INTRODUCTION:

Herbal medicine has become a globally and economically important ingredient and has been used worldwide from the past several years^1,2. Nowadays in Indonesia, various alternative therapies are developed based on natural ingredients one of which is propolis which has numerous advantages and is relatively safe. Propolis is a non-toxic natural substance produced by bees from various plant sources. Propolis is a resin substance produced by bees.

It can be used as anti-inflammatory agent, immunostimulant, and as a local anesthesia for wound healing. Thus, it has been considered very useful for clinical use. The ability of propolis as an anti-inflammatory and antibacterial agent make it a new option for curing in tooth infection. It has been used since long ago as traditional medicines, biocosmetics, and health foods³.

Propolis has a wide biological activity spectrum including antibacterial, anti-fungal, anti-virus, anti-protozoan, antioxidant, anti-cancer, anti-tumor, anti-inflammatory, immunomodulator, wound healing driving and anesthesia agent⁴. Propolis induces formation of new bone by means of increasing osteoblast. Osteoblast produces collagen, proteoglycan, and glycoprotein for production and growth of new bone in surface area, and for the formation of the bone in cartilage⁵.

Orthodontic tooth movement occurs after bone remodeling, due to pressure to the teeth to move into their desired place. Pressure given to teeth will induce bone apposition and resorption. Teeth movement during orthodontic treatment produces a response towards periodontal ligament and alveolar bone. Main components playing role in teeth movement are collagen fibers and cells, blood vessel, and tissue fluid. Osteoblast, osteoclast, fibroblast, and cementoblast participate in apposition, including new bone and cementum formation. Osteoclast also functions in bone and cementum resorption^6,7.

Bone remodeling is a cycle, consisting of bone resorption due to osteoclast activity in pushing side and osteoblast induces new bone formation in pulling side⁸. Bone apposition occurred during orthodontic teeth movement is a biological process involving acute inflammation. Histological study showed that first phase of resorption occurred at 3-5 days, followed by recovery at 5-7 days. The process was then followed by final phase of bone apposition between 7 and 14 days^9-11.

MATERIALS AND METHODS:

Ethical Approval:

When reporting experiments on animal subjects, the authors should indicate whether the procedures comply with the ethical standards of the committee responsible for human experiments. The papers including animal experiments should be conducted with the approval of local animal welfare committees or the human subject, respectively.

This study received Ethical Clearance Approval, No. 210 /HRECC.FODM/IX/2017 related to animal subjects from the Ethics Research Committee, Faculty of Dental Medicine, University of Airlangga. The research formed an analytical observation that included the use of a laboratory experiment and randomized post-test only control group design.

Study area:

The studies were carried out within the framework of the dissertation of the research topic “Development of alternative medical and methods to improve the bone remodeling animals and evaluate orthodontic tooth movement.” Cavia cobaya with a separator rubber were studied.

Experimental animal:

RUNX-2 and ALP in maxilla animals were recorded on the basis of clinical examination, and the results of a complete immunohistochemical staining.

Research design:

The conducted research was true experimental research. The design of this study was a post-test only randomized control group design¹².

Treatment Management:

This study used Cavia cobaya sample with various criteria that includes male, age ± 3-5 months, body weight 250-400mg, physical health is characterized by active characteristics, the laboratory for examining environmental factors is a factor that influences the state of the laboratory environment for Cavia cobaya examination so that adequate radiation and free air circulation are given. From the results of the calculation of the sample formula and the formula for the possibility of dead animals the minimal sample number for each group was seven. Previous studies used 3% and 5% propolis producing significantly effective result. Both studies were the base used for this experiment^13,14. Propolis used was from Apis mellifera that originated from Malang city. Propolis was extracted into gel by adding 5% HPMC. Treatment performed to animal model was carried out as following; materials were first prepared, then insulin syringe needle was blunted using scissors so as not to wound gingiva sulcus. Separator was fitted between incisors in the upper mandible using separator pliers and then left for 2 weeks to trigger orthodontic teeth movement. Teeth were cleaned periodically using cotton pellet and tweezer. Propolis gel was given to gingiva sulcus using blunt needle daily starting from 3^rd day after separator was fitted until the 17^th day of treatment. Design of this study was randomized post-test only control group design. Twenty-eight Cavia cobaya were divided into 4 groups of K- (without treatment), K+ (giving separator rubber), P1(giving separator rubber and 3% propolis), P2 (giving separator rubber and 5% propolis). RUNX-2 and ALP expression were evaluated using immunohistochemical staining.

Statistical analysis:

Data was analyzed using Statistical Package for Social Science (SPSS) version 17.00 (IBM SPSS, Chicago, USA).

Analysis of RUNX-2 variance was performed (P<0.05) based on Brown-Forsythe statistical test, significant difference between group based on multiple comparisons Games-Howell test. Analysis of ALP variance was performed (P<0.05) based on based on one-way ANOVA statistical test, significant difference between group based on multiple comparisons LSD test.

RESULTS AND DISCUSSION:

Bone remodeling in orthodontic tooth movement is a biological process involves an acute inflammatory response to the periodontal tissue. Histological research shows 3-5 days is the first stage of resorption and 5-7 days is the recovery period followed by the final stage of bone remodeling between 7-14 days⁹. An inflammatory response can protect the body from either injury or infection. The factors of inflammation response can be due to damaged cell areas. There are mediators and cells controlling inflammatory response. One of these molecules was cytokines.

An examination of the maxilla incisive Cavia cobaya of RUNX-2 and ALP on day 17

Figure 1: Evaluation of RUNX-2 expression observed using anti RUNX-2 immunohistochemical staining. Positive expression was marked with brown color at periodontal ligament tissue macrophage in the pulling area. Slides were observed at 400x magnitude for 8 observation fields in Cavia cobaya pulling side. Arrow = positive RUNX-2 expression. K- = negative control, K+ = positive control given ortho pressure with 5% HPMC, P1 = ortho pressure treatment group given 3% propolis, P2 = ortho pressure treatment group given 5% propolis.

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Table 1: Comparison of mean RUNX-2 expression from each group

Group	n	RUNX2				P
Group	n	x	SD	Min	Max	P
K-	7	6.29^a	1.11	5	8	0.000*
K	7	10.71^b	2.43	8	14
P1	7	12.14^b	3.49	9	17
P2	7	17.00^c	1.53	14	19

*) significant at =0.05 based on Brown-Forsythe statistical test

^abcsame superscript letter indicates no significant difference between group based on multiple comparisons Games-Howell test

Table 1 and Figure 1 describe data of RUNX-2 expression evaluation. Brown-Forsythe statistic test showed significant difference between groups in the RUNX-2 expression in pulling area during teeth relapse process (p<0.05). Statistical test was then continued using multiple comparisons Games-Howell test.

Table 1 shows that there was significant difference between treatment groups in this study. K (+) and K (-) showed significant different (p<0.005), whereas the most prominent difference was found in P1 and P2 (p=0.001). This means that treatment significantly affected RUNX-2 expression in the pulling side.

RUNX-2 participates in the differentiation of mesenchymal stem cells with osteoblast. During osteoblast differentiation, RUNX-2 functions as osteoblast marker and transcriptional factor with runt domain bind to nuclear in a number of enhancers and promoters. RUNX-2 together with other proteins interact directly or indirectly with various molecules in bone remodeling to form signaling network¹⁵.

Runx-2 plays role in osteoblast maturation and novel bone formation. CAPE has been proven to increase RUNX2 expression. CAPE active content has strong potential for antioxidative activity, anti-apoptosis effect, and modulation of RUNX-2 and RANKL/OPG¹⁶. Flavonoid plays role in new bone formation by stimulating osteoblast maturation. Flavonoid also affects osterix and RUNX-2 expression, which then stimulates osteoblast differentiation¹⁶.

Figure 3: Evaluation of ALP expression observed using anti ALP immunohistochemical staining. Positive expression was marked with brown color at periodontal ligament tissue osteoblast in the pulling area. Slides were observed at 400x magnitude for 8 observation fields in Cavia cobaya pulling side. Arrow = positive ALP expression. K- =negative control, K+ = positive control given ortho pressure with 5% HPMC, P1 = ortho pressure treatment group given 3% propolis, P2 = ortho pressure treatment group given 5% propolis.

Table 2: Comparison of mean ALP expression in the pulling area from each group

Group	n	ALP				P
Group	n	x	SD	Min	Max	P
K-	7	9.86^a	2.91	6	14	0.000*
K	7	11.29^ab	1.60	9	13
P1	7	12.71^b	1.11	11	14
P2	7	23.14^c	2.97	18	27

*) significant at =0.05 based on one-way ANOVA statistical test

^abcsame superscript letter indicates no significant difference between group based on multiple comparisons LSD test

Table 2 and Figure 2 describe data of ALP expression evaluation. ALP expression in the pulling area showed significant difference from each treatment group based on one-way ANOVA (p<0.05), which then continued using LSD statistical test.

Table 2 shows comparison of ALP expression between groups. Significant difference was found between K (+) and K (-) (p<0.005), but the most prominent difference was found in P1 and P2 (p=0.001) that were given propolis extract treatment. This showed that propolis affected ALP expression in the pulling side significantly.

Previous study which used normal ortho pressure showed there was significant change (p <0.05) of alkali phosphatase activity on day 7, 14, and 21 of treatment in both right and left sides of mesial and distal between treatment and control. Highest enzyme activity was found on day 14 of initial phase, but it then dropped significantly as treatment progressed, especially in mesial side ¹⁷. CAPE content in propolis was able to raise ALP enzyme expression produced by osteoblast during bone formation and growth ¹⁸.

CONCLUSION:

Dose 5% Apis Mellifera Propolis extract increased RUNX-2 and ALP expression during remodeling process in orthodontic tooth movement.

CONFLICTS OF INTEREST:

All authors state that they have no conflicts of interest.

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Received on 01.05.2020 Modified on 10.07.2020

Research J. Pharm. and Tech. 2021; 14(5):2363-2366.

DOI: 10.52711/0974-360X.2021.00417