Ozonized solutions favor the repair of experimentally induced skin wounds in rats

This study aimed to evaluate and compare the effects of ozonized solutions on tissue wound repair in rats. Treatments consisted of ozonized water (GA), 0.9% sodium chloride (GCL), ozonized oil (GO), and 0.2% allantoin cream (GAL). The morphometric evaluation showed that wounds of the GA group presented a higher degree of retraction (p<0.05) at three and eight days of treatment (37.96 and 84.81%, respectively). Picrosirius red staining showed that groups GA and GO presented higher deposition (p<0.05) of type I collagen at 15 and 22 days of treatment, respectively. The neovascularization was higher in wounds of group GO on days 3, 8, and 15 (p<0.05), with higher VEGF immunostaining. (p<0.05). Thus, ozonized water enhances wound retraction and assists in the maturation and remodeling phase, while ozonized oil promotes higher neovascularization during tissue repair and higher deposition of type I collagen from the third week of treatment.

INDEX TERMS:
Ozonized water; wound healing; ozonized oil; ozone therapy; skin wounds; rats

Authorship

Rafael C. Sanguanini

Graduate Program in Animal Science (PPGCA), Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.Universidade Federal de GoiásBrazilGoiânia, GO, BrazilGraduate Program in Animal Science (PPGCA), Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.

http://orcid.org/0000-0002-5677-8634

Mariana F. Bento

Graduação em Medicina Veterinária (Bolsista PIBIC/CNPq), Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.Universidade Federal de GoiásBrazilGoiânia, GO, BrazilGraduação em Medicina Veterinária (Bolsista PIBIC/CNPq), Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.

Evelyn de Oliveira

http://orcid.org/0000-0001-6998-0834

Emmanuel Arnhold

Departamento de Zootecnia, Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.Universidade Federal de GoiásBrazilGoiânia, GO, BrazilDepartamento de Zootecnia, Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.

Mariana B.R. Faleiro

Faculdade de Odontologia (FO), Universidade Federal de Goiás (UFG), Av. Universitária esquina com 1ª Avenida s/n, Setor Leste Universitário, Campus Colemar Natal e Silva, Goiânia, GO 74605-220, Brazil.Universidade Federal de GoiásBrazilGoiânia, GO, BrazilFaculdade de Odontologia (FO), Universidade Federal de Goiás (UFG), Av. Universitária esquina com 1ª Avenida s/n, Setor Leste Universitário, Campus Colemar Natal e Silva, Goiânia, GO 74605-220, Brazil.

Leandro G. Franco

Departamento de Medicina Veterinária, Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.Universidade Federal de GoiásBrazilGoiânia, GO, BrazilDepartamento de Medicina Veterinária, Escola de Veterinária e Zootecnia (EVZ), Universidade Federal de Goiás (UFG), Avenida Esperança s/n, Campus Universitário Samambaia, Goiânia, GO 74690-900, Brazil.

Moema P.C. Matos

Veridiana Maria B.D. Moura ^**Corresponding author: vdmoura@ufg.br

*Corresponding author: vdmoura@ufg.br

Conflict of interest statement.- The authors have no competing interests.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables | Formulas

Figures (4)
Tables (5)
Formulas (1)

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Fig.1.
Wound on the back of rats of groups ozonized water (GA), 0.9% sodium chloride (GCL), ozonized oil (GO), and 0.2% allantoin cream (GAL) on days zero (upper) and eight (lower) after injury induction. Wounds of similar size to day zero in all groups; the GA rat wound exhibits greater retraction at 8 days after injury induction when compared to that of the other groups.

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Fig.2.
Photomicrographs of the skin of rats from different groups at 15 days after wound induction, showing newly formed vessels (arrows). (A) Group ozonized water (GA) showing moderate neovascularization. HE, obj.40x. (B) Group 0.9% sodium chloride (GCL) showing discreet neovascularization. HE, obj.40x. (C) Group ozonized oil (GO) showing marked neovascularization. HE, obj.40x. (D) Group 0.2% allantoin cream (GAL) showing discreet neovascularization. HE, obj.40x.

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Fig.3.
Photomicrographs of rat skin showing type I and III collagen fibers at 8, 15, and 22 days after induction in groups ozonized water (GA), 0.9% sodium chloride (GCL), ozonized oil (GO), and 0.2% allantoin cream (GAL). Picrosirius red, obj.40x.

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Fig.4.
Photomicrographs of the skin of rats from different groups at 8 days after wound induction. (A) Group ozonized water (GA) showing moderate anti-VEGF immunostaining. IHC, obj.40x. (B) Group 0.9% sodium chloride (GCL) showing discreet anti-VEGF immunostaining. IHC, obj.40x. (C) Group ozonized oil (GO) showing marked anti-VEGF immunostaining. IHC, obj.40x. (D) Group 0.2% allantoin cream (GAL) showing moderate anti-VEGF immunostaining. IHC, obj.40x.

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Table 1.
Means, medians and ranks sums, respectively, referring to the percentage of skin wound retraction in rats submitted to different treatments at 3, 8, 15, and 22 days after the surgical procedure

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Table 2.
Means, medians and ranks sums, respectively, of neovascularization in the skin wound of rats at 3, 8, 15, and 22 days after induction and treatment with ozonized water (GA), sodium chloride (GCL), ozonized oil (GO), and allantoin (GAL)

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Table 3.
Means of collagen fibers in rat skin wounds at 8, 15, and 22 days after induction and treatment in groups GA, GCL, GO, and GAL

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Table 4.
Mean percentage of type I and III collagen fibers in relation to total collagen in wounds at 8, 15, and 22 days after induction in groups GA, GCL, GO, and GAL

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Table 5.
Means, medians and ranks sums, respectively, of VEGF immunostaining in the skin wound of rats at 3, 8, 15, and 22 days after induction in groups GA, GCL, GO, and GAL

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Fig.1. Wound on the back of rats of groups ozonized water (GA), 0.9% sodium chloride (GCL), ozonized oil (GO), and 0.2% allantoin cream (GAL) on days zero (upper) and eight (lower) after injury induction. Wounds of similar size to day zero in all groups; the GA rat wound exhibits greater retraction at 8 days after injury induction when compared to that of the other groups.

Fig.2. Photomicrographs of the skin of rats from different groups at 15 days after wound induction, showing newly formed vessels (arrows). (A) Group ozonized water (GA) showing moderate neovascularization. HE, obj.40x. (B) Group 0.9% sodium chloride (GCL) showing discreet neovascularization. HE, obj.40x. (C) Group ozonized oil (GO) showing marked neovascularization. HE, obj.40x. (D) Group 0.2% allantoin cream (GAL) showing discreet neovascularization. HE, obj.40x.

Fig.3. Photomicrographs of rat skin showing type I and III collagen fibers at 8, 15, and 22 days after induction in groups ozonized water (GA), 0.9% sodium chloride (GCL), ozonized oil (GO), and 0.2% allantoin cream (GAL). Picrosirius red, obj.40x.

Fig.4. Photomicrographs of the skin of rats from different groups at 8 days after wound induction. (A) Group ozonized water (GA) showing moderate anti-VEGF immunostaining. IHC, obj.40x. (B) Group 0.9% sodium chloride (GCL) showing discreet anti-VEGF immunostaining. IHC, obj.40x. (C) Group ozonized oil (GO) showing marked anti-VEGF immunostaining. IHC, obj.40x. (D) Group 0.2% allantoin cream (GAL) showing moderate anti-VEGF immunostaining. IHC, obj.40x.

Table 1. Means, medians and ranks sums, respectively, referring to the percentage of skin wound retraction in rats submitted to different treatments at 3, 8, 15, and 22 days after the surgical procedure

Group	Period
Group	3 days	8 days	15 days	22 days*
GA	37.6/37.9/35.3^a	84.9/84.8/27.2^a	100/100/14.0^a	100^a
GCL	33.4/33.1/29.5^a	78.1/80.2/20.4^ab	99.5/100/12.3^a	100^a
GO	22.6/24.4/16.8^b	69.8/72.7/14.0^b	98.2/100/11.8^a	100^a
GAL	21.9/22.6/16.3^b	62.4/66.7/12.3^b	98.2/100/11.8^a	100^a

^a,b,ab
Within the same period, a value is only statistically different from another if there are no equal letters. A value identified as “a” is different from a value identified as “b”, but not from a value identified as “ab”, by the Kruskal-Wallis test (p<0.05); *At 22 days after the surgical procedure, all rats showed 100% wound retraction. Thus, the tests were not applied because all values are equal and therefore, there is no difference to be tested; GA = ozonized water group, GCL = sodium chloride group, GO = ozonized oil group, GAL = allantoin group.

Table 2. Means, medians and ranks sums, respectively, of neovascularization in the skin wound of rats at 3, 8, 15, and 22 days after induction and treatment with ozonized water (GA), sodium chloride (GCL), ozonized oil (GO), and allantoin (GAL)

Group	Period
Group	3 days	8 days	15 days	22 days
GA	9.33/9/5.16^ab	18.66/19/6.50^ab	10.00/10/6.00^ab	6.33/6/2.00^b
GCL	8.33/8/3.50^b	14.66/14/2.33^b	9.33/9/4.50^b	8.66/9/7.66^a
GO	11.00/11/9.50^a	20.33/20/9.00^a	14.00/13/10.66^a	8.66/9/7.66^a
GAL	10.66/10/7.83^ab	20.00/20/8.16^a	9.33/9/4.50^b	8.50/8.5/7.00^a

^a,b,ab
Within the same period, a value is only statistically different from another if there are no equal letters. Thus, a value identified as “a” is different from a value identified as “b”, but not from a value identified as “ab”, by the Kruskal-Wallis test (p<0.05).

Table 3. Means of collagen fibers in rat skin wounds at 8, 15, and 22 days after induction and treatment in groups GA, GCL, GO, and GAL

Group	Period
Group	8 days	15 days	22 days
GA	4.71^a	20.54^a	22.87^a
GCL	6.67^a	15.51^a	21.06^a
GO	3.66^ab	10.73^a	25.12^a
GAL	1.29^b	10.17^a	24.03^a

^a,b,ab
Within the same period, a value is only statistically different from another if there are no equal letters. Thus, a value identified as “a” is different from a value identified as “b”, but not from a value identified as “ab”, by the Duncan test (p<0.05); GA = ozonized water group, GCL = sodium chloride group, GO = ozonized oil group, GAL = allantoin group.

Table 4. Mean percentage of type I and III collagen fibers in relation to total collagen in wounds at 8, 15, and 22 days after induction in groups GA, GCL, GO, and GAL

Period	Collagen fiber	Group
Period	Collagen fiber	GA	GCL	GO	GAL
8 days	I	77.33^a	75.93^a	50.10^a	54.83^a
8 days	III	22.67^A	24.07^A	49.90^A	45.17^A
15 days	I	93.46^a	88.83^ab	84.60^ab	82.10^b
15 days	III	6.54^B	11.17^AB	15.40^AB	17.90^A
22 days	I	84.59^b	86.49^ab	90.20^a	84.41^b
22 days	III	15.41^A	13.51^AB	9.80^B	15.59^A

^a,b,ab
Within the same period, groups followed by the same lowercase letter have statistically equal means (considering type I collagen fibers); ^A,B,AB within the same period, groups followed by the same uppercase letter have statistically equeal means (considering type III collagen fibers); For these comparisons, Duncan test was used (p<0.05); GA = ozonized water group, GCL = sodium chloride group, GO = ozonized oil group, GAL = allantoin group.

Table 5. Means, medians and ranks sums, respectively, of VEGF immunostaining in the skin wound of rats at 3, 8, 15, and 22 days after induction in groups GA, GCL, GO, and GAL

Group	Period
Group	3 days	8 days	15 days	22 days
GA	1.33/1/6.50^a	2.66/3/6.67^ab	1.00/1/3.00^b	0.66/1/5.33^b
GCL	1.00/1/4.50^a	2.00/2/3.00^b	1.66/2/6.66^ab	0.33/0/3.66^b
GO	1.66/2/8.50^a	3.00/3/8.50^a	2.33/2 /9.66^a	1.66/2/10.0^a
GAL	1.33/1/6.50^a	3.00/3/7.83^ab	1.66/2/6.66^ab	1.00/1/7.00^ab

^a,b,ab
Within the same period, a value is only statistically different from another if there are no equal letters. Thus, a value identified as “a” is different from a value identified as “b”, but not from a value identified as “ab”, by the Kruskal-Wallis test (p<0.05); GA = ozonized water group, GCL = sodium chloride group, GO = ozonized oil group, GAL = allantoin group.

D W R = \frac{(I W A - W A^{N})}{I W A} \times 100

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Colégio Brasileiro de Patologia Animal - CBPA Pesquisa Veterinária Brasileira, Caixa Postal 74.591, 23890-000 Rio de Janeiro, RJ, Brasil, Tel./Fax: (55 21) 2682-1081 - Rio de Janeiro - RJ - Brazil
E-mail: pvb@pvb.com.br

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Ozonized solutions favor the repair of experimentally induced skin wounds in rats

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[1] *Corresponding author: vdmoura@ufg.br