The role of ozonated Aloe vera oil in full-thickness skin defects: Macrophage count and epithelization length parameter

Introduction

Soft tissue wound refers to the damage or loss of tissue continuity, possibly caused by internal factors such as drugs, changes in circulation and metabolic processes, infection, and oxygen transportation failures. External factors include extreme temperatures, injuries, radiation, and the presence of chemical substances¹. The body's physiological response, in terms of wound healing, results from various interrelated processes, including coagulation, inflammation, deposition, extracellular matrix differentiation, fibroplasia, epithelialization, contraction, and remodeling¹. Furthermore, the remediation course is mediated by various cells, cytokines, matrices, and growth factors, through three phases - inflammation, tissue proliferation, and remodeling². The process of acute cutaneous wound healing consists of three stages. The first stage is the inflammatory phase, which occurs following the injury and lasts until day-3. The second stage is the proliferation phase that generally takes place from day -7 until day-14, and the third stage is the remodeling phase that can last for several months or even years. The result of a previous study explained that only at specific doses did ozonated oil show a significant increase in the initial phase of wound healing³.

Macrophages play critical roles in all phases of adult wound healing, which are inflammation, proliferation, and remodeling. As wounds heal, the local macrophage population transitions from predominantly pro-inflammatory (M1-like phenotypes) to anti-inflammatory (M2-like phenotypes). Thus, local macrophages retain pro-inflammatory characteristics⁴.

Aloe vera is a thorny plant that grows in tropical and subtropical climates. It has been used for millennia to improve cutaneous wound healing, with this now being known to be due to its ability to increase the proliferation and migration of fibroblasts and human primary epidermal keratinocytes (HPEKs) flow cytometry analysis revealed that cell surface expressions of β1-, α6-, β4-integrin, and E-cadherin increased in HPEKs treated with Aloe vera gel. These increases may contribute to cell migration and wound healing⁵. Many active ingredients, including vitamins, enzymes, minerals, sugars, lignin, salicylic acids, and amino acids, have been identified from these species. However, therapeutic effects have not correlated well with each component. Recently, a study exploring the biological effects of a single element from Aloe species was conducted. Choi et al. reported that a 5.5 kDa glycoprotein isolated from Aloe vera gel by activity-guided sequential fractionation was found to enhance keratinocyte proliferation and migration, and thereby accelerate wound healing⁵. However, it is currently believed that biological activities associated with Aloe arise from the synergistic actions of multiple compounds rather than from a single component⁵.

Ozone therapy is an alternative medicine with disinfecting properties and strong oxidative stress induction capacity. These characteristics have been implicated in the stimulation of protective mechanisms for cells and organs by increasing the efficacy of endogenous oxygen-free radicals scavenging⁶. Moreover, ozone therapy inactivates bacteria and damages cell walls through the oxidation of phospholipids and lipoproteins, and also inhibits fungal growth, damages viral capsids, and interferes with the bacterial reproduction cycle⁷. This therapy could be applied as an adjuvant or alternative to standard treatments in patients with various types of wounds, particularly when used topical⁶. Previous studies have highlighted the benefits of ozonated sesame oil in wound healing⁷.

Therefore, this study aimed to evaluate the effect of Aloe vera oil provided with different dosages of ozone in accelerating the healing rate comparing two-phases; inflammation ( day 3) and proliferation (day 7) of full-thickness defect wounds in Sprague Dawley rats as an adjuvant treatment based on macrophage count and new epithelialization length.

Methods

This was an experimental study using a post-test only control group design. The outcome data were recorded after the duration of the treatment. A total of 50 adult male Sprague Dawley rats were distributed randomly into ten groups, with five in each. A total of ten groups were created, where the controls, P1 (received Aloe vera oil) and P2 (received gentamicin), respectively, for three days. Furthermore, P3, P4, and P5 was administered ozonated Aloe vera Oil at a dose of 600, 1200, and 1800 mg/ml of ozone, respectively for three days. Therefore, P6 (received Aloe vera oil) and P7 (received gentamicin), respectively, for seven days, while P8 P9 and P10 received ozonated Aloe vera Oil at a dose of 600, 1200, and 1800 mg/ml ozone, respectively for seven days. A full-thickness defect measuring 1 cm in diameter was created on the back of the rat. The rats were sacrificed in each group on the third (P1, P2, P3, P4, P5) (inflammation stage of wound healing ) and seventh day (P6, P7, P8, P9, P10) (proliferation stage wound healing), respectively, to obtain the macrophage count and length of new epithelialization (Figure 1).

Figure 1. Study flow chart.

Full-thickness wounds

Full-thickness wounds of one-centimeter diameter were made on the back of the rat (Figure 2). The procedure involved anesthetizing the Rats with a mixture of Ketamine (100 mg/mL; Ketamin Cas Number: 6740-88-1 Ikapharmindo, Indonesia) -Xylazine (20 mg/mL; Xyla Cat. No.: IX2 Interchemie werken "De Adelaar" BV Local Distributor: PT Tekad Mandiri Citra). A Mixture dose of 80 mg/kg Ketamine; 10 mg/kg Xylazine via the intraperitoneal route, followed by the careful placement of the rat in the left lateral decubitus position. The hair on the dorsum area was shaved, and povidone-iodine was used to clean the skin, followed by ethanol 70%. The wound area was marked, and the lesion was made by cutting the skin using a scalpel to the base of the panniculus carnosus. The fatty and connective tissues were cleaned, and the site irrigated with normal saline (if necessary), before wrapping with a band-aid. Furthermore, the non-adhesive cottonoid was applied to covering the wound. The rats were then placed in a clean and warm cage with adequate lighting to recover from the anesthesia.

Figure 2. Full-thickness defect induction.

A: Image of the wound creation process on the back of rat; B: Example of full-thickness defect wound (round shape in this study); C: Placement of non-adhesive segment of Hansaplast plaster on wound model; D: Elastomull bandage was used to wrap the thorax and dorsum area of rat, and tightness was adjusted to ensure proper breathing (adequate chest expansion) and movement of extremities before returning to the cage.

Results

Analysis of macrophage count

Table 2 shows the differences in the mean macrophage count of treatment groups 1-10, and the highest mean value was recorded on day-3, in P5(administered 1800 mg of ozonated Aloe vera oil) as 131.96. Meanwhile, the lowest (79.96) was measured on day-3, in P2, with gentamicin treatment (Figure 3). The result of the data normality test shows the absence of normal data distribution (p=0.028 or p<0.05). Hence, data transformation was performed to generate normally distributed data (with a p-value range of 0.065 to 0.884).

Table 2. Data normality test result of macrophage count.

Group	Mean ± SD	Median (min – max)	p	Transf.
P1	93.08 ± 20.89	85.6 (78.4 – 129.6)	0.028	0.065*
P2	79.96 ± 11.93	78.2 (63.0 – 95.8)	0.848*	0.759*
P3	88.04 ± 34.18	80.2 (40.6 – 131.8)	0.864*	0.521*
P4	108.92 ± 13.49	106.2 (96 – 131.8)	0.117*	0.186*
P5	131.96 ± 25.95	141.4 (102.2 – 161.8)	0.395*	0.325*
P6	69.08 ± 13.08	68.6 (54.8 – 87.4)	0.834*	0.884*
P7	90.28 ± 36.21	78.6 (56.6 – 132.6)	0.170*	0.216*
P8	90.28 ± 10.67	90.2 (73.4 – 102)	0.553*	0.401*
P9	85.96 ± 17.71	78.2 (72 – 116)	0.104*	0.201*
P10	83.16 ± 25.30	94.8 (51.2 – 108.0)	0.276*	0.222*

* Normal (p > 0.05); ^£ Shapiro-wilk

Figure 3. Histopathological findings of macrophage count by hematoxylin-eosin staining 100x magnification in all groups (P1 – P10).

All normally distributed data were tested using the one-way ANOVA to ascertain the presence of significant differences in mean macrophage count between the ten groups. Table 3 shows that the data obtained were not homogenous, at p=0.028, or p<0.05. However, one-way ANOVA test resulted in a p-value of <0,001, indicating the presence of a significant difference between groups. Therefore, a Post hoc Games-Howell test was then conducted to observe the variation in the mean macrophage count of two groups. Table 4 shows the results of the Post hoc Games-Howell test, and a significant difference (p<0,05) was observed between the group provided with gentamicin (P2) and 1800 mg/ml of ozonated Aloe vera oil (P5) on day-3, at 79.96 and 131.96, respectively. Also, there was substantial variation between the groups administered 1200 mg/ml (P4) and 1800 mg/ml (P5) of ozonated Aloe vera oil sacrificed on day 3, with those euthanized Aloe vera oil on day 7 (P6), at 168.92, 131.96 and 69.08, respectively.

Table 3. Homogeneity and one-way ANOVA test results of macrophage count.

Group	Mean ± SD	P	Homogenous
P1	93.08 ± 20.89	0.029*	0.028
P2	79.96 ± 11.93
P3	88.04 ± 34.18
P4	108.92 ± 13.49
P5	131.96 ± 25.95
P6	69.08 ± 13.08
P7	90.28 ± 36.21
P8	90.28 ± 10.67
P9	85.96 ± 17.71
P10	83.16 ± 25.30

* Significant (p < 0.05); ** Homogenous (p > 0.05)

Table 4. Post hoc Games-Howell result for the macrophage count.

Group	P2	P3	P4	P5	P6	P7	P8	P9	P10
P1	0.935	1.000	0.807	0.293	0.429	1.000	1.000	0.999	0.996
P2	–	1.000	0.107	0.046*	0.898	1.000	0.891	0.999	1.000
P3		–	0.899	0.580	0.992	1.000	1.000	1.000	1.000
P4			–	0.755	0.038*	0.911	0.399	0.410	0.658
P5				–	0.015*	0.569	0.140	0.125	0.293
P6					–	0.968	0.306	0.711	0.988
P7						–	1.00	1.000	1.000
P8							–	1.000	0.997
P9								–	1.000

* Significant (p < 0.05)

Analysis of epithelialization

Table 5 shows the variation in the mean length of new epithelialization between the ten treatment groups, with the highest value of 2451.40, recorded in group P10 provided with 1800 mg/ml of ozonated Aloe vera oil, on day-7. Meanwhile, the least (799.49) was reported on day-3, in group P3 administered 600 mg/ml of ozonated Aloe vera oil (Figure 4). The data normality test result shows the normal distribution in the ten treatment groups, with p-value ranging from 0.083 to 0.970. Therefore, all normally distributed data were tested using the one-way ANOVA to investigate any significant differences in mean macrophage count between the ten groups. Based on the normality test, Table 6 demonstrated non-normal distributed data, with p=0.013 or p<0.05. However, one-way ANOVA test showed a p-value of <0.001, indicating the absence of any significant differences between treatment groups. Therefore, a Post hoc Games-Howell test was conducted to evaluate the variation in the mean length of new epithelialization between two groups.

Table 5. Data normality test result of new epithelialization length (in micrometres).

Group	Mean ± SD	Median (min – max)	p
P1	1179.20 ± 235.65	1142.72 (950.12 – 1523.65)	0.606*
P2	1175.14 ± 343.37	1111.27 (804.10 – 1563.76)	0.370*
P3	799.49 ± 272.93	789.97 (466.58 – 1141.83)	0.907*
P4	1487.41 ± 871.70	1230.43 (699.53 – 2962.25)	0.182*
P5	981.11 ± 236.15	871.14 (769.91 – 1275.64)	0.158*
P6	1160.88 ± 276.51	1078.29 (867.30 – 1595.27)	0.636*
P7	1367.64 ± 105.20	1314.88 (1254.88 – 1506.94)	0.427*
P8	2074.48 ± 531.74	1981.17 (1333.25 – 2604.28)	0.462*
P9	1984.09 ± 653.72	1675.41 (1397.27 – 2726.21)	0.083*
P10	2451.40 ± 296.22	2303.98 (2157.46 – 2866.08)	0.403*

* Normal (p > 0.05); ^£ Shapiro-wilk

Figure 4. Histopathological findings of epithelial tissue length by a microscopic procedure using 40x magnification in all groups (P1 – P10).

Table 6. Homogeneity and one-way ANOVA test results of new epithelialization length.

Group	Mean ± SD	p	Homogenous
P1	1179.20 ± 235.65	<0.001*	0.013
P2	1175.14 ± 343.37
P3	799.49 ± 272.93
P4	1487.41 ± 871.70
P5	981.11 ± 236.15
P6	1160.88 ± 276.51
P7	1367.64 ± 105.20
P8	2074.48 ± 531.74
P9	1984.09 ± 653.72
P10	2451.40 ± 296.22

* Significant (p < 0.05)

The result of the Post hoc Games-Howell test (Table 7) shows a substantial increase in the length of new epithelialization, particularly in groups treated using 1800 mg/ml of ozonated Aloe vera oil (P10) for seven days (2451.40 μm), compared to those administered Aloe vera oil therapy (P6) and gentamicin therapy (P7), at 1160.88 µm and 1367.64 µm, respectively. Furthermore, ozonated Aloe vera oil therapy proved effective in each group of Sprague Dawley rats with full-thickness skin defect, based on the increased epithelialization.

Table 7. Post hoc Games-Howell result of new epithelialization length.

Group	P2	P3	P4	P5	P6	P7	P8	P9	P10
P1	1.000	0.447	0.996	0.920	1.000	0.798	0.168	0.390	0.002*
P2	–	0.665	0.997	0.978	1.000	0.942	0.191	0.425	0.005*
P3		–	0.773	0.967	0.579	0.079	0.043*	0.130	<0.001*
P4			–	0.928	0.994	1.000	0.928	0.981	0.485
P5				–	0.970	0.185	0.082	0.220	0.001*
P6					–	0.825	0.163	0.382	0.002*
P7						–	0.312	0.601	0.008*
P8							–	1.000	0.897
P9								–	0.869

* Significant (p < 0.05).

This process occurred during the wound healing process with gentamicin (P7), with no significant difference (1367.64 µm), compared to P6 treated with Aloe vera oil (1160.88 µm), P8 with 600 mg/ml of ozonated Aloe vera oil (2074.48 µm), and P9 with 1200 mg/ml of ozonated Aloe vera oil (1984.09). Therefore, the administration of ozonated Aloe vera oil at specific doses has a significant positive effect on the healing response of full-thickness defect wounds in Sprague Dawley rats, leading to faster new epithelialization length development.

Discussion

The process of acute cutaneous wound healing generally consists of three stages, including the inflammatory phase experienced immediately after the injury, which extends to day-7. The second stage is proliferation, observed to day-14, and the third is the remodeling phase, known to last for several months or even years⁶. Previous studies have demonstrated a significant increase in the initial phase of wound healing in response to moderate doses of ozonated oil (1500 mg/dl)⁶. The current study showed the effectiveness of 1800 mg/ml ozonated Aloe vera oil in promoting the wound healing response of full-thickness defects in Sprague Dawley.

According to Valacchi et al.⁶, ozone is an efficient bactericidal, anti-fungi, and anti-viral agent, hence the propensity to reduce infection and improve skin unification during wound healing. The effect of ozonated oil on wound closure rate depends on the peroxide content⁶, as ozone possesses powerful oxidative characteristics in both gas and liquid phases, therefore producing microbicidal effects needed to damage the cell wall and cytoplasmic membrane of bacteria and fungi. This cascade of events leads to increased cell permeability, consequently facilitating the entry of ozone molecules¹¹.

The use of ozonated oil also prevents superinfections and stimulates tissue reconstruction through increased cell proliferation and the formation of new vascularization⁶. Also, an increase in oxygen tension at the wound site elevates the tendency of granulation tissue formation, which has implications on the wound closure rate⁴. The augmented expression of transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF), known to play critical roles in the wound healing process, has also been achieved in clinical and experimental wound healing research with ozone therapy¹².

The results of data analysis showed differences in the mean macrophage count between groups treated with 600, 1200, and 1800 mg/ml of ozonated Aloe vera oil and the control, administered Aloe vera oil or gentamicin alone. The evaluation parameter was higher in groups provided with 1200 mg/ml and 1800 mg/ml of ozonated Aloe vera oil for three days compared to those given gentamicin for three days and Aloe vera oil for seven days. This outcome was probably due to the increased oxidative role of ozone during the early phase of the inflammatory process¹³.

Ozone has been widely recognized as an anti-viral, bactericidal, and anti-fungal agent, and used in clinical therapy for its beneficial wound healing effects. This element is not able to penetrate the cell but is able to bind unsaturated fatty acids, and consequently form reactive oxygen species (ROS)⁶.

One form of ROS compound is hydrogen peroxide, known as a non-radical oxidant and implicated as an ozone mediator in several biological processes, including wound therapy¹⁴. Besides, ROS plays a crucial role in these processes by decreasing blood flow and forming a thrombus.

ROS are known to assist in the transportation of neutrophils through the blood vessels, therefore serving as a protection against bacteria by signaling the immunocytes, including monocytes, to fight pathogenic germs¹⁵. Furthermore, ROS stimulates endothelial cells to form new blood vessels, form an extracellular matrix (including collagen synthesis), and keratinocyte proliferation¹⁵, although excessive production leads to oxidative stress. This condition causes adverse effects on the wound healing process, hence the need for antioxidants to achieve a dynamic balance¹⁵.

The mean value of total macrophage produced on day 4 – 7 was lower than day 1 – 3. According to previous research, this variation was affiliated with the presence of vitamins, enzymes, proteins, carbohydrates, minerals (calcium, sodium, magnesium, zinc, and iron), and amino acids in Aloe vera, which play a major role in treating inflammation¹⁶. Also, various anti-inflammatory agents, including salicylic acid, Indomethacin, Manosa-6-phosphate, B sitosterol, as well as components of Lignin, Saponin, and Anthaquinone consisting of Aloin, Barbaloin, Anhtranol, Anthracene, Aloetic acid, Aloe-emodin, have been identified as essential ingredients of antibiotics and pain killer drugs. Meanwhile, Aloe vera is also rich in vitamins, polysaccharides, and other components with health benefits. The anti-inflammatory constituents can stimulate the immune system, leading to cell regeneration, and the consequent acceleration of healing in the inflammatory phase¹⁶.

Based on the results of new epithelialization length measurement, ozone induces a notable effect on the wound healing process. Furthermore, the administration of 1800 mg/ml ozonated Aloe vera oil for seven days had a significant positive mean difference compared to the other groups.

Various studies have been conducted to observe the role of topical Aloe vera on the wound healing process. Oryan et al.¹⁷ examined a 2×2 cm wound on the back of rats treated with Aloe vera gel, and the parameters evaluated include wound surface, size shrinkage, and epithelialization. Teplicki et al.⁸ demonstrated increased healing in terms of proliferation and migration of fibroblasts and keratinocytes after treatment. Aloe vera shows effectiveness through increased cell migration, keratinocyte function, and epithelialization repair⁸.

The current study results indicate the fastest wound healing rate in the group treated with the highest dose of ozonated Aloe vera oil (1800 mg/ml). This was followed by others, in the order of 600 mg/ml and 1200 mg/ml, the positive control administered gentamicin, and then Aloe vera oil, respectively. Therefore, further research is required to examine the effect of ozonated Aloe vera oil to ascertain the most effective dose for wound healing.

This study has several limitations, including the lack of uniformity in the hygiene level of each rat, which possibly influenced the wound healing process. Also, the wound tissue sampling was not conducted identically, therefore making it impossible to determine the exact phase of wound healing encountered on day seven, as the process in Sprague Dawley rats is perhaps different from humans.

Conclusion

Based on the results and discussion, ozonated Aloe vera oil applied to full-thickness skin defect in Sprague Dawley rats resulted in higher macrophage count and longer new epithelialization length than the control group.

However, uniformity in the hygiene level of each rat is important in order to accelerate wound healing processes and minimize infection. Also, there is a need for further research to investigate the exact component in Aloe vera oil responsible for the treatment progression. Furthermore, subsequent studies are also to determine the effective dose to facilitate implementation is not only experimental animals, but also humans, to observe the antioxidant effect of ozonated Aloe vera oil.

Data availability