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 Table of Contents  
ORIGINAL RESEARCH
Year : 2019  |  Volume : 11  |  Issue : 3  |  Page : 122-126

The effects of different durations of zinc oxide–Turmeric dressing application on wound toward neovascularization and expression of macrophage marker antibody and Cyclooxygenase-2: An In vivo study


1 Department of Clinical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
2 Department of Dental Materials, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
3 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Brawijaya University, Malang, Indonesia
4 Department of Oral and Maxillofacial Pathology, Faculty of Dental Medicine; Graduate Student of Basic Medical Science Program, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
5 Department of Pediatric Dentistry, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

Date of Web Publication25-Jun-2019

Correspondence Address:
Dr. Asti Meizarini
Department of Dental Materials, Faculty of Dental Medicine, Universitas Airlangga, Mayjen Prof. Dr. Moestopo Street 47, Surabaya 60132
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_31_19

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  Abstract 

Background: Zinc oxide–turmeric dressing was known to have better anti-inflammatory properties than zinc oxide–eugenol dressing, but the optimal application duration is yet to be discovered. Aims: This study aimed to determine the optimal application duration of zinc oxide–turmeric dressing through molecular observation of neovascularization and expression of MAC387 and cyclooxygenase-2 (COX-2) during wound healing process in rat model. Materials and Methods: Twenty male Wistar rats were randomly divided into four groups (n = 5), Full-thickness excision of 6x6 mm was made on the dorsal area of all subjects and following the excision: the control (C) group was left undressed, while treatment groups was dresed with zinc oxide turmeric dressing for 3 days (T3 group), 5 days (T5 groups), and 7 days (T7 groups) prior to being sacrificed on day 7. All subjects were sacrificed on day 7. Hematoxylin-eosin staining and immunohistochemistry staining were performed to identify the neovascularization and MAC387 and COX-2 expressions. Data obtained were analyzed with analysis of variance and least significant difference test. Results: There was a significant higher neovascular number, higher MAC387 expression, and lower COX-2 expression in T5 and T7 groups compared to the C group (P < 0.05). Conclusions: The application of zinc oxide–turmeric wound dressing for 5 and 7 days could increase both neovascularization and MAC387 expression and also decrease COX-2 expression on the observation day in day 7. The optimal application duration is 5 days.

Keywords: Cyclooxygenase 2, Macrophages, Neovascularization, Periodontal dressings, Turmeric extract, Wound healing


How to cite this article:
Aryati, Meizarini A, Riawan W, Puteri A, Kuntari S. The effects of different durations of zinc oxide–Turmeric dressing application on wound toward neovascularization and expression of macrophage marker antibody and Cyclooxygenase-2: An In vivo study. J Int Oral Health 2019;11:122-6

How to cite this URL:
Aryati, Meizarini A, Riawan W, Puteri A, Kuntari S. The effects of different durations of zinc oxide–Turmeric dressing application on wound toward neovascularization and expression of macrophage marker antibody and Cyclooxygenase-2: An In vivo study. J Int Oral Health [serial online] 2019 [cited 2019 Sep 15];11:122-6. Available from: http://www.jioh.org/text.asp?2019/11/3/122/261263


  Introduction Top


Surgical intervention is often required to achieve the best result of dental treatment. It is necessary to protect the wound from mechanical trauma and maintain its stability from the high activity of mastication in the oral cavity after surgery. These could be achieved by the usage of the wound dressing.[1]

Wound dressing in the oral cavity or periodontal pack application was aimed to facilitate healing by preventing pain-induced trauma during mastication, to minimize postoperative infection and hemorrhage, and to protect clot detachment from the forces applied during speaking or chewing.[2] The combination of zinc oxide with eugenol dressing is widely used as periodontal dressing due to its anti-inflammatory properties, but the eugenol itself was previously reported to cause contact allergy at low dose and cytotoxicity in fibroblasts and osteoblast-like cell at the high dose.[3],[4] These findings urge the discovery of alternative material to replace eugenol, and one of the potential materials is turmeric extract. The previous in silico and in vivo study conducted by Meizarini et al.[5] showed that zinc oxide–turmeric extract dressing produces higher anti-inflammatory effect compared to zinc oxide–eugenol dressing. Besides, turmeric extract was found to have low cytotoxicity.[6]

The duration of classical wound dressing application could range from 3 to 14 days after surgery, but as a general rule based on the usual timetable of healing and clinical experience, it is stated to be kept in the wound site for 7 days.[1],[2] There is a possibility of the dressing broken and loosen on day 3 or 5, right before the 7 days postsurgery control visit. Although it is best to repack the dressing, not all the patients could come to the clinic prior to the control appointment.[7] In addition, a long-term application of wound dressing was proven to increase the inflammation due to plaque accumulation underdressing.[1] These encourage the authors to determine the optimal duration of zinc oxide–turmeric extract wound dressing application when observed on the 7 days postsurgery control visit.

Wound healing process could be observed from the expression of macrophage in the wound site, as it has an important role in wound healing through releasing factors to attract the inflammatory cells toward wound sites, stimulating the formation of granulation tissues, and enhancing the migration of cells.[8] With in situ macrophage proliferation, the alternate pathway will be activated, resulting in faster collagen production than normally occurs.[9] MAC387 is one of the markers used to observe the macrophage expression and found to be more specific than the CD68 marker.[10] Cyclooxygenase-2 (COX-2) is known to be increased during inflammation which results in the increase of prostaglandin release which further aggravates the inflammation. Reducing the expression of COX-2 will accelerate the wound healing process.[11] Curcumin, as the active substance in turmeric extract, is known to have anti-inflammatory activities by inhibiting COX-2 expression.[12],[13] Neovascularization is also one of the possible factors to be used to observe the wound healing process, as neovascularization provides nutrients for the tissue to repair the damaged area and regenerate itself.[14] Previous studies confirmed that curcumin could increase proangiogenic factors, which is important to promote the angiogenesis process.[15] Thus, neovascularization and MAC387 and COX-2 expressions were observed in this study to find the effect of zinc oxide–turmeric extract dressing on the wound healing process.

The aim of this study was to determine the optimal application duration of zinc oxide–turmeric dressing through the molecular observation of neovascularization and expression of MAC387 and COX-2 during wound healing process on the 7 days postsurgery in rat model.


  Materials and Methods Top


This study was approved by the Health Research Ethical Clearance Commission from Faculty No. 139/HRECC.FODM/VIII/2017.

This laboratory experimental research used post-test only control group design. This study has been done in 2 months. The sample size was determined using sample size formula by Lemeshow et al. with 5 samples per group for the result.[16]

Twenty male Rattus norvegicus (Wistar rats) of 3 months old, weighing 20–300 g, were randomly divided into four groups (n = 5) based on the treatment received: C group received no treatment; T3 group was being excised followed by zinc oxide–turmeric dressing application which would be undressed on day 3; T5 group was being excised followed by zinc oxide–turmeric dressing application which would be undressed on day 5; and T7 group was being excised followed by zinc oxide–turmeric dressing application which would be undressed on day 7. Prior to the treatment, the subjects were adapted for a week with food and drink given ad libitum.

0.3 g zinc oxide powder (Merck KGaA, Darmstadt, Germany) and 0.3 g turmeric liquid extract (Balai Materia Medica, Batu, Indonesia) were mixed for 60 s using a stainless-steel spatula on a mixing pad until homogenous consistency reached prior to being applied in the surface of the wound excision.

After a week of adaptation period, each Wistar rat of treatment groups (T3, T5, and T7) was intramuscular anesthetized using 10% ketamine (KEPRO, ZA, Denmark) and 2% xylazine (Interchemie Werken, Venray, Holland) combination at 1:1 ratio (0.1 ml/100 g rat body weight). The skin on the vertebral thoracic region was swapped with 70% alcohol and shaved, and 6 mm × 6 mm symmetrical squares were drawn as the excision outline. Full-thickness excision with sufficient depth up to the fascia area (2 mm) was made using surgical blade no. 15 (Swann Morton, Sheffield, England) with the help of chirurgical tweezers. After the saline solution was used to clean the wound excision, C group was covered with Hypafix (BSN, Hamburg, Germany), while zinc oxide–turmeric extract wound dressing was then applied to T3, T5, and T7 prior to being covered with Hypafix. For T3 group, the dressing was undressed on day 3 and recovered with Hypafix. The same treatment was applied to T5 group on day 5 while for T7 group was done on day 7. On day 7, all subjects were sacrificed to obtain the sample by excising the tissues around the wound area with 5 mm of extended length from each margin and subepithelial (4 mm) in depth.

The samples were embedded in paraffin blocks prior to being fixed with 10% neutral buffered formalin for 48 h. A section of 4 μm in thickness was made using microtome prior to being placed on a poly-L-lysine slide surface (Biocare, USA), heated at 30°C–35°C on a hot plate for 24 h, and washed to achieve deparaffinization condition. Further processed was done to make histopathological anatomy microscope slides.

Hematoxylin-eosin (HE) (Sigma-Aldrich, St. Louis, USA) staining was performed to observe the neovascular formation. Immunohistochemistry (IHC) examination was done in accordance with the avidin–biotin complex system (IHC Kit, D-BioSys, The Netherlands) using specific monoclonal Macrophage Marker Antibody (MAC387) sc-66204 and monoclonal COX-2 antibody sc-19999 of Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). The samples were then observed under ×400 magnification for HE examination slides and ×1000 magnification for IHC examination slides using a light microscope (Nikon Eclipse E 100, Tokyo, Japan). The calculation was done in 10 specific fields of view for HE slides and 20 specific fields of view for IHC slides and performed by two researchers with 95% clinical agreement. Pictures were taken with a Sony ILCE α6000 camera (Sony, Tokyo, Japan).

Statistical analysis

The data were analyzed by means of the Statistical Package for the Social Sciences version 21 (IBM, New York, USA), and the results were expressed as mean ± standard deviations (SD). One-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test was applied in order to assess the statistical significance of the differences between the study groups at P < 0.05.


  Results Top


The statistical analysis of neovascularization and MAC387 and COX-2 expressions was performed using ANOVA followed by an LSD test because data distribution was found to be normal and homogeneous. The mean and SD of those variables is shown in [Table 1].
Table 1: Comparison of mean±standard deviation of neovascularization and MAC387 and cyclooxygenase-2 expressions

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The result showed that there is an increase of neovascular number found, respectively, in C, T3, T5, and T7 groups with a significant difference in both T5 and T7 to C group, while no significant difference was found in T3 to T5 group and T5 to T7 group [Figure 1]. The expression of MAC387 in T5 and T7 groups was found to be significantly higher compared to C and T3 groups [Figure 2], while the expression of COX-2 in both the groups was found to be significantly lower compared to C and T3 groups [Figure 3]. No significant difference was found in T5 and T7 groups for both variables.
Figure 1: The hematoxylin-eosin staining of neovascularization (×400) for C, T3, T5, and T7 groups. Yellow arrows denote the stained neovascular

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Figure 2: The immunohistochemistry staining of MAC387 expression (×1000) for C, T3, T5, and T7 groups. Yellow arrows denote the stained macrophages

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Figure 3: The immunohistochemistry staining of cyclooxygenase-2 expression (×1000) for C, T3, T5, and T7 groups. Yellow arrows denote the expressed cells

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  Discussion Top


Wound healing process was divided into several phases: coagulation and hemostasis phase (after initial injury), inflammatory phase (day 1–3), proliferative phase (day 4–14), and remodeling phase, although overlapping between phases is evident.[17] This study specifically observed the proliferative phase as the samples were obtained from day 7.

In this study, it showed that 5- and 7-day application of zinc oxide–turmeric dressing could significantly decrease COX-2 expression in day 7 compared to 3 days of application duration and without any treatment. COX-2 is known to have a role in initiating inflammation, which its expressions are induced by growth factors and cytokines released in response to injury.[18] It also initiates inflammation. It starts to increase 3 h after initial injury, then peaks at 3 days, and almost normalized at 7 days.[19] By downregulating COX-2 expression, it will suppress the biosynthesis of prostaglandins from arachidonic acid resulting in inflammatory inhibition, which will accelerate the wound healing process.[20],[21] The inhibition of COX-2 expression possibly accomplished through the ability of curcumin, the active substances of turmeric extract, to suppress nuclear factor kappa B (NF-κB) activation and pro-inflammatory gene expression through preventing the phosphorylation of inhibitory factor I-kappa B kinase. By downregulating NF-κB activation, the COX-2 expression will subsequently decrease.[21] The anti-inflammatory effect of curcumin extract has also been proven clinically in human by Farjana et al., who managed to administer curcumin extract gel to gingivitis patients thrice a day for 21 days and resulted in tremendous decrease of inflammation despite no mechanical debridement done to the patient.[22]

Macrophage usually appears in wound site 48–72 h after the injury to play its role in the phagocytosis process, producing various cytokines, growth, and angiogenic factors to upregulate fibroproliferation and angiogenesis.[17],[23] In general, it can be broadly classified as M1 phenotypes, which were often found in the early phase of wound healing to act as pro-inflammatory, bactericidal, and phagocytic, and M2 phenotypes, which were found in later phase as prorepair to act as an anti-inflammatory, matrix producing, proangiogenesis, and prowound healing.[24] However, there was a limitation of MAC387 marker as it has no ability to distinguish the detected macrophages as M1 or M2. A previous study confirmed the ability of combination zinc oxide–turmeric dressing application in increasing macrophage expression if compared to the undressed wound,[13] while the present study showed that in day 7, only groups dressed with zinc oxide-turmeric dressing for 5 and 7 days has a significant higher number of MAC387 compared to the undressed control group. Although several studies have confirmed that curcumin inhibited the pro-inflammatory M1 phenotype, it also upregulates anti-inflammatory M2 phenotype, which will improve the wound healing process. This previous study also indicated that curcumin could switch M1 phenotype to be M2 phenotype by inhibiting TLR4-MAPK/NF-κB pathway.[25]

As previously stated, macrophage also has proangiogenic properties.[23] Thus, the increase of MAC-387 will inevitably affect the neovascularization which was in accordance with the result of this study, as T7 group was observed to have a significant higher neovascular number in day 7 compared to T3 group. Although there is no significant difference, there was also an increase of neovascular number in the T5 group compared to the T3 group. By stimulating macrophage expression, curcumin will indirectly upregulate the secretion of vascular endothelial growth factor and fibroblast growth factor, eventually promoting angiogenesis.[19]

The increase of both neovascular number and MAC387 expression and the decrease of COX-2 expression were observed in day 7 in other longer duration groups compared to the T3 group, indicating that the longer the application duration of zinc oxide–turmeric dressing, the greater the effect. With a longer duration of application, the active substance in the dressing will have a longer time to contact and permeate to the wound site, which will improve the wound healing process. From this study, although 7-day application of zinc oxide–turmeric extract wound dressing gave the best result with a higher neovascular number, the optimal duration was 5 days because the difference was not significant statistically.

With regard to its anti-inflammatory effect through decreasing COX-2 expression and its wound healing promotion through increasing neovascularization and MAC387 expression, the usage of combination of zinc oxide and turmeric extract, as alternative material to traditional zinc oxide and eugenol wound dressing, is promising. Clinical prove of curcumin gel for gingivitis treatment supports further the usage of this wound dressing combination on periodontal tissue as postoperative periodontal packs in the future.[22] Its optimal duration of 5-day application will come practical in clinical used compared to traditional zinc oxide and eugenol wound dressing which needs to be kept for 7 days.[2] Nevertheless, this study was still limited to its effect in anti-inflammation and early wound healing process. Future study to observe its effect in re-epithelization until the healing process completed is necessary to conduct before its usage in clinical use.


  Conclusions Top


The application of zinc oxide–turmeric wound dressing for 5 and 7 days could increase both neovascularization and MAC387 expression and also decrease COX-2 expressions in day 7 compared to 3-day application and without dressing. The optimal application duration is 5 days.

Financial support and sponsorship

This study was supported by the Excellence in Higher Education Institution Basic Research Grant of 2017 No. 004/ADD/SP2H/LT/DRPM/VIII/2017 from the Ministry of Research, Technology and Higher Education of Indonesia.

Conflicts of interest

There are no conflicts of interest.

 
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