1. Introduction
Burn wounds represent a major public health issue with significant morbidity and mortality. They are associated with prolonged hospitalization, disfigurement, disability, and social stigma, while in severe cases their outcome can be fatal, as according to the World Health Organization (WHO) approximately 11 million burn injuries and 180,000 deaths related to severe burns is estimatedtooccur annually worldwide [
1,
2,
3,
4]. They are among the most common injuries of the skin and depending on the depth of skin injury, they are classified as superficial/epidermal (1st degree), partial-thickness (2nd degree), and full-thickness (3rd degree) burns [
5].
Wound healing occurs as a natural response of the body to restore its injured tissues. It is a dynamic and complex process consisting of four interconnected and often overlapping phases that include haemostasis, inflammation, proliferation, and remodeling. Characterized by various biophysiological functions and different predominant cells, the proper sequence, duration, and intensity of these phases are crucial for the complete healing of the wounds [
6].With several biological events and biochemical components involved in each phase, the breakdown of this well-coordinated sequence results in incomplete wound healing
, affecting ultimately the functional recovery of the wound [
6].
To facilitate the healing process, various wound dressings have been developed over the years. Wound dressings should have the ability to restore theskin barrier function, prevent infection, manage fluid loss, control the excess of exudates, and reduce scar formation [
7]. The selection of suitable materials that can promote wound healing is of high importance for designing effective wound dressings.
Recently, marine polysaccharides have emerged as valuable biomaterials for the development of advanced wound dressing systems [
8,
9,
10,
11]. Marine polysaccharides are highly biocompatible, biodegradable, and abundant biopolymers with diverse structures and functionalities [
12,
13]. Exhibiting unique physicochemical properties and a broad spectrum of biological activities, they are attracting considerable attention for their utilization as safe biomaterials in various novel biomedical systems [
14,
15,
16,
17,
18,
19]. Due to their ability to absorb wound fluids and maintain a moist environment in the wound area, they are considered ideal wound dressing components promoting various phases of endogenous wound healing in different types of wounds when formulated in films, fibers, and hydrogels [
9,
20].
Among them, ulvans, amply present in the cell walls of green macroalgae of the order Ulvales,arecomplex anionic sulfated polysaccharides that are mainly composed of sulfated rhamnose, glucuronic and iduronic acids, and xylose [
21,
22,
23]. Exhibiting chemical analogy to glycosaminoglycans, intrinsic cytocompatibility, and significant antioxidant, anti-inflammatory, antiviral, antihyperlipidemic, and antitumor activity, ulvansare attractiveas biomaterial for the development of hybrid composites, particles, nanofibrous matrices, hydrogels, multilayer films, membranes, and polyelectrolyte complexes [
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36]. Due to the presence of rare sugars, such as sulfated rhamnose and iduronic acid that may enhance their wound healing potential, theyare considered a promising biopolymer for the development of novel wound dressing systems for the treatment of various skin inflammations [
11,
37,
38].
Carrageenans are the main components of the cell walls of many red seaweeds. They are anionic sulfated polysaccharides composed of alternating units of D-galactose and 3,6-anhydro-D-galactose linked by α-(1,3) and β-(1,4) glycosidic bonds and depending on the sulfation degree and the 3,6-anhydro-D-galactose content, they are classified into three principal forms, namely kappa (κ)-, iota (ι)-, and lambda (λ)-carrageenans[
10,
15,
39,
40,
41,
42,
43]. Due to their low cost, resemblance to native glycosaminoglycans, and significant biological activities (antioxidant, anti-hyperlipidemic, anticoagulant, antiviral, antitumor, and immunomodulatory), they are widely used in the food, cosmetics, and pharmaceutical sectors [
39,
40,
44,
45]. Among them, κ-carrageenansexhibiting enhanced gelling properties, are considered a promising biopolymer for tissue regeneration and wound healing applications [
10,
44,
46,
47].
Carrageenans at concentrations ranging from 0.5 to 1.5% w/w haveshown potential wound healing enhancement both in vivo [
48,
49] and in vitro [
50]. However, carrageenans have never been tested at higher doses. Additionally, ulvans have shown in vitro antioxidant and antibacterial properties at concentrations of 5%, 7.5% and 10% w/w [
33].
The aim of the present study was to investigate the healing efficacy of ulvan and carrageenan at concentrations between 1.5 to 10% w/w, to define the optimal beneficial dose for the successful treatment of second-degree burns in mice and compare the healing potential of the two polysaccharides.
Thus, in the present work, we have investigated the burn wound healing potential of ulvan and carrageenan, formulated in gels for topical application on second-degree burn wounds. Gels of 4% polyacrylamide, C13-14 isoparaffin and laureth-7, incorporatingulvan or carrageenan at 1.5, 5.0, and 10% w/w, were prepared and evaluated for their wound healing efficacy on the burn-inflamed skin of SKH-hr2 hairless female mice. To evaluate and compare the therapeutic effect of the two polysaccharides, histopathological analyses and clinical evaluation of the skin were performed. Wound area, skin texture, and hemoglobin levels were determined using photo-documentation, while biophysical skin parameters, such as transepidermal water loss (TEWL), hydration, and skin thickness, were also assessed. Moreover, the wound healing efficacy of the biopolymer-based gels was evaluated by recording in vivo the changes in the skin of mice before, during, and at the end of the treatment, by FT-IR spectroscopy.
4. Discussion
Clinical evaluation demonstrated that the mice treated with the 10% w/wcarrageenan gel showed significantly enhanced wound healing in comparison to all other groups. The mice in this group approached almost complete wound closure, earlier than the other groups, from day 21 (
Figure 1). Mice treated with the 5% w/w ulvan gel also demonstrated a significant healing effect, ranking second to the group which was treated with the 10% w/w carrageenan gel, asthey approached total wound healing on day 23 (
Figure 1).
During the experiment, the wound area was measured on various days and the healing rate was calculated. The measurement of wound areas revealed that mice treated with the 10% w/w carrageenan gel exhibited significantly smaller wounds compared to all other groups starting from day 6.This statistically significant difference in wound area was maintained for the vehicle group until the end of the experiment (
Figure 2A).
The 10% w/w carrageenan gel accelerated the healing process significantly in the first 15 days of the experiment, having the greatest slope compared to all other treatments (
Figure 2B,
Table 2). The 5% w/w ulvan gel appeared to accelerate the healing process during the second period, from day 15 to 26 (
Figure 2B). It seems that the process of wound healing can be divided into two distinct phases, each progressing at a different rate. The initial phase is characterized by a faster rate (days 1-15), followed by the second phase, which exhibits a slightly slower progression (days 15-26).
Histopathological examination and haemoglobin measurements indicated that mice treated with the 10% w/w carrageenan gel and the 5% w/w ulvan gel exhibited reduced inflammation levels compared to the other groups. This observation highlights the significance of dosage in relation to therapeutic effectiveness. The excipient, on the other hand, displayed more intense inflammation than all the other treatments, including the control. It is, therefore, not unlikely, that the utilization of this gelling agent might have attenuated the therapeutic efficacy of the polysaccharides (
Figure 3 and
Figure 4,
Table 4).
Concerning the biophysical parameters, the 10% w/w carrageenan and the 5% w/w ulvan treatments resulted in significantly higher hydration values, whereas TEWL and skin thickness were not restored to their initial levels in any of the groups(
Figure 5).
The optimum efficacy of the 10% carrageenan gel compared to the 5%ulvan gel was also observed in the FT-IR spectra of the mice skin. As noted in the spectrum of the mice treated with the 10% carrageenangel (
Figure 7),the increase in the intensity of the
vNH and
vOHabsorption bands at approximately 3500 cm
-1 and 3300 cm
-1 indicated the formation of aminoglycans. Additionally, the band at 1772 cm
-1, assigned to Ig-COO
-, suggested the presence of inflammation. Negative peaks of amide I and amide II bands in the subtractive spectrum revealed that, although the histopathological data were similar, the proteins did not attain the natural α-helix secondary conformation after the application of the 5% ulvan gel.A crucial observation was the increased intensity of the band in the spectral region between 1200-1000 cm
-1, where the
vC-O-C of sugar rings and bridges absorb, confirming the development of collagen and elastin (
Figure 6 and
Figure 7).
Apparently, the dosage is closely related to the therapeutic effectiveness. Carrageenan exhibited optimal performance at the highest of the tested concentrations (10% w/w), while ulvan at an intermediate concentration (5% w/w) (
Figure 1,
Figure 2 and
Figure 3 and 7,
Table 3 and
Table 4).
It has been suggested that carrageenan could be used in films as a wound healing agent [
10],and its wound healing efficacy has been proven byvarious
in vitro [
50,
58] and in vivo studies [
48,
49]. The beneficial effects of ulvan in burn wounds have been also reported in an
in vivo study where the combination of ulvan with gelatin formulated in electrospunnanofibers showed a significant burn healing effect [
54].
Despite the uniform burns inflicted at the beginning of the experiment, mice treated with the vehicle exhibited a poorer clinical outcome compared to the control. With the vehicle, keloids and oedema were developed during the healing process, a fact that complicated the removal of necrotic tissue. Apparently, the gelling agent (polyacrylamide, C13-14 isoparaffin, and laureth-7) is responsible for the observed secondary effects.It seems that the use of polyacrylamide, C13-14 isoparaffin, and laureth-7 on an open wound may cause this reaction. According to the literature, the gelling agent is not toxic to normal skin, except for a single case of allergic contact dermatitis [
59].
In a future study, the efficacy of the optimal dose of the carrageenan gel could be examined using an alternative gelling agent. It may also be important to explore the potential synergistic effects of the two polysaccharides, carrageenan at 10% w/w and ulvan at 5% w/w, either in combination or not, with a therapeutic drug.
Author Contributions
Conceptualization, J.A., E.I., V.R. and MCR.; methodology, D.S., A.V., S.K.; investigation, D.S., A.P., M.K., I.S.,A.V. and S.K.; resources, E.I., V.R. and M.R.; writing—original draft preparation, D.S, A.P., A.V., J.A, S.K. and MCR; writing—review and editing, D.S., E.I., V.R. and MCR.; visualization, D.S., J.A. and M.K.; supervision, J.A. and MCR.; project administration, D.S..; All authors have read and agreed to the published version of the manuscript.
Figure 1.
Representative images of mice burn wound areas receiving the different treatmentson days 1, 3, 6, 15, 21, 23 and 26.
Figure 1.
Representative images of mice burn wound areas receiving the different treatmentson days 1, 3, 6, 15, 21, 23 and 26.
Figure 2.
(A) Histogram of wound area reduction in relation to time. (B) Wound healing rate(*p0.05, **p0.01, ***p0.001).
Figure 2.
(A) Histogram of wound area reduction in relation to time. (B) Wound healing rate(*p0.05, **p0.01, ***p0.001).
Figure 3.
Hematoxylin and eosin-stained sections of mice skin at the burn wound area for mice receiving different treatments (100 x).
Figure 3.
Hematoxylin and eosin-stained sections of mice skin at the burn wound area for mice receiving different treatments (100 x).
Figure 4.
Histograms of (A) haemoglobin, and (B) skin texture of mice receiving different treatments in relation to time (*p0.05, **p0.01).
Figure 4.
Histograms of (A) haemoglobin, and (B) skin texture of mice receiving different treatments in relation to time (*p0.05, **p0.01).
Figure 5.
Histograms of (A) hydration, (B) transepidermal water loss (TEWL), and (C) skin thickness of different treatments at the beginning and the end of the experimental procedure (*p0.05, **p0.01, ***p0.001, ****p0.0001).
Figure 5.
Histograms of (A) hydration, (B) transepidermal water loss (TEWL), and (C) skin thickness of different treatments at the beginning and the end of the experimental procedure (*p0.05, **p0.01, ***p0.001, ****p0.0001).
Figure 6.
FT-IR spectra of mice skin treated with carrageenan and ulvan at various concentrations during the final stage of wound healing.
Figure 6.
FT-IR spectra of mice skin treated with carrageenan and ulvan at various concentrations during the final stage of wound healing.
Figure 7.
FT-IR spectra of miceskin during wound healing: (1) spectrum of miceskin treated with the 5% w/w ulvan gel, (2) spectrum of miceskin treated with the 10% w/w carrageenan gel, and (3) spectrum resulting from the subtraction of spectrum 1 from spectrum 2.
Figure 7.
FT-IR spectra of miceskin during wound healing: (1) spectrum of miceskin treated with the 5% w/w ulvan gel, (2) spectrum of miceskin treated with the 10% w/w carrageenan gel, and (3) spectrum resulting from the subtraction of spectrum 1 from spectrum 2.
Table 1.
Classification of mice according to the administeredgels and their concentrations (w/w).
Table 1.
Classification of mice according to the administeredgels and their concentrations (w/w).
Mice classification |
Treatment |
Control |
no treatment |
Vehicle |
Pure gel with the excipients (gel with PIL) |
Ulvan 1.5% |
1.5% ulvan gel |
Ulvan 5% |
5% ulvan gel |
Ulvan 10% |
10% ulvan gel |
Carrageenan 1.5% |
1.5% carrageenan gel |
Carrageenan 5% |
5% carrageenan gel |
Carrageenan 10% |
10% carrageenan gel |
Table 2.
Scoring system for the histopathological evaluation of burn wound healing.
Table 2.
Scoring system for the histopathological evaluation of burn wound healing.
|
absence |
mild |
moderate |
heavy |
Inflammation |
0 |
1 |
2 |
3 |
Oedema |
0 |
1 |
2 |
3 |
Hyperkeratosis |
0 |
1 |
2 |
3 |
Wound thickness |
0 |
1 |
2 |
3 |
Ulceration |
0 |
1 |
2 |
3 |
Necrosis |
0 |
1 |
2 |
3 |
Parakeratosis |
0 |
1 |
2 |
3 |
Table 3.
Wound reduction equations, as derived from linear regression analysis, for the different treatments and concentrations (w/w) regarding the first half (days 1-15) and the second half (days 15-26) of the healing process.
Table 3.
Wound reduction equations, as derived from linear regression analysis, for the different treatments and concentrations (w/w) regarding the first half (days 1-15) and the second half (days 15-26) of the healing process.
|
Day 1–Day 15 |
Day 15–Day 26 |
Control Vehicle Ulvan 1.5% Ulvan 5% Ulvan 10% Carrageenan 1.5% Carrageenan 5% Carrageenan 10% |
Y = -13.44*X + 274.8 |
Y = -4.574*X + 112.1 |
Y = -12.83*X + 286.8 |
Y = -5.839*X + 140.6 |
Y = -13.76*X + 293.0 |
Y = -5.841*X + 140.9 |
Y = -13.51*X + 281.8 |
Y = -4.301*X + 101.5 |
Y = -12.06*X + 283.6 |
Y = -6.215*X + 148.8 |
Y = -14.20*X + 288.3 |
Y = -3.866*X + 92.23 |
Y = -12.23*X + 276.3 |
Y = -5.588*X + 132.6 |
Y = -15.59*X + 271.6 |
Y = -2.656*X + 62.78 |
Table 4.
Histopathological results according to different treatments: evaluationof inflammation (Inf), oedema (Oed), hyperkeratosis (HPKe), wound thickness (WTh), ulceration &necrosis (Ulce&Ne) and parakeratosis (PKe).
Table 4.
Histopathological results according to different treatments: evaluationof inflammation (Inf), oedema (Oed), hyperkeratosis (HPKe), wound thickness (WTh), ulceration &necrosis (Ulce&Ne) and parakeratosis (PKe).
Treatment |
Inf |
Oed |
HPKe |
WTh |
Ulce&Ne |
PKe |
Total Score |
Control |
2.0 |
3.0 |
3.0 |
3.0 |
0 |
1.0 |
12.0 |
Vehicle |
3.0 |
3.0 |
2.7 |
3.0 |
0 |
0.7 |
12.4 |
Ulvan 1.5% |
2.0 |
2.3 |
2.3 |
2.3 |
0 |
0.3 |
9.2 |
Ulvan 5% |
1.7 |
1.0 |
2.0 |
1.7 |
0 |
0 |
6.4 |
Ulvan 10% |
2.7 |
2.0 |
2.0 |
2.0 |
0 |
0 |
8.7 |
Carrageenan 1.5% |
3.0 |
3.0 |
2.0 |
2.0 |
0 |
0 |
10.0 |
Carrageenan 5% |
3.0 |
3.0 |
3.0 |
3.0 |
0 |
0 |
12.0 |
Carrageenan 10% |
1.3 |
1.0 |
1.0 |
1.3 |
0 |
0 |
4.6 |