of excisional wounds
Days SHAM PVA BioMem PVA/LP 0.2 %
2 Edema
1 (1–2) 2 (2–3) 3 (2–3)* Inflammatory infiltrate
2 1.5 (1–2) 2 3 (2–3)*, #
7 2 (1–2) 2 (2–3) 3 (1–3)*, # 14 1 (1–2) 1 (1–2) 2 (1–2)*, #
On days 2,7, and 14 after surgery, animals were sacrificed and samples of neoformed tissue were removed to perform histological analyses. Hematoxylyn-eosin-stained sections were employed to estimate edema and inflammatory infiltrate. Data represent the median and range of scores from three independent experiments: (0) absent, (1) mild, (2) mod-erate, and (3) intense to excisional wounds
*P< 0.05 indicates statistical difference compared with the SHAM group and#P< 0.05 with the PVA group (n= 5 animals/group/time point/ experiment, Kruskal-Wallis test followed by Dunn’s test)
0 20 40 60 80
2nd day
* #
SHAM PVA BioMem PVA/LP 0.2% (v/v)
Neutrophils
x 10
3 /mg of tissue
Neutrophils
x 10
3 /mg of tissue
a
0 3 6 9 12
Days
27 14
* #
*# SHAM
PVA
BioMem PVA/LP 0.2% (v/v) BioMem PVA/LP 1% (v/v)
b
Fig. 5 Myeloperoxidase activity. Animals were sacrificed on day 2 after surgery and samples of excisional (a) or incisional (b) wounds were removed to determine myeloperoxidase activity. Data are mean of three independent experiments and are expressed as mean ± standard error mean (S.E.M.) of neutrophils number × 103/mg of tissue. *P< 0.05 indicates statistical difference compared with the SHAM group and #
P< 0.05 with the PVA group (n = 5 animals/group/time point/ experiment, ANOVA-Bonferroni test)
Table 2 Semi-quantitative evaluation of microscopic flogistic signs of incisional wounds
Days SHAM PVA BioMem PVA/LP 0.2 %
2 Edemas
1.5 (1–2) 2 (1–2) 3 (2–3)* Inflammatory infiltrate
2 1.5 (1–2) 1.5 (1–2) 3 (1–3)* #
7 2 (1–2) 2 (1–3) 2 (1–3)
14 1 (1–2) 1 (1–2) 2 (1–3)* #
On days 2, 7, and 14 after surgery, animals were sacrificed and samples of neoformed tissue were removed to perform histological analyses. Hematoxylyn-eosin-stained sections were employed to estimate edema and inflammatory infiltrate. Data represent the median and range of scores from three independent experiments: (0) absent, (1) mild, (2) mod-erate, and (3) intense to incisional wounds
Previously, the PVA/LP 0.2 % (v/v) biomembrane was shown to improve the healing performance of incisional and excisional wounds (Figueiredo et al.2014). The incisional wound model is used to investigate the properties inherent to the implant after subcutaneous insertion.
This approach allows for evaluating neoformed tissue macro- and microscopically, as well as assessing immunolog-ical aspects without the influence of additional factors, such as microorganisms (Figueiredo et al.2014, Kyariakides et al. 2001). The excisional wound model, in contrast, involves a significant loss of skin and subcutaneous tissue. Therefore, the healing process can be monitored based on the wound area, the histological organization of connective tissue, and re-epithelialization (Davidson1998).
It was interesting to observe the presence of neutrophils even on the later stage of tissue remodeling, suggesting that the inflammatory phase of the healing process continued dur-ing the proliferatdur-ing and remodeldur-ing phases. Neutrophil infil-tration normally lasts for only a few days, after tissue injury and at the beginning of the healing process, characterizing the inflammatory phase. Infected wounds face prolonged inflam-matory phase with intense neutrophilic infiltrate that may de-lay healing (Li et al.2007). In this context, the results present-ed here suggest that wounds treatpresent-ed with PVA or BioMem
PVA/LP 0.2 % (v/v) were not subject to an infectious process since the healing process was improved.
It is also suggested that the predominance of mononuclear leukocytes infiltrate on days 7 and 14 in the animals treated with BioMem PVA/LP 0.2 % (v/v) was due to the stimulus given in the early inflammatory phase of wound healing (day 2) and do not typify as continuous stimulus. These findings are corroborated by the reduction in myeloperoxidase activity from day 2 to days 7 and 14 after surgery. It is therefore concluded that LP displayed pro-inflammatory properties.
In an independent study, a water-soluble latex extract in-duced in vitro NO release by macrophages (Al Seddek et al. 2009). These authors also observed an in vivo increase in peritoneal macrophages 4 days after treatment. In the present study, an increase in NO was observed in incisional wounds (day 2), also suggesting the activation of macrophages in ad-dition to activated neutrophils. NO was not consistently quan-tified in excisional wounds. This would explain, at least in part, the intensified inflammation induced in the earlier in-flammatory phase of PVA/LP 0.2 % (v/v) treated wounds. According to the literature, increases in fibroplasia and colla-gen deposition are stimulated by NO (Madigan et al.2011). This is concordant with incisional wounds treated with BioMem PVA/LP 1 % (v/v), i.e., fibroplasia but not collagen
0 500 1000 1500 2000 2500
2nd day
SHAM PVA BioMem PVA/LP 0.2% (v/v)
* #
a
TNF (pg/mL)
0 500 1000 1500 2000
2nd day
* #
SHAM PVA BioMem PVA/LP 0.2% (v/v)
c
IL-1
β
(pg/mL)
0 200 400 600 800 1000
2nd day
SHAM PVA BioMem PVA/LP 0.2% (v/v)
* #
b
TNF (pg/mL)
0 500 1000 1500 2000
2nd day
SHAM PVA BioMem PVA/LP 0.2% (v/v)
d
IL-1
β (pg/mL)
Fig. 6 TNF and IL-1βlevels. On day 2 after surgery, animals were sacrificed and samples of incisional (a,c) and excisional wounds (b,d) were removed to determine cytokines levels by ELISA. Data are mean of three independent experiments and are expressed as mean ± standard
error mean (S.E.M.) of cytokine/mL from supernatant/mg tissue. *P< 0.05 indicates statistical difference compared with the SHAM group and#
deposition was favored, as revealed at day 14. It is possible that the higher levels of TNF and IL-1βdetected on day 2 after surgeries may have been stimulated not only by macro-phages as the first activated cells, but also later by recruited neutrophils, to a lesser extent.
A biomembrane made with the natural latex extracted from the rubber tree Hevea brasiliensis also stimulated healing (Andrade et al. 2011). The implant induced a large amount of neoformed tissue, but the membrane was not reabsorbed during the cicatricial process, which is an undesired effect. The inflammatory phase was stimulated by leukocyte infiltration and IL-1β release. The involvement of NO was not documented. The healing process was characterized by fibroplasia but not collagenesis. In comparison, BioMem PVA/LP 0.2 % (v/v) seems to display better performance. It is worth noting that any adverse effect was documented in animals receiving the biomembrane (PVA or PVA plus LP) in terms of macroscopic observation, animal behav-ior (discomfort or touchiness), or interference on data collecting. These are secondary data but also important to be documented since side-effects and other undesired
0 200 400 600 800
*
*
a
RPMI LPS LP
Macrophages (MØs)
TN
F
(ρ
g/
m
L
)
0 50 100 150 200
*
*
b
RPMI LPS LPMacrophages (MØs)
IL
-1β (ρ
g/
m
L
)
Fig. 7 TNF and IL-1βlevels. Macrophages culture was treated with RPMI, LPS (1μg/mL), or LP (500μg/mL), and the supernatants obtained were used to determine cytokines levels by ELISA. Data are expressed as mean ± standard error mean (S.E.M.) of TNF (a) and IL-1β(b) levels. *P< 0.05 indicates statistical difference compared with RPMI (n= 5 wells /group, ANOVA-Bonferroni test)
0 50 100 150 200
SHAM PVA
BioMem PVA/LP 0.2% (v/v) BioMem PVA/LP 1% (v/v) *#
7 2
Days
NO
3
-/N
O2
-(m
M
)
Fig. 8 Nitrite levels. On days 2, 7, and 14 after surgery, animals were sacrificed and samples of incisional wounds were removed to determine nitrite levels by Griess reaction. Data are mean of three independent experiments and are expressed as mean ± standard error mean (S.E.M.) of nitric oxide (NO3/NO2) levels (μM). *P< 0.05 indicates statistical difference compared with the SHAM group and#P< 0.05 with the PVA group (n= 5 animals/group/time point/experiment, ANOVA-Bonferroni test)
0 20 40 60 80 100
Degranulated Mast Cell (%)
2 7 14
Days *
SHAM PVA BioMem PVA/ LP 0.2% (v/v)
a
0 15 30 45 60 75
Degranulated Mast Cell (%)
2 7 14
Days
*
*# *# SHAM PVA
BioMem PVA/LP 0.2% (v/v) BioMem PVA/LP 1% (v/v)
b
features are frequently limiting to develop new products for human health.
In the past quarter century, biomaterials of synthetic or biological origin have evolved significantly as wound dress-ings (Ovington2007). However, the higher cost of producing synthetic drugs remains an obstacle to develop new wound dressings (Macdonald and Asiedu2010). Therefore, the search to identify new wound healing agents in medicinal plants, with lower toxicity and cost, is currently of great interest.
The search for both new active chemicals on wound healing processes and for inert matrices capable of absolving/retaining and delivering these active compounds are intense. While we have found and proposed a protein-based biomembrane, the literature concerned describes differ-ent and interesting/promising materials for the matrix but al-most all associated to natural products (secondary metabolites or their chemical derivatives) that as a rule displays anti-inflammatory activity, affecting the natural and necessary first phase of the wound healing. Instead, our proposed system efficiently modulates the inflammatory phase in such a way it is warranted and improves the whole healing process mak-ing it briefer.
Conclusion
PVA/LP is a biomembrane composed of 1 % polyvinyl alco-hol supplemented with latex proteins extracted from C. procera. The biomembrane, applied topically as a wound dressing, enhanced wound healing and was completely reabsorbed. The PVA component was considered inert while the mechanisms underlying LP activity was characterized as pro-inflammatory without any adverse effects observed. The results reported here will certainly stimulate new studies to evaluate PVA/LP performance in a realistic, clinical setting. However, to establish the underlying mechanism how LP could modulate the inflammatory mechanism during wound healing will deserve much more efforts.
Acknowledgments This study is part of the consortium Molecular and Functional Biotechnology of Plant Latex and supported by the following Brazilian agencies: CNPq (RENORBIO and Universal), CAPES, and FUNCAP.
Compliance with ethical standards All applicable international, na-tional, and/or institutional guidelines for the care and use of animals were followed. All the experimental procedures performed in studies involving animals were approved by the institutional committee for animal use and care following approval under the number 39/2014.
Conflict of interest The authors declare that they have no potential financial conflicts of interest.
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