Comparative Study of Morphometric and Fourier Transform Infrared Spectroscopy Analyses of the Collagen Fibers in the Repair Process of Cutaneous Lesions

Abstract

Objective: Compare the efficacy of light-emitting diode (LED) and therapeutic ultrasound (TUS), combined with a semipermeable dressing (D), at forming collagen in skin lesions by morphometry and Fourier transform infrared spectroscopy (FT-IR).

Materials and Methods: Surgical skin wounds (2.5 cm) were created on 84 male Wistar rats divided into four groups (n=21): Group I (Control), Group II (LED), Group III (LED+D), and Group IV (US+D). On days 7, 14, and 21, the tissue samples were removed and divided into two pieces, one was used for histological examination (collagen) and the other for FT-IR.

Results: The histomorphometric analysis showed no significant differences among groups for collagen deposition at 7 days. However, at 14 days, more deposition of collagen was noted in the groups LED (p<0.05) and LED+D (p<0.001) than in the control. At 21 days, the groups LED, LED+D, and US+D presented significantly greater deposition of collagen when compared with the control. The FT-IR spectra, at 14 days, LED+D had greater amounts of type I collagen, a better organization of fibers, and greater difference of mean separation between the groups, not observed at 7 and 21 days.

Innovation: The histomorphometric and FT-IR analyses suggest that the association of semipermeable dressing to LED therapy and to TUS modulates biological events, increasing fibroblast/collagen response and accelerating dermal maturation.

Conclusion: The histomorphometric and FT-IR analyses showed that LED therapy is more efficacious than TUS, when combined with a semipermeable dressing, and induced the collagen production in skin lesions.

Veruska Cronemberger Nogueira, PhD

Introduction

Collagen is the most abundant structural protein in the body and a biopolymer, which is a triple helical ribbon formed by three polypeptide chains, each one made up to frequent the Gly-X-Y sequence structured as α-helices, where proline and hydroxyproline are often in X and Y positions, respectively. These three α-helices are organized to form the characteristic structure of type I collagen; a three-dimensional (3D) aggregation of triple helices in collagen fibrils.^1,2 It is critical for the formation of the extracellular matrix of connective tissue.^3,4 It is important to quantify this protein in skin wounds because its formation is associated with the tissue repair process.^3–5

The macroscopic and histomorphometric analyses provide qualitative and quantitative benefits in assessing the evolution of tissue repair process. However, new analytical techniques have been studied for a better understanding of tissue repair process.^4,6,7

Among the optical spectroscopic techniques, Fourier transform infrared spectroscopy (FT-IR) stands out for its ability to identify biological tissue components through the characterization of vibrational modes of molecular radicals.⁸ It produces spectral images of histological sections and identifies biomolecular changes in tissues by studying the variance of the vibrational bands of biomolecules.⁹

FT-IR is based on molecular characteristics associated with various physiological and pathological conditions, which may be called molecular histopathology.³

This analytical tool can be used to record information, identifying the structural characteristics of collagen networks, such as the degree of fiber organization and the secondary structure of collagen (α-helices), which could lead to differentiation of the types of collagen in the healing process.^3,10,11

The treatment of cutaneous wounds is dynamic and depends on the evolution of the stages of the repair process. One important method currently used to treat a wound is the application of a dressing, which facilitates the healing process of skin lesions protecting the area and helping the healing wound. An ideal dressing is able to ensure optimal healing principles.^12–14

There is a wide variety of industrialized dressings, each one suitable for a different type of skin wound.^12,13,15 Among these dressings, the semipermeable film stands out. It consists of polyurethane polymer and has selective permeability, which provides a humid environment that is advantageous for healing, enabling fewer dressing changes in superficial wounds without exudate.^14,16 Light-emitting diode (LED) therapy and therapeutic ultrasound (TUS) represent alternative therapeutic interventions for the treatment of wounds.^5,7,17

Recent research has shown that the photomodulation caused by LED therapy acts on the cell and its permeability, and on the mitochondria, stimulating the synthesis of ATP, which provides energy for the synthesis of proteins such as collagen and elastin.^18,19

On the other hand, scientific reports show that TUS enhances the macrophagic response at the injury site, reducing the inflammatory cells and stimulating the formation of granulation tissue and the migration of fibroblasts, which synthesize the extracellular matrix to support newly formed tissue.^13,20,21

Clinical Problem Addressed

Recent studies have used a semipermeable film dressing and have demonstrated that the therapeutic effects of TUS, the application of which is limited to wound edges, can be extended to the wound bed due to the low attenuation properties of the films, resulting in an increased speed of healing, positively impacting the quality of scar tissue.^16,22,23

In a previous study, our results (unpublished data) showed that the association of LED therapy with a semipermeable film dressing application could improve the outcome of the healing process. The association of the dressing determined the modulation of reepithelialization, neoangiogenesis, collagen formation and also promoted the rapid replacement of collagen type III fibers by type I fibers and the remodeling of these fibers in the injured area.²⁴

Currently there is not much information about the biomolecular alterations that occur during the evolution of the repair process when applied with different therapies. Therefore, this study aimed to compare two technical analyses, histomorphometry (the gold standard) and FT-IR (noninvasive), to evaluate the molecular alterations and the histological characteristics observed with the association of LED or ultrasound with a semipermeable dressing in the healing process of surgical wounds in rats.

Materials and Methods

This study was approved by the Ethics Committee of the Faculty Integral Differential (FACID), Teresina, Piauí, Brazil (No. 246/2009), and was carried out according to the Arouca Law No. 11.794/2008, following the guidelines and regulations for animal research. It used 84 male Wistar rats (Rattus norvegicus), 60 days and±250 g. The animals were kept in individual cages with food and water available ad libitum and a 12-h light/12-h dark cycle of 12 h each.

Surgical procedure

All of the animals were anesthetized intramuscularly with ketamine hydrochloride (10%, 0.1 mL/100 g/kg) associated with the same dose of xylazine chloride (2%). An area in the dorsolateral region, measuring 6×4 cm, was shaved and cleaned with iodine alcohol (4%). A 2.5-cm diameter circular skin wound (full-thickness) was produced, using a punch with a No. 4 blade on its lower end, until complete removal of the tissue. Surgical wounds were carried out in a standardized manner, in the same region of the skin and with an equal area and depth (until the reticular dermis) for all animals.

Semipermeable dressing, LED therapy, and ultrasonic irradiation

All of the therapies used in this study started at 48 h after the operation, when there was a significant reduction of inflammatory exudates; the skin was cleaned with 0.9% sodium chloride solution.

A Tegaderm^® film roll was used (3M of Brazil Ltda.), consisting of a semipermeable, transparent polyurethane moisture-retaining film, with properties that stimulate tissue repair. This type of film is recommended for uninfected open skin wounds and must be renewed every 7 days or when the material becomes saturated.^16,22,23

The dressing was applied according to need and surpassing the edges of the wound. A gentle pressure was applied to the film to ensure perfect adhesion. To remove the dressing, it was lifted at one end and a hand was placed under the bandage to support the skin, while the bandage was carefully pulled parallel to the skin in the direction of the fur.

An LED was produced for this research by the Institute of Research and Development of UNIVAP (IP&D, Brazil), with a spectral band of 640±20 nm, output power of 30 mW, and a total dose of 16 J/cm². A clinical ultrasound (Sonopulse Special; IBRAMED) was used for the sonic irradiation. It was pulsed with a fundamental frequency of 3 MHz, had a power density of 0.5 W/cm², a pulse repetition frequency of 16 Hz at 50%, an effective irradiation area of 3.5 cm,² and a 2-min application time in direct contact with circular motions.^7,21,25,26 The manufacturer calibrated the equipment before and after the experiment, ensuring that there was no loss of strength during the treatment of the experimental groups.

The animals were randomly divided into four groups of 21 animals each, according to the treatment protocol: Group I (control, no treatment), Group II (LED therapy, at a single point on the wound bed, total dose of 16 J/cm², LED), Group III (LED combined with a dressing, at a single point on the wound bed, total dose of 16 J/cm², LED+D), and Group IV (ultrasound combined with a dressing on the wound bed, US+D). They were subdivided according to the observation time of 7, 14, and 21 days (n=7/group), corresponding to different stages of the repair process. The remaining applications were carried out on alternate days, following the described protocols, in such a way that there were 3 applications in the 7-day groups, 7 applications in the 14-day groups, and 10 applications in the 21-day groups.

At the end of each experimental time, seven animals from each group were euthanized and the skin of the region of interest was excised and stored according to the type of analysis (histological or FT-IR).

Histological and morphometric analysis

The anatomical piece was removed with a sterile scalpel, with a 1-cm margin around the wound. It was then hemisectioned, identified, and one part was stored in 10% formalin and sent for histological processing, taking into account that the histopathological analysis is considered to be the gold standard for the assessment of the evolution of tissue repair.²⁷

Longitudinal histological sections, perpendicular to the skin surface of 5 μm, were obtained and then stained with Masson's trichrome (TM) to identify the collagen fibers. The histological analysis and morphometry were then carried out, based on digital images of three regions of the surgical wound (right margin, central region, and left margin) of each animal, using a Leica^® DMLB2 optical system (40×).

Quantitative and qualitative analysis of fibrillar collagen

For the quantitative and qualitative study of the volume of tissue occupied by fibers containing collagen, histological sections from all of the groups were stained with TM. These techniques stain the fibrillar collagen an intense blue when observed under standard light (unpolarized).

Six digital images were obtained from the histological sections for each staining technique, using a uniform randomized sampling for each slide, to observe the entire length of newly formed tissue in the repair process.

For this purpose, a system consisting of a Nikon Eclipse E600^® microscope (400×magnification) coupled to a Nikon DXM1200F^® digital video camera was used. The high-resolution digital images were captured using the Nikon program (ACT-1 version 2.62 2000^®) and a computer coupled to the system.

For the quantitative analysis, the TM images were processed using Image Pro-Plus 4,1 for Windows (Media Cybernetics-Silver) to select, according to the color scale, only the fibers containing collagen, visualized by staining in blue (Fig. 1). This enabled the calculation of the following areas:

• (a) Reference compartment in the reticular dermis, which corresponded only to regions identified as tissue, a result of the injury repair process.

• (b) Area occupied by fibrillar collagen within this compartment.

Figure 1.

Illustrative photographs of the photomicrograph of the papillary dermis showing the collagenous fibers distributed over the scar tissue (A). The yellow dotted square is a segmented image with collagenous fiber selection (B). Masson trichrome staining, 400×. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

The ImageJ software was used to calculate the following: (i) the reference area, shown by dotted lines in Fig. 1A (A_R) and (ii) the area of collagen fibers, the white region (A_CF). The fraction of the volume occupied by a structure in a 3D space can be approximated by its area in a 2D space, multiplied by the volume thickness. Therefore, it is possible to calculate the percentage fraction of the volume of collagen fibers in a wound using the following equation²⁸:

where A_L is the area of the final wound (measured on the animal), and T is the thickness of the wound.

The qualitative analysis of the slides stained with TM enabled the differentiation of collagenous fibers according to density. The thick fibers were classified as type I and the thin fibers as type III.

Statistical analyses

The histomorphometric data were statistically analyzed using ANOVA followed by the Tukey test, with a 95% confidence interval. The analyses were carried out using GraphPad Prism 5.0 software (Windows platform).

FT-IR analyses

The other fraction of the sample (for the FT-IR analysis) was immediately placed in a 1.2-mL Nalgene^® cryogenic tube and stored in liquid nitrogen (−196°C), until the completion of the experimental reading, to preserve the tissue structures of the fragments.

The samples were deposited in a dry state using a 5301 Eppendorf concentrator. The spectra of the samples were recorded in the range of 4,000–900 cm⁻¹, with 64 scans and a spectral resolution of 4 cm⁻¹, using a Spectrum 400 coupled to an ATR module with power control (PerkinElmer). The spectra were inserted into the software Origin (v8.5) and subjected to signal processing as described in the following: (i) the mean spectrum was calculated for each sample, (ii) the spectra were smoothed using the adjacent mean to remove the background noise, (iii) the spectra were normalized vectorially, and finally, (iv) all of the spectra were centered on the mean for statistical analysis through principal component analysis using the Minitab program. The first six principal components (PC1–PC6) were used in the linear discriminant analyses with the aid of the cross validation method.^6,29–31 The linear discriminant analysis was carried out for each group, as a function of experimental time, to differentiate the separation in the repair phases. The second derivative of the spectra was used to show change in the bands according to the treatment.

Results

Morphometric analysis

Regarding the percentage of collagen fibers present in the repair tissue of the surgical lesions, there was no significant difference between groups at day 7, despite the trend of an increase in the mean and standard deviation in the LED+D group (1.76±125) compared with the other groups (Fig. 2).

Figure 2.

Percentage of collagen fibers in surgical lesions of the experimental groups in 7 days. Accompanying mean value±standard deviation.

At 14 days, the LED (10.27±1.11, p<0.05) and LED+D (15.99±1.27, p<0.001) groups had greater deposition of collagen fibers when compared with the control group (7.65±980). The LED+D also showed a significant difference (p<0.01) when compared with the LED and US+D (9.36±1.49) groups (Fig. 3).

Figure 3.

Percentage of collagen fibers in surgical lesions of the experimental groups in 14 days. The p-values are indicated by the * accompanying mean value±standard deviation (*p<0.05; **p<0.01, and ***p<0.001).

At 21 days, all of the treatment groups presented statistically significant results when compared with the control group (12.35±3.44), with p<0.001 in LED+D (34.16±999), p<0.01 in US+D (29.56±2.00), and p<0.05 in LED (27.43±3.33) (Fig. 4).

Figure 4.

Percentage of collagen fibers in surgical lesions of the experimental groups in 21 days. The p-values are indicated by the * accompanying mean value±standard deviation (*p<0.05; **p<0.01, and ***p<0.001).

The qualitative analysis of the gradation of the remodeling of collagenous fibers in the slides stained with TM showed that at 7 days of treatment, there was early-stage repair in groups LED, LED+D, and US+D. This repair was characterized by young and bulky fibroblasts and the presence of scattered and thin collagenous fibers, originating from the wound margins, while the control group presented a high quantity of granulation tissue, characteristic of the initial phase of the process.

At 14 days, the healing in the treated groups had developed further, as could be observed from the presence of mature fibroblasts, and thicker and more organized collagenous fibers. These characteristics are associated with the proliferative phase of the tissue repair process, and it is worth noting that in the LED+D group, there was a predominance of thick fibers (type I) when compared with the other groups. On the other hand, at the same experimental time, the control specimens had collagenous fibers, which were not very thick and were randomly arranged, characterizing the initial phase of the repair process. At 21 days, the collagenous fibers of the treated groups were completely organized and remodeled, with a predominance of thick fibers, when compared with the control, which still presented immature and less compact fibers.

FT-IR analysis

Figure 5 shows the average FT-IR spectra of the groups at 14 days, because at this experimental time was observed a greatest tissue differentiation, maximizing the differences between the treated groups. To identify the main vibrational bands contributions, the spectra were divided in seven regions and the band assignments are shown in Table 1.^3,26,32–38

Figure 5.

Mean Fourier transform infrared spectroscopy spectra of the experimental groups 14 days in 7 spectral ranges: A (3,600–3,000 cm⁻¹), B (2,990–2,800 cm⁻¹), C (1,710–1,482 cm⁻¹), D (1,480–1,355 cm⁻¹), E (1,274–1,189 cm⁻¹ and 1,185–1,132 cm⁻¹), F (1,189–1,120 cm⁻¹), and G (990–955 cm⁻¹). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Table 1.

Vibrational bands in the spectral ranges 7 A (3,600–3,000 cm⁻¹), B (2,990–2,800 cm⁻¹), C (1,710–1,482 cm⁻¹), D (1,480–1,355 cm⁻¹), E (1,274–1,189 cm⁻¹ and 1,185–1,132 cm⁻¹), F (1,189–1,120 cm⁻¹), and G (990–955 cm⁻¹)

Regions	Peak position (cm−1)	Major assignment	References
A	3,600–3,000	Vibrational modes of νs (O-H) and vibrational modes of νs (O-H) and νs N-H, which is mainly due to the O-H stretching of the hydroxyl group and N-H (amide A) of proteins in the main chain (3,200 cm−1)	32
B	2,990–2,800	C-H stretching vibrations of >CH2 and –CH3 of lipids and proteins in the organic matrix	32–34
C	1,710–1,482	νs >(C=O)-RNA/DNA, attributed to the C=O stretching and the nucleic acids RNA and DNA, which comprise amide I (νs C=O and δs N-H) and amide II (δs N-H and νs C-N)	3,32,34,35,39
D	1,480–1,355	δa (C-H) of CH2 and CH3 represents the deformation (C-H) of CH2 and CH3, consisting of proteins and carbohydrates	3,32,33,36
E	1,274–1,189	Amide III, by the C-N stretching and N-H deformation	35
E	1,185–1,132	νs (C-O), νs (C-C), νs (C-O-C), and (C-O-P) represents a region that is considered mixed, characterized by the symmetric stretching of PO2 in nucleic acids, and the C-O-C, C-O-P stretching in mono- and polysaccharides	36
F	1,189–1,120	C-O, C-OH, C-O-C stretching, consisting of proteins, carbohydrates, and cyclic ethers	3,32,37,38
G	990–955	symmetric stretching of PO4	34

ν_s, symmetric stretching; δ_a, asymmetric deformation; δ_s, symmetric deformation.

The spectral second derivatives were performed at an experimental time of 14 days, as depicted in Fig. 6. The spectral contribution at 1,656 cm⁻¹ is given by molecular radical α-like helix and at 1,647 cm⁻¹ for unordered structure.^3,39 The control group had the greatest contribution of molecular radical from unordered structure, while LED+D had the lowest contribution. Indeed there was a balance between the α-like helix and unordered structure values; it means that groups with the highest α-like helix showed lowest unordered structure contributions.

Figure 6.

Spectra of the second derivative in time of the experimental groups 14 days. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

The α-like helix is related with the organized structure present in type I collagen, which could be correlated with the final stage of regeneration of the tissue. However, the contribution of unordered structure indicates less degree of organization of the structures of the collagenous fibers,^3,39 which represents the type III collagen that is formed more rapidly in the beginning of the healing process. This process has defined phases, but each individual needs different time to complete the full process.

However, if specific treatment is applied to a group, the member could have a similar response as a function of the treatment benefits. Figure 7 shows the results of the linear discriminant analysis for the groups studied and expresses the differences in the evolution of the repair process according to the treatment. For the well-defined phases, the linear discriminant analysis (LDA) highest value indicates that each animal was classified at the correct experimental time, indicating the treatment benefits. The control groups presented 43% of discrimination among the experimental time, showing that each animal has its own time and that it is not possible to define a clear separation between the healing phases for this group. On the other hand, LED and TUS groups combined with a dressing showed stages of better defined repairing, with separation of animals at the experimental times. These results indicate the influence of the treatment in the group, changing the individual time for group time. In addition, the wound dressing in the treatment potentiates the LED treatment.

Figure 7.

Linear discriminant analysis of the experimental groups.

Discussion

The variation in the distribution (morphological anisotropy) of the collagenous fibers enables one to understand the relationship between the structure and function of skin tissues under normal and pathological conditions.^3,10 The effect of the treatment used to accelerate tissue repair can be assessed by quantifying and differentiating the collagenous fibers in the dermis at various stages of the healing process.^5–7,40 Thus, the results of this study show the applicability of FT-IR for the analysis and monitoring of the process of tissue repair in the experimental model using rats.

The data obtained in this study enabled the observation that, through the analysis of the volume fraction of the collagenous fibers, the semipermeable dressing, ultrasonic irradiation, and phototherapy promote collagen synthesis, which confirms several experimental studies that have researched the individual effects of dressings, US, and LED therapy on the production of dermal collagen.^{7,12–14,21,25}

At the experimental period of 7 days, none of the therapeutic protocols presented statistically significant qualitative or quantitative results, although there was a slight tendency in morphometric values for the group LED+D. It is worth emphasizing that during this stage, the process of tissue repair is at the end of the inflammatory phase, and the presence of inflammatory infiltrate is still visible. This result does not corroborate studies that suggest that phototherapy acts on cell permeability and mitochondria, by stimulating the synthesis of ATP and favoring the onset of the deposition of thin collagen fibrils.^18,19,41

At 14 days, the qualitative histological analysis showed a predominance of thick fibers (type I) in the group LED+D, when compared with the other groups, which is characteristic of the process of fibrillogenesis.^4,40,42 Moreover, in this experimental period, the animals in the groups treated with phototherapy (LED and LED+D) had statistically significant morphometric results regarding the deposition of collagenous fibers, when compared with the control. Considering the data obtained at this experimental time for the LED group, this result is probably due to the action of phototherapy, making energy available to the cells that were damaged by the surgical process, thus accelerating the repair process during its secondary phase.^{20,21,25,43,44} The most significant results were observed with the combination of LED and a semipermeable dressing (LED+D). It is noteworthy that the application of the dressing positively influenced the tissue repair process by accelerating it. One explanation for this result may be the presence of the film that properly controls the moisture of the wound environment. Moreover, its porous nanofiber structure was excellent at avoiding dehydration in the area and simultaneously maintained the cellular microenvironment, effectively protecting against bacterial infections.^13,16,23,24

At the experimental time of 21 days, all of the therapeutic protocols used in this study (LED, LED+D, and US+D) presented statistically significant results with increased formation of collagen fibers and an advanced stage of remodeling of these fibers, with a predominance of thick and organized fibers (demonstrating their maturity) when compared with the control. The remodeling of collagen, by the thickening of the fibers, is probably due to the replacement of type III collagen by type I.⁴⁵

Therefore, these results confirm studies that suggest that US and LED therapies, used individually, increase the formation of collagenous fibers and complete reepithelialization of the injured area.^7,18,20,44

In this study, it was possible to verify that the combination of the semipermeable dressing with LED therapy accelerated the tissue repair process. This would reduce the postsurgical time for healing, which is a very important factor in clinical procedures.

The FT-IR results regarding the molecular composition of the specimens of all experimental groups provided similar information to the histological analysis, indicating the neoformation of collagen in the dermis. FT-IR provided information about the parameters of the organization of fibers and about the secondary structure of collagen, revealed by the curve fitting of amide I in the spectral range 1,700–1,600 cm⁻¹.^3,39

For the definition of the collagen bands using the FT-IR vibrational spectroscopy, bands of protein amide I and II (1,710–1,482 cm⁻¹) and amide III (1,274–1,189 cm⁻¹)^3,34,35 were identified. Amide I and II bands are sensitive to changes in the secondary structure of protein, and structural information regarding the type of collagen and degree of organization is achieved primarily in the bands relating to amide I.^3,39,46

The types of collagen fibers can be differentiated by the parameters of the secondary structure. Thus, type I collagenous fibers can be differentiated from type III by their higher α-helix values.^3,39,46 This biopolymer has regions already identified as amide I, between 1,700–1,600 cm,⁻¹ due to C=O stretching, and in the region 1,480–1,350 cm⁻¹, due to the deformation of CH₂ and CH₃; amide II between 1,600–1,500 cm⁻¹, due to C=N stretching and N=H deformation, and amide III between 1,300–1,180 cm⁻¹, by the C-N stretching and N-H deformation, which are the main components in the formation of tissue repair.^3,34,35

At 7 days, the spectra were similar for all treatment groups compared with the control. However, at 14 days, the LED+D group had higher amounts of type I collagen (higher α-helix value) and more organized collagenous fibers, confirming the results of the histological analysis. This indicated a higher volume fraction and thicker fibers in this group compared with the others and suggests a predominance of mature fibers.^3,39

In this study, at 14 days, the morphometric analysis had better results for the experimental protocols with LED therapy, especially when combined with the dressing. However, FT-IR analysis showed that US therapy combined with the dressing was the second best treatment option, considering the greater amount of α-helix that characterizes type I collagen as well as the greater organization of collagen fibers, when compared with isolated LED therapy.^12–14,13

From 21 days, when the repair process is the final stage, characterized by the organization and remodeling of collagen fibers, it was possible to observe similarity in both the histological and spectral analyses between the treated specimens and the control, which suggests that all groups that received some type of therapy were at a more advanced stage of the tissue repair process.

Considering the previous results, the linear discriminant analysis was chosen. The results showed that the group LED+D had greater separation between the experimental times, confirming the histomorphometric data and showing the action of the dressing when combined with LED in accelerating the process. The other treated groups (US+D and LED) had a lower degree of differentiation, although the delimitation of the experimental times was visible. Moreover, in the control group, the separation of animals did not produce conclusive results, considering that over 55% of the animals were improperly separated, which indicates a delay in the evolution of the tissue repair process when compared with the treated groups.

Innovation

The quantitative analysis and the computational morphometry of histological sections of the dermis can be complemented by FT-IR, which provides information about the biodistribution and type of collagen, which highlights, through the results of this study, the positive effects of the combination of the semipermeable dressing with LED therapy and US in the acceleration of tissue repair, with earlier formation of type I collagen and better organization of collagen fibers.

However, further studies are needed to investigate the pathophysiological, biochemical, and/or molecular mechanisms of LED therapy and US combined with the prohealing effect of the semipermeable dressing. The in vivo analysis of the tissue repair process of the groups studied would enable the application of this knowledge in clinical practice.

Conclusion

The experimental results of this study, which compared morphometry analysis with FT-IR, suggest that the combination of a semipermeable dressing with LED therapy and TUS modulates biological events, increasing the fibroblast/collagen response and accelerating dermal maturation. We emphasize that among the therapies studied, the LED therapy showed the best results, early stimulating the production and organization of collagen fibers in experimental cutaneous lesions.

Abbreviations and Acronyms

FT-IR

Fourier transform infrared spectroscopy

LDA

linear discriminant analysis

LED

light-emitting diode

PC

principal components

TM

Masson's trichrome

TUS

therapeutic ultrasound

US

ultrasound

Author Disclosure and Ghostwriting

No competing financial interests exist. No ghostwriters were used to write this article.

About the Authors

Veruska Cronemberger Nogueira, Fellow PhD degree, Postgraduate Program in Biomedical Engineering, Vale do Paraiba University (UNIVAP), Sao Jose dos Campos-SP, Brazil. Conception, design, and intellectual and scientific content of the study. Leandro Raniero, Assistant Professor, IPD, UNIVAP, Sao Jose dos Campos-SP, Brazil. Conception, design of the study, and critical revision. Guilherme Bueno Costa, Graduate student, UNIVAP, Teresina-PI, Brazil. Acquisition of data. Nayana Pinheiro Machado de Freitas Coelho, Fellow PhD degree, Postgraduate Program in Biomedical Engineering, UNIVAP, Sao Jose dos Campos-SP, Brazil. Acquisition of data. Fernando Cronemberger Miranda, Assistant Professor, UESPI, Teresina-PI, Brazil. Histopathological examinations. Emília Ângela Loschiavo Arisawa, Associate Professor, IPD, UNIVAP, São José dos Campos-SP, Brazil. Scientific and intellectual content of the study, interpretation of data, and critical revision.