Effect of ultrasound and dexpanthenol on collagen organization in tegumentary lesions

Abstracts

OBJECTIVE: To analyze the effect of ultrasound (US), dexpanthenol (d-P) and a combination of these treatments (US+d-P) on collagen fiber organization in tegumentary lesions in rats by birefringence analysis. METHODS: Wistar rats (50) were anesthetized (Thionembutal - Sodic = 50mg/Kg), 1cm² of dorsal region skin was removed, and the animals were divided into five groups: control (C), gel (G), US (3 MHz, 0.1 W/cm2, 1 minute, continuous), d-P (10%) and US+d-P. After daily treatment for 7 and 14 days, 6µm thick sections of lesioned areas were stained in picrosirius and measurements of the collagen birefringent area (µm²) were obtained using polarized light microscopy (Zeiss Axiolab-ZEISS- Germany) with histological image analysis software (KS 400 2.0 - Kontrol Eletronics, Munique, Germany). The means were compared by ANOVA followed by the Tukey test (p<0.05). RESULTS: The US+d-P group showed a significantly greater (p<0.001) birefringent area (1586.43±162.14) than the other experimental groups: C (139.36±35.35), US (317.55±129.9) and d-P (192.41±3657) by the 7th day of treatment, indicating acceleration of the wound healing process. By the 14th day of treatment, the US+d-P, US and d-P groups presented greater birefringence than the control group, but did not differ from each other. CONCLUSION: The combination of treatments (US+d-P) accelerated collagen fiber synthesis and organization in the early stages of cutaneous repair.

ultrasound; dexpanthenol; collagen; physical therapy

OBJETIVO: Analisar o efeito do ultrassom (US), do dexapantenol (d-P) e da associação dos tratamentos (US+d-P) na organização de fibras colágenas na lesão tegumentar em ratos por meio da análise da birrefringência. MÉTODOS: Foram utilizados 50 ratos Wistar, anestesiados com Thionembutal Sódico (50mg/Kg), dos quais foi retirado 1cm² de pele da região dorsal, divididos em cinco grupos: controle (C), gel (G), US (3 MHz, 0,1 W/cm², 1 minuto, modo contínuo), d-P (10%) e US+d-P. Após sete e 14 dias de tratamento diário, foram removidos segmentos dessas áreas e obtidos cortes de 6µm de espessura que, posteriormente, foram corados em Picrosirius. Os cortes foram observados em microscopia de polarização utilizando um software responsável pela medida de birrefringência das fibras colágenas (KS400 2.0 - Kontrol Eletronics). As médias das áreas birrefringentes (µm²) de cada grupo foram submetidas à análise de variância pela ANOVA, seguida do teste de Tukey (p<0,05). RESULTADOS: A média de área birrefringente do grupo US+d-P (1586,43±162,14) foi maior (p<0,001) que a dos grupos experimentais (C: 139,36±35,35, US: 317,55±129,9 e d-P: 192,41±36,57) no 7º dia de tratamento, indicando uma aceleração na síntese e organização das fibras colágenas na região lesionada. No 14º dia de tratamento, os grupos US+d-P (2858,47±510,17), US (1779,94±482,78) e d-P (2546,88±304,45) apresentaram área birrefringente maior que a do grupo C, porém não diferiram entre si. CONCLUSÃO: A associação dos tratamentos (US+d-P) acelerou a síntese e a organização das fibras colágenas apenas no estágio inicial de reparo tegumentar.

ultrassom; dexapantenol; colágeno; fisioterapia

ORIGINAL ARTICLE

^IFaculty of Health Sciences, Universidade Metodista de Piracicaba (UNIMEP), Piracicaba, SP, Brasil

^IIFaculty of Odontology of Piracicaba, Universidade Estadual de Campinas (UNICAMP), Piracicaba, SP, Brasil

^IIIPharmacy and Biochemistry, Diadema Campus, Universidade Federal de São Paulo (UNIFESP), Diadema, SP, Brasil

^IVUniversity Center Hermínio Ometto of Araras (UNIARARAS), Araras, SP, Brasil

Correspondence

ABSTRACT

OBJECTIVE: To analyze the effect of ultrasound (US), dexpanthenol (d-P) and a combination of these treatments (US+d-P) on collagen fiber organization in tegumentary lesions in rats by birefringence analysis.

METHODS: Wistar rats (50) were anesthetized (Thionembutal - Sodic = 50mg/Kg), 1cm² of dorsal region skin was removed, and the animals were divided into five groups: control (C), gel (G), US (3 MHz, 0.1 W/cm2, 1 minute, continuous), d-P (10%) and US+d-P. After daily treatment for 7 and 14 days, 6µm thick sections of lesioned areas were stained in picrosirius and measurements of the collagen birefringent area (µm²) were obtained using polarized light microscopy (Zeiss Axiolab-ZEISS- Germany) with histological image analysis software (KS 400 2.0 - Kontrol Eletronics, Munique, Germany). The means were compared by ANOVA followed by the Tukey test (p<0.05).

RESULTS: The US+d-P group showed a significantly greater (p<0.001) birefringent area (1586.43±162.14) than the other experimental groups: C (139.36±35.35), US (317.55±129.9) and d-P (192.41±3657) by the 7^th day of treatment, indicating acceleration of the wound healing process. By the 14^th day of treatment, the US+d-P, US and d-P groups presented greater birefringence than the control group, but did not differ from each other.

CONCLUSION: The combination of treatments (US+d-P) accelerated collagen fiber synthesis and organization in the early stages of cutaneous repair.

Keywords: ultrasound; dexpanthenol; collagen; physical therapy.

Introduction

Collagen is the most abundant protein in animal organisms and it is also the main protein structure of the connective tissue. There are several types of collagens, although types I, II, III and IV are the most common¹. Collagen type I the is most prevalent in the dermis, although types III, IV and V can also be observed on a smaller scale.

Collagen molecules aggregate through crosslinking, and this highly organized net of chemical interactions turns the collagen into an organic crystal, which explains why it is resistant to tensions and pressures².

Variations in the organization and molecular order of collagen bundles may be detected and measured during the repair process by analysis of their anisotropic properties (birefringence)³.

Total birefringence is dependent on the geometry of the molecules, the partial volume (concentration), the aggregation level, the orientation of the bundle components and the oscillatory strength of all electronic transitions in the molecule, as well as the collagen crosslinkings^3-5.

The repair process of a wound has three phases that continually overlap: inflammatory, proliferative and remodeling, and it involves several biochemical and cellular alterations⁶. Regardless of the tissue, collagen is the most important component in the repair process.

Skin lesions are treated with vitamins such as dexapanthenol or d-panthenol (d-P), which is a pro-vitamin of the B complex that, when topically applied, is converted into pantothenic acid, a natural skin constituent⁷. Ebner et al.⁸ observed in vitro and in vivo that fibroblast activation with d-P leads to an acceleration of the epithelization of wounds, creating an epithelium with a high organizational level of epidermal structures.

As a therapeutic modality, ultrasound (US) has been used both in the tissue repair process and in the treatment of wounds^9-12. Studies have demonstrated an increase in collagen synthesis, fibroblast proliferation and local microcirculation with ultrasound treatment^10,13,14, although according to ter Haar14, in many of these effects, the mechanism of action is unknown. Some studies have demonstrated a risk of alterations in healthy skin¹⁵ from the continuous use of 3 MHz US at intensities from 1 to 2 W/cm2 as well as in vitro¹⁶ skin samples irradiated above 2.5 W/cm2.

Due to the difficulty of transdermal drug permeation, the use of physical accelerators such as US in a therapy called phonophoresis^17,18 has been studied. The research group responsible for the above-mentioned study also verified that not only did US accelerate the tegumentary repair process but its combination with d-P¹⁹ further accelerated that process. Their study also showed increased re-epithelization of the wound after seven-days of treatment, although it did not quantitatively study collagen fibers, which justifies the present study. The present study tried to verify if these treatments alone and/or in combination could alter the organization of collagen, which is of great importance in the healing process.

Since US requires a coupling agent, the addition of d-P to that agent may accelerate collagen synthesis in lesions thus treated. Therefore, the aim of this study was to analyze the effect of US, d-P and a combination of these treatments in tegumentary lesions in rats by histomorphometric analysis of collagen fiber birefringence.

Methods

Animals

This study was approved by the Committee for Ethics in Animal Experimentation of the Universidade Federal de São Carlos (UFSCar), São Carlos, SP, Brazil (protocol nº 020/2006). Fifty male Wistar rats, weighing approximately 250g each, were maintained in the Biotery of the Universidade Metodista de Piracicaba (UNIMEP), Piracicaba, SP, Brazil, with a controlled light/dark cycle (12h/12h) and water and chow ad libitum in individual cages in which the litter was changed daily.

Experimental procedures

After anesthesia with sodium phenbarbital (50mg/Kg), the animals were trichotomized in the dorsal area, from which 1cm² of skin was removed, including the hypodermis, using a scalpel and a hollow template. All animals received only one application, in the lesioned area, of polyvinyl-pyrrolidone-iodine (PVPI) 10.0g with 10% stabilizer and softener after the surgery.

Experimental groups

The animals were divided into five experimental groups (n=10): control (C), gel (G), gel + ultrasound (US), gel + dexapanthenol (d-P), gel + US+ dexapanthenol (US+d-P). Five animals from each group received treatment for seven days and the other five animals for 14 days; euthanasia was carried out on the 8th and 15th day, respectively. The daily treatment of all animals occurred only in the morning period and was carried out by the same researchers.

C group

The animals of group C received no treatment after the surgical procedure.

Gel group

Carbopol 940^® was the gel used; it consists of carbomer, propylene glycol, triethanolamine, methylparaben and distilled water. Two grams of gel were applied daily over the entire lesion with a spatula.

d-P group

This group received gel plus 10% d-P, with 2g applied daily over the lesion, as in the previous group.

US and US+d-P groups

US (Sonomaster Dual, KW Eletrônica Ltd., Amparo, Brazil) was applied daily to the lesion of the US and US+d-P groups. The parameters were: a 3 MHz frequency^11,20, a 0.1 W/cm² intensity¹¹, continuous emission mode21 and an application time of 1 min/cm²² with the transducer being moved slowly and continually until the end of the application²³, and an ERA of 4 cm² ± 30%.

The US intensity was calibrated at 3 MHz before and during the experiment using distilled and degassed water in an US scale (UPM-DT-10 Ohmic Instruments, Easton, USA). Ultrasonic wave transmissivity in Carbopol 940^® gel and d-P gel was verified according to the method of Guirro, Guirro and Ferreira²⁴, considering a variation of 10% in US and 0.07% in scale. The procedure was repeated five times for each gel and none of the preparations showed a statistically significant attenuation (data not shown).

Histological processing

Twenty-four hours after the final day of treatment, the animals were euthanized by cervical displacement and the lesioned area was removed including 2 mm of the surrounding intact skin. Cross-sectional segments of the central area of these lesions were fixed in 10% buffered formalin solution and histologically processed for embedding in paraplast. From each segment three 6µm histological slices stained with Picrosirius 1% (Sirius red in saturated solution of picric acid) were prepared on microscope slides for blinded analysis. Using polarization microscopy (Zeiss Axiolab, ZEISS, Germany) in conjunction with histological image analysis software (KS 400 2.0 - Kontrol Eletronics, Munique, Germany), the birefringence of the collagen fibers was quantified by a sole observer in five areas (10,000 µm² each) per slice that were distributed throughout the entire lesion.

Statistical analysis

GraphPad Prism 4.0 (GraphPad Software Ind, San Diego CA, USA) was used for statistical analysis. The Kolmogorov-Smirnov test was used to verify the normality of data distribution. Afterwards, the analysis of variance (ANOVA) test was used, which was followed by the Tukey test. A value of p<0.05 was considered statistically significant. The obtained data were submitted to polynomial regression in order to evaluate the birefringent areas in light of the different treatments and treatment lengths.

Results

The US+d-P group showed a greater birefringent area (p<0.001) on the 7^th day of treatment than the other groups (Table 1 and Figure 1). On the 14^th day, the d-P group as well as the US+d-P group showed greater birefringent areas than the groups receiving isolated treatments (US). The US group was significantly different (p<0.001) from both the C and G group, in which there was no intergroup difference.

The data were also analyzed by means of polynomial regression. All groups showed an increase of the mean birefringent area during the treatment time, although the US, d-P and US+d-P groups showed a significant improvement (p<0.001) that did not occur in the C and G groups (p>0.05), as demonstrated in Figure 1.

Figure 2 shows bright areas corresponding to the birefringence of collagen fibers in the injured area of animals in the experimental groups treated for seven and 14 days. On the 14^th day, the tissue was more mature and, consequently, the collagen fibers were brighter.

Discussion

The regeneration of wounds is a highly organized process involving cellular interactions, the formation of granulation tissue and the deposition of proteoglycans and collagen, which is the main component of a mature scar⁶.

To provide therapeutic effects in the tissue repair process, such as in the interaction between cells and matrix, physical and pharmacological resources have been used, including the separate application of US^9-12 and d-P^7,8 in isolated treatments.

On the 7^th day of treatment in the present study, the d-P group's collagen fiber birefringent area did not differ from the C group's according to the Tukey test (Table 1). However, on the 14^th day of treatment, the mean birefringent area of the d-P group was significantly greater (p<0.001) than the C group's (Table 1, Figure 2). It is probable that d-P promoted an increase in collagen synthesis by means of fibroblast activation, which confirms the results of Ebner et al.⁸. Since the synthesis and remodeling rate of the collagen is slow, the effect only appeared on the 14^th day.

According to Young and Dyson¹¹, US treatment accelerates inflammatory reaction and cellular proliferation in the initial stages of epidermal regeneration in rats, as well as increases collagen deposition and the tensile strength of the wound. Demir et al.¹³ used US at intensities of 0.1 W/cm² and 0.5 W/cm² and observed inflammatory phase acceleration in the wound repair. Using the same parameters as the present study but with pulsed US, these authors observed a decrease in the length of the inflammatory phase time. In the proliferative phase, there was an increase in hydroxyproline and fibroblast numbers, as well as stimulated collagen synthesis.

Similar results were observed in the present study in that there was no significant difference in the birefringent areas (Table 1 and Figure 1) of the US group treated for seven days compared to the other groups. However, on the 14^th day, which is in the proliferative phase, there was an increase in comparison to the C group.

The combination of treatments (US+d-P) has been studied by our research group in vitro²⁵ as well as in tegumentary regeneration¹⁹. In a qualitative analysis, Polacow et al.¹⁹ observed stimulated re-epithelialization of wounds and increased fibroblast numbers, as well as better organized healing tissue after a seven-day-treatment. However, they did not study the effects of this combined treatment on the synthesis and organization of collagen.

In the present study, under the same experimental conditions, the results showed that on the 7^th day of treatment the US+d-P group had a significantly greater birefringence area (p<0.001) than either of the isolated treatments, which demonstrates accelerated maturation of the collagen fibers present in the injured tissue (Table 1). Our results reinforce the beneficial effect of this association, because if there are more fibroblasts¹⁹, there is also increased synthesis of extracellular matrix, which confirms that fibroblasts direct this process since they liberate growth factors that contribute to wound healing^10,12,26. The cavitation effect of ultrasound increases the influx of calcium in the monocytes, acting as a second messenger and promoting the synthesis and liberation of factors that contribute to wound healing^11,27.

According to KoeKe et al.²⁸, who studied the effect of US associated with 10% hydrocortisone, phonophoretic transdermal delivery positively influences the repair process of tendons when compared to isolated treatments with US and hydrocortisone.

On the 14^th day of treatment, the US+d-P group showed greater birefringence, although it did not differ from the group treated with d-P alone, since, during that treatment period (from seven to 14 days) the wounds were in the remodeling phase and the synthesis rate and deposition of collagen were undergoing reductions in the injured tissue.

Data were submitted to polynomial regression (Figure 1) in order to evaluate the birefringent areas with respect to the different treatment lengths, and the results of this analysis showed that all the experimental groups except C and G showed a significant increase (p<0.001) in birefringence on the 14^th day compared to the first seven days of treatment. This reveals the natural maturity of collagen fibers in this phase of the repair process, which corresponds to the beginning of the remodeling phase. However, the combined treatment was very effective until the 7^th day. Future studies involving this combination of treatments and including histological and biochemical analyses of the lesions during the inflammatory phase (around the 3^rd day) should be developed in an attempt to understand its mechanism of action.

Lasers, another physical therapeutical resource, have also been used for treating wounds^29,30. However, as Vogt et al.³¹ observed, the healing process in a humid environment is faster than in a dry environment, which has also been confirmed by several other studies reporting that fibroblasts and endothelial cells increase with the isolated use of linkage gel^9,19. This study demonstrates that if d-P is added to this gel, added benefit may be obtained, especially when used in conjunction with US.

Although the present study approached a very restricted aspect of tegumentary regeneration, the results complement other studies that have analyzed this association of treatments, which seems promising for the treatment of lesions, but needs further basic study.

The results confirmed our hypothesis that the combination of treatments (US+d-P) accelerates collagen synthesis in tegumentary lesions, suggesting that it may accelerate the healing process in an early stage of treatment.

Conclusion

Isolated treatment with d-P and US in rat skin lesions, as well as a combination of the two treatments, stimulated collagen maturation. The combined treatment (US+d-P) was more effective in the first phase of the healing process (seven days) in that it accelerated the maturation of collagen fibers, which propitiates faster healing.

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Effect of ultrasound and dexpanthenol on collagen organization in tegumentary lesions

Gustavo N. Guimarães^{I, II}; Maria S. M. Pires-De-Campos^I; Gislaine R. Leonardi^III; Helena H. K. Dib-Giusti^IV; Maria L. O. Polacow^I

Publication Dates

• Publication in this collection
  28 July 2011
• Date of issue
  June 2011

History

• Reviewed
  07 Oct 2010
• Received
  07 May 2010
• Accepted
  14 Feb 2011