Effects of 3% binahong (Anredera cordifolia) leaf extract gel on alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)

Ethical considerations

All experimental procedures in this study were carried out in accordance with the Institutional Animal Care and Usage Committee (ARRIVE) guidelines 2.0. Ethical clearance had been approved by the Health Research Ethics Committee (KEPK) at the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia with letter numbers: 0206/KEPH-FMIPA/2021 and 0202/KEPH-FMIPA/2021.

Animals

Sample size

The sample size in this study was calculated based on previous research of a similar nature.³ The effect size (Cohen’s d) between the experimental group and the control group from the previous research was 3.31.³ The minimum sample size obtained from the calculations was three animals and the final sample size was adjusted to four animals per group (with a total of 12 groups) or 48 animals in total. There was a 10% addition from the minimum sample size to anticipate sample exclusion during the experiment.

Rats

Animals used in this study were 48 male Wistar rats (Rattus norvegicus) of two to three months old and with an average body weight of 200-250 grams. The rats should also be in good health and had never received any research treatment previously. Rats with abnormalities or those that died before the experiment ended were excluded from the study. The rats that died were not replaced as long as the minimum sample size was still maintained. The rats were acquired and housed at the Animal House in the Faculty of Mathematics and Natural Sciences in Universitas Sumatera Utara, Medan, North Sumatra, Indonesia. Before administering the treatment, the rats were acclimatized for seven days to assure that the rats could adapt to their surroundings and were allowed ad libitum feeding and drinking.

Study design and groups

The study conducted was an in vivo experiment with a post-test only control design. The rats were randomized by simple random sampling and allocated into twelve groups by the Animal House lab technicians: group I to IV were given 3% Binahong leaf extract gel, group V to VIII were given base gel as the negative control, and group IX to XII were given Gengigel^® as the positive control (Figure 1). The treatments were given for 14 days as that was the approximate amount of time needed for the socket wound to close completely.² The residual socket volume (RSV) and fibroblast proliferation were observed on the 3^rd, 7^th, and 14^th day post-extraction, while the osteoblast and osteocyte proliferation (cells) were observed on the 7^th, 14^th, and 28^th day post-extraction. All researchers were blinded to the box assignation of the rats, except for the researchers who were in charge of applying the gel to the socket wounds.

Figure 1. Group allocation; each group is differentiated from one another based on its treatment and observation day and is comprised of four Wistar rats.

Tooth extraction

Wistar rats were anesthetized intraperitoneally using a combination of 91 mg/kgBW ketamine (Agrovet market, Peru) and 9.1 mg/kgBW xylazine (Interchemie, Netherlands) with an anesthetic dose of 0.1 mL/100 g rat weight to alleviate the pain induced by the procedure.³¹ After that, the mandibular left incisor was extracted using an artery clamp (Wells Spencer, London) with a luxation movement.³² After the extraction, the socket was irrigated with distilled water to clean the socket from debris. The extraction was performed by the same veterinarian who was blinded to the group allocation.

Binahong leaf extract gel and base gel preparation

The Binahong leaf extract used in this study was obtained from the Pharmacognosy Laboratory, Faculty of Pharmacy, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia. A total of 400 g fully opened Binahong leaves and aged approximately 12 weeks were selected and obtained from Simpang Perdagangan village, Tigabinanga district, Karo regency, North Sumatra, Indonesia in April 2017. Tigabinanga district is located at coordinate 3.0729, 98.2415. It has an altitude of 490 to 750 meter above sea level.³³ Karo regency has a tropical climate, with rainy season (August-January, March-May) and dry season (February, June, July).³⁴ Binahong leaves were extracted by the maceration method using 80% ethanol (Smart Lab Indonesia, Indonesia) as the solvent. The Binahong leaves were initially grinded into powder with an electric blender (Philips HR2115, Indonesia) and then soaked in 80% ethanol in a closed container and distilled for five days at room temperature. After five days, the 80% ethanol solvent was replaced with new solvent and soaked again for two days. Subsequently, a final filtering process was carried out. Then, a water bath was used to evaporate the solvent until the extract dried.³⁵

The base gel was made by adding 10 mL of hot distilled water to a mortar, then 0.125 g of carbopol (Merck, Germany) was added and the mixture was stirred with a pestle. Next, 1.5 g of triethanolamine (TEA) (Merck, Germany) and 2 g of glycerin (Merck, Germany) were added and the mixture was stirred until it was homogeneous. In a second mortar, a mixture of 10 mL distilled water, 0.125 g of hydroxypropyl methylcellulose (HPMC) (Merck, Germany), nipagin (Merck, Germany), and nipasol (Merck, Germany) were stirred until homogeneous.³⁰ The second mortar mixture was poured into the first mortar and the mixture was stirred until it was homogeneous. To obtain 20 g of 3% Binahong leaf extract gel, 0.6 g of Binahong leaf extract was added to the base gel and the mixture was stirred until it was homogenous (Figure 2). The gel was reproduced every 3 to 4 days to ensure its freshness.

Figure 2. The composition of 3% Binahong leaf extract gel.

Application of gels

The socket wounds in group I to VI were applied with 3% Binahong leaf extract gel; group V to VIII were given base gel; and group IX to XII were given Gengigel^® (Ricerfarma, Italy). In every application, 0.1 mL of gel was applied directly to the socket wound using a 1 mL syringe (One Med Health Care, Indonesia) with a bent needle irrigation tip (Ivoclar Vivadent, ∅: 1,2 mm, Liechtenstein) until the gel covered the entire wound surface to make sure that the gel would be directly in contact with the wound. Applications were made twice a day in the morning at 8.00 a.m.-10.00 a.m. and in the afternoon at 4.00 p.m.-6.00 p.m. for 14 days. The time was chosen to ensure consistent schedule for the gel application each day and the application was conducted by a researcher who was aware of the group allocation.

Clinical measurement of residual socket volume

Measurements of the socket volume were made with a digital caliper (Digital Caliper, China), a pair of compasses (Joyko^®, Indonesia), and a periodontal probe (Kohler, NR-3182, Germany) and were performed on the rats in group III, VII, XI, so that repeated measurements could be made on the same samples. The measurements were performed by the same researcher blinded. The average socket volume was calculated for each male Wistar rat. To determine the residual socket volume for each rat, the socket volume obtained from the measurements on the 3^rd day, 7^th day, and 14^th day were each divided by the socket volume on the 1^st day. Socket volume = mesiodistal width × buccolingual width × probing depth.³⁶

Histopathological analysis

After reaching the 3^rd day (group I, V, IX), the 7^th day (group II, VI, X), the 14^th day (group III, VII, XI), and the 28^th day (group IV, VIII, XII) post-extraction, the rats from the respective groups were sacrificed by cervical dislocation. This method was chosen to ensure rapid termination and lower chance of tissue contamination. The mandible of the rats was removed from the skull and the socket wound tissue was excised. Fresh tissue was fixed in 10% Buffered Neutral Formalin (BNF) solution (Milestone Medical, Italy) with a pH of 6.8-7.0 and the ratio of tissue to the BNF solution was 1:10.³⁷ The tissue containers were labelled and fixation was carried out for 12-48 hours, after which the tissue was immersed in 10% EDTA solution (Merck, Germany) for 10 days for decalcification.³⁸ The tissue was then cut using a scalpel with a thickness of 4 mm and the tissue was placed in tissue cassettes and put into a basket.³⁷ The baskets were loaded into an automatic processor machine and then transferred to a dehydration machine for tissue dehydration.³⁷ The next stage was embedding, in which the tissue was placed in a mould and submerged in liquid paraffin.³⁷ The cooled paraffin blocks were cut with a thickness of 4-5 μm using a microtome machine and the slices were placed in a water bath at 45°C.³⁷ The tissue slices would be collected on clean glass slides. The slides were labelled and placed in an incubator at 37°C to dry overnight.³⁷ The slides were then stained with haematoxylin-eosin (Merck, Germany) to see osteoblasts and osteocytes and with Masson's Trichrome (Merck, Germany) to see fibroblasts microscopically.³⁷ The stained tissue slides were then observed under an electric microscope (Primo Star, Carl Zeiss, Germany) with a magnification of 400 times in 10 viewing fields. Fibroblasts when observed under microscope are spindle-shaped with an ovoid nucleus and are commonly found in connective tissue.⁴ Osteoblasts when active are spindle-shaped or cuboidal, large and broad with abundant basophilic cytoplasm. When inactive, osteoblasts are flat, spindle-shaped, and lined up along the bone matrix surface.^10,11,39 Meanwhile, osteocytes are smaller than osteoblasts, branched out, and are trapped in lacunae among the matrix formed by osteoblasts.^11,39

The calculation of the mean cell count was carried out by two observers blinded to avoid bias and was aided with a tally counter (SXH, 5136, China) and a calculator (Casio, ƒχ-82MS, Japan). The results of the fibroblast, osteoblast, and osteocyte cell counts from 10 viewing fields were averaged.⁴⁰

Statistical analysis

The data were first analysed with Shapiro-Wilk normality test to ascertain the distribution of the data. Data that were normally distributed would be analysed with parametric tests. The RSV data were analysed using repeated measures ANOVA and one-way ANOVA test, while the histopathological data of the mean cell count of fibroblasts, osteoblasts, and osteocytes were analysed using one-way ANOVA test. The multiple comparison tests were performed with Least Significant Difference (LSD) test. The results were considered as significant if the p-value was below 0.05. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS), version 21 (IBM^® Inc., USA) by a researcher who was aware of the group allocation.

Clinical measurement of residual socket volume

The residual socket volume (RSV) was measured on the 3^rd, 7^th, and 14^th day post-extraction. The RSV in all treatment groups decreased over time and based on the repeated measures ANOVA test, there were significant differences in the RSV on the 3^rd, 7^th, and 14^th day in the Binahong group, the Gengigel^® group, and the base gel group (p < 0.05) (Figure 3A). In the Binahong group, significant RSV differences were observed between the 3^rd and 7^th day, between the 3^rd and 14^th day, and between the 7^th and 14^th day, while in the Gengigel^® group and the base gel group, significant RSV differences were only observed between the 3^rd and 14^th and 7^th and 14^th day (p < 0.05). There was no difference in the RSV between the 3^rd and 7^th day in the Gengigel^® group and the base gel group (Table 1).

Figure 3. Clinical and histopathological analysis of alveolar bone healing between different treatment groups.

Results were shown as the means of the residual socket volume (clinical observation) and the number of fibroblast, osteoblast, and osteocyte cells (histopathological observation).

Based on the one-way ANOVA test, there was a significant difference in the RSV among the treatment groups on the 7^th and 14^th day (p < 0.05). On the 7^th day, there were significant RSV differences between the Binahong group and the Gengigel^® group, between the Binahong group and the base gel group, and between the Gengigel^® group and the base gel group, while on the 14^th day, the significant RSV differences were only between the Binahong group and the base gel group and between the Gengigel^® group and the base gel group (Table 2).

Histopathological analysis

Fibroblast proliferation was examined on the 3^rd, 7^th, and 14^th day post-extraction. There was an increase trend in the number of fibroblasts from the 3^rd to 7^th day and a decrease from the 7^th to 14^th day (Figure 3B). Fibroblasts can be seen among the blue-stained collagen fibre (Figure 4). Based on the one-way ANOVA test, there were significant differences in the number of fibroblasts on the 3^rd, 7^th, and 14^th day in the Binahong, Gengigel^®, and base gel groups (p < 0.05). In the Binahong group, there were significant differences in the number of fibroblasts between the 3^rd and 7^th day and between the 7^th and 14^th day, while in the Gengigel^® group, there were significant differences in the number of fibroblasts between the 3^rd and 7^th day and between the 3^rd and 14^th day. In the base gel group, the significant differences were only observed between the 3^rd and 7^th day (Table 1).

Figure 4. Histopathological observation of fibroblasts in the alveolar bone healing of sockets.

The observation was conducted on the 3^rd, 7^th, and 14^th day post-extraction socket wounds stained with Masson’s Trichrome. (Abbreviation: Fb: Fibroblasts (×400)).

Significant differences in the number of fibroblasts among the Binahong group, Gengigel^® group, and the base gel group were observed on the 3^rd, 7^th, and 14^th day (p < 0.05). On the 3^rd day, there were significant differences in the number of fibroblasts between the Binahong group and the base gel group and between the Gengigel^® group and the base gel group. On the 7^th day, there were significant differences between the Binahong group and the Gengigel^® group, between the Binahong group and the base gel group, and between the Gengigel^® group and the base gel group. On the 14^th day, a significant difference was only observed between the Gengigel^® group and the base gel group (Table 2).

The proliferation of osteoblasts was examined on the 7^th, 14^th, and 28^th day post-extraction. In the Binahong group and the Gengigel^® group, there was an increase trend in the number of osteoblasts from the 7^th to 14^th day and a decrease from the 14^th to 28^th day, although in the base gel group, the number of osteoblasts kept rising over time (Figure 3C). Osteoblasts can be seen along the alveolar bone matrix (Figure 5). Based on the one-way ANOVA test, there were significant differences in the number of osteoblasts among the 7^th, 14^th, and 28^th day in the Binahong group and the base gel group (p < 0.05), while the Gengigel^® group showed no differences in the number of osteoblasts among those days. In the Binahong group, there were significant differences between the 7^th and 14^th day and between the 7^th and 28^th day, while in the base gel group, there were significant differences between the 7^th and 14^th day, between the 7^th and 28^th day, and between the 14^th and 28^th day (Table 1).

Figure 5. Histopathological observation of osteoblasts and osteocytes in the alveolar bone healing of sockets.

The observation was conducted on the 7^th, 14^th, and 28^th day post-extraction socket wounds stained with haematoxylin-eosin. (Abbreviations: Ob: osteoblasts; Oc: osteocytes (×400)).

The differences in the number of osteoblasts among the Binahong group, Gengigel^® group, and the base gel group were only observed on the 14^th day post-extraction (p < 0.05). On the 14^th day, significant differences were observed between the Binahong group and the base gel group and between the Gengigel^® group and the base gel group (Table 2).

Osteocyte proliferation was examined on the 7^th, 14^th, and 28^th day post-extraction. In the Binahong group and the base gel group, there was a trend of increase in the number of osteocytes over time, although in the Gengigel^® group, the increase only happened from the 7^th to 14^th day and was followed by a decrease from the 14^th to 28^th day (Figure 3D). Osteocytes can be seen in the alveolar bone matrix inside lacunae (Figure 5). Based on the one-way ANOVA test, there were significant differences in the number of osteocytes among the 7^th, 14^th, and 28^th day post-extraction only in the Binahong group and the base gel group (p < 0.05). No difference was observed in the Gengigel^® group among those days. In the Binahong group, there were significant differences between the 7^th and 14^th day and between the 7^th and 28^th day, while in the base gel group, significant differences were observed between the 7^th and 14^th day, between the 7^th and 28^th day, and between the 14^th and 28^th day (Table 1).

Differences in the number of osteocytes among the treatment groups were only observed on the 14^th and 28^th day post-extraction (p < 0.05). On the 14^th day, there were significant differences between the Binahong group and the base gel group and between the Gengigel^® group and the base gel group. On the 28^th day, significant differences were observed only between the Binahong group and the base gel group (Table 2).

Underlying data

Zenodo: The dataset of ‘The effects of 3% Binahong (Anredera cordifolia) leaf extract gel on the alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)’. https://doi.org/10.5281/zenodo.5189362.⁵⁹

This project contains the following underlying data:

Zenodo: Rat tooth extraction and 3% Binahong (Anredera cordifolia (Ten.) STEENIS) leaf extract gel. https://doi.org/10.5281/zenodo.5202954.⁶⁰

This project contains the following underlying data:

Reporting guidelines

Zenodo: ARRIVE checklist for ‘The effects of 3% Binahong (Anredera cordifolia) leaf extract gel on the alveolar bone healing in post-extraction tooth socket wound in Wistar rats (Rattus norvegicus)'. https://doi.org/10.5281/zenodo.5203068.⁶¹

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).