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 Table of Contents  
ORIGINAL RESEARCH
Year : 2020  |  Volume : 12  |  Issue : 5  |  Page : 463-469

Characteristic assay of incorporation of carbonated hydroxyapatite–propolis as an alternative for alveolar bone loss therapy on periodontitis: An in vitro study


1 Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia
2 Master of Clinical Dentistry Program, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia

Date of Submission27-Nov-2019
Date of Decision17-Mar-2020
Date of Acceptance17-Mar-2020
Date of Web Publication21-Oct-2020

Correspondence Address:
Dr. Suryono Suryono
Department of Periodontology, Faculty of Dentistry, Universitas Gadjah Mada, Jl. Denta, Sekip Utara, Yogyakarta.
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_315_19

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  Abstract 

Aim: To examine the characteristics of carbonated hydroxyapatite (CHA) incorporated with propolis in the concentrations of 5%, 7.5%, and 10%. Materials and Methods: In this in vitro study, the CHA bone graft (GamaCHA) material was immersed in the propolis solution with the concentrations of 5%, 7.5%, and 10% by serial dilution method for 24h at room temperature. Absorbance readings of loading and release assay were carried out using UV-Vis spectrophotometers at 289nm. Antibacterial testing was done by calculating the inhibition zone of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The microtetrazolium (MTT) assay was used for viability test of fibroblast NIH 3T3 cell culture by measuring the optical density in the enzyme-linked immunosorbent assay (ELISA) reader. The data of the study were analyzed with one-way analysis of variance (ANOVA) (loading, release, antibacterial P. gingivalis, and viability test) and Kruskal–Wallis (antibacterial A. actinomycetemcomitans). Results: The results showed that CHA incorporated with 10% propolis had the highest loading percentage, the largest diameter of bacterial inhibition zone, and the highest fibroblast viability (P < 0.05). However, the difference in the concentrations of propolis incorporated with CHA did not affect the percentage of propolis release (P > 0.05). Conclusion: CHA incorporated with 10% propolis solution showed highest loading percentage, antibacterial activity, and viability.

Keywords: Antibacterial Assay, Carbonated Hydroxyapatite, Loading, Propolis, Release, Viability Assay


How to cite this article:
Suryono S, Kusumawati I, Devitaningtyas N, Sukmawati AN, Wijayanti P. Characteristic assay of incorporation of carbonated hydroxyapatite–propolis as an alternative for alveolar bone loss therapy on periodontitis: An in vitro study. J Int Oral Health 2020;12:463-9

How to cite this URL:
Suryono S, Kusumawati I, Devitaningtyas N, Sukmawati AN, Wijayanti P. Characteristic assay of incorporation of carbonated hydroxyapatite–propolis as an alternative for alveolar bone loss therapy on periodontitis: An in vitro study. J Int Oral Health [serial online] 2020 [cited 2020 Nov 27];12:463-9. Available from: https://www.jioh.org/text.asp?2020/12/5/463/298793




  Introduction Top


Pathogenic bacteria cause periodontitis, which is an inflammatory disease. Some of the anaerobic gram-negative bacteria that cause periodontitis are Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Periodontitis can damage the periodontal ligament, cementum, and alveolar bone, further leading to bone damage. In fact, untreated damage of periodontal tissue can cause tooth loss.[1] Periodontal tissue damage caused by periodontitis requires adequate therapy. One of the objectives of periodontal therapy is to recover from the damage in the alveolar bone, cementum, and periodontal ligaments caused by this disease. Periodontal regeneration is carried out by using a number of techniques, such as bone graft, guided tissue regeneration, and growth factors.[2]

Surgical periodontal therapy with bone graft material is expected to regenerate the bone tissues and induce new attachments. Synthetic alloplastic material is one of the bone graft materials that does not cause additional morbidity like the autograft material does nor cause disease transmission.[3] Carbonated hydroxyapatite (CHA) is an alloplastic bone graft material that has the same composition as that of a natural bone. In addition, it also has biocompatibility and osteoconductive capacity similar to that of a natural bone.[4] Apart from being a bone graft material, CHA is also proven to serve as a drug delivery system. As it is derived from a synthetic material, the alloplastic bone graft material has a nonvascularized structure, which minimizes the risk of bacterial adhesion in the bone graft material applied to the defected area. The contamination of bone graft material may directly occur in the oral cavity during surgery or after surgery.[5] Therefore, it is necessary to add an antibacterial agent to address this issue. Propolis is a natural material that potentially functions as an antibacterial agent, antioxidant, anti-inflammatory, and immunomodulator, which is beneficial for the regeneration of bone.[2] Propolis also has a minimum cytotoxic effect on gingival fibroblasts.[6] In fact, the biological effects of propolis are mainly present due to the presence of flavonoid compounds and caffeic acid phenethyl ester (CAPE).

Controlled delivery is expected to provide a local therapeutic effect on the areas with bone graft.[7] The addition of propolis in the periodontitis experiment can reduce the loss of alveolar bone.[8] A previous study proved that 10% propolis gel could stimulate gingival healing process in Sprague Dawley rats.[9] In fact, the incorporation of CHA with propolis as an antibacterial agent should emphasize on the release of antibacterial agent. The concentrations of propolis incorporated with CHA determine the antibacterial power of the material. The release of propolis incorporated with the bone graft material is expected to occur gradually in a long time.[10] The objective of this study was to examine the characteristics of CHA incorporated with propolis in the concentrations of 5%, 7.5%, and 10% by calculating the percentage of propolis loading and release, antibacterial assays against P. gingivalis and A. actinomycetemcomitans, and viability assay of NIH 3T3 fibroblasts.


  Materials and Methods Top


Study design

This in vitro study is true experimental research. This study was conducted in Laboratorium Riset Terpadu, Faculty of Dentistry, Universitas Gadjah Mada; Microbiology Laboratory, Faculty of Dentistry, Universitas Airlangga; and Parasitology Laboratory, Faculty of Medicine, Universitas Gadjah Mada, during 6 months (February–July 2019). All the procedures were followed in accordance with the ethical standards of experimentation by Faculty of Dentistry Ethics Committee, Universitas Gadjah Mada (003/KKEP/FKG-UGM/EC/2019).

Sampling criteria

A simple random sampling method was used. Inclusion criteria for loading assay were 10mg of GamaCHA, propolis solution within expired date. Exclusion criteria included GamaCHA and propolis solution with expired date. Inclusion criteria for antibacterial assay were sterilized incorporation CHA-propolis with ethylene oxide sterilization and bacteria with ATCC 33277. Bacteria contaminated with another strain were excluded from the study. Inclusion criteria for viability test were fibroblast cell line NIH 3T3. Exclusion criteria for viability test were contaminated cultured cell. The number of sample needed in this study was four. Pilot study had the loss of follow-up of 5%. To anticipate the loss of follow-up in this study, the sample size for each group was increased 5% by adding two samples in each group. So the sample size for this study was six in each group. The total sample size needed in this study was six.

Study material preparation

Making propolis-incorporated carbonated hydroxyapatite

The serial dilution method was used to obtain the propolis solution with the concentrations of 5%, 7.5%, and 10%. Thereafter, the CHA bone graft material (GamaCHA) was immersed in the propolis solution for 24h at room temperature. The procedure was carried out in six replicates for each concentration of propolis.

Propolisloading percentage (loading test): The samples of two types of 1 mL propolis solution (one that was used to immerse the CHA bone graft material and the other that was not used to immerse the graft specimen) were taken with the help of micropipette and put into two different UV-vis cuvettes, respectively. Sterile distilled water was used as the blank solution. Absorbance readings were taken at a wavelength of 289nm.

Propolis release percentage (release test): The specimens of bone graft material with the propolis content were dried in an incubator at 37°C, which was covered with a filter paper. All of the dried specimens were then immersed in the phosphate-buffered saline (PBS) solution, whose pH was 7.4. After an hour, the PBS solution and specimens were centrifuged at 2000 rotation per minute for 5min. The PBS solution was taken and put in a UV-vis cuvette, whereas the new PBS solution was added in the graft material specimens in the microcentrifuge tube. The absorbance of the solution was measured by using a UV-vis spectrophotometer at a wavelength of 289nm. This treatment was repeated at the specific intervals of 3, 6, 24, 48, and 72h. The release percentage of propolis was calculated by using the same formula used for calculating the loading percentage of propolis.

Antibacterial power: The cultures of P. gingivalis and A. actinomycetemcomitans were prepared by taking McFarland (108 CFU/mL) standard into reference. Then, the cultures were taken in the same quantities and filled into two inoculation needles to be dissolved in the 10-mL liquid brain heart infusion (BHI) media. The suspension was swabbed on the surface of the Mueller Hinton Agar plate in a petri dish. Thereafter, holes were made as per the diameter of the bone graft materials. The bone graft materials incorporated with the propolis solution were then implanted in each of the holes and incubated in an anaerobic incubator at 37°C. The inhibition zone around the agar surface was measured with a caliper after being incubated for 24h [Figure 1].
Figure 1: Inhibition zone formula

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Viability in fibroblast cells: This study used NIH 3T3 fibroblast cell line. Propolis in the concentrations of 5%, 7.5%, and 10% was used as the test material in the study. The test material was placed in a plate, and the cell culture was incubated for 24h at 37°C in each treatment. The microtetrazolium (MTT) assay on the cell culture was performed by measuring the optical density by the enzyme-linked immunosorbent assay (ELISA) reader. The number of viable cells is indicated by the purple color intensity in the culture. Higher color intensity shows higher cell viability.

Observational parameters

In this study, loading and release data were calculated using the following formula:



where

A = absorbance of propolis solution before being incorporated with CHA

B = absorbance of propolis solution after being incorporated with CHA

The data from this calculation were in percentage.

The antibacterial assay was measured using the following formula:



where

Dv = vertical diameter

Ds = well diameter

Dh = horizontal diameter

The data from this calculation were inhibition zone as measured in millimeter.

The results from fibroblasts cell line viability were calculated using the following formula:



The acquired data were in percentage.

Statistical analysis

The data were statistically analyzed using the Statistical Package for the Social Sciences (SPSS) software for Windows, version 21.0 (SPSS, Chicago, Illinois). Descriptive statistics as well as advanced statistics were used [Table 1]. One-way analysis of variance (ANOVA) with a significance level of 0.05 for loading, release, antibacterial P. gingivalis, and viability test, followed by a post hoc least significant difference (LSD) Kruskal–Wallis analysis for antibacterial A. actinomycetemcomitans test, followed by Mann–Whitney U test.
Table 1: Descriptive and statistical analysis on all variables

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  Results Top


Loading

The results of one-way ANOVA test showed P = 0.00 (P < 0.05) with a significance level of 0.05, which means that significant differences were observed among the concentration groups [Table 1]. Thereafter, the data were processed by multivariate comparison analysis by using post hoc LSD test, and the results showed significant differences among the concentration groups [Table 2]. The data of loading percentage of propolis are present in [Figure 2]. The highest loading percentage was found in group C (32.08%), whereas group A showed the lowest loading percentage (10.63%). Hence, the concentration of propolis solution used for immersion affects the percentage of propolis loading.
Table 2: Within-group comparison between treatment group in loading assay by least significant differences

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Figure 2: Percentage of propolis loading

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Release

The results of one-way ANOVA test showed P = 1.00, with a significance level of 0.05, which means that the differences in the mean of propolis release among the concentration groups were not statistically significant [Table 1]; releasing percentage of propolis into PBS solution after incubation during several time points is shown in [Figure 3]. The lines are intersected on several observation times. The release profiles among all groups are quite similar. The highest release percentage was found at the first hour and then gradually decreased until the first 24h. Releasing of propolis was still detected until 72h. This implies that the concentration of propolis solution used for immersion does not affect the percentage of propolis release.
Figure 3: Percentage of propolis release

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Antibacterial assay

[Figure 4] presents the mean diameter of inhibition zone against P. gingivalis and A. actinomycetemcomitans bacteria. CHA incorporated with 10% propolis had the largest inhibition zone diameter as compared to CHA incorporated with 5% and 7.5% of propolis. On the contrary, as a negative control, the CHA material did not have any antibacterial power. Statistical tests conducted by using Kruskal–Wallis test for A. actinomycetemcomitans bacteria showed a value of P = 0.00 (P < 0.05), which means that there were significant differences among the treatment groups [Table 1]. Thereafter, the data were processed by multivariate comparison analysis by using the Mann–Whitney U test, whose results showed significant differences among the treatment groups [Table 3]. Statistical tests conducted by using one-way ANOVA test for P. gingivalis bacteria showed a value of P = 0.00 (P < 0.05), which means that there were significant differences among the treatment groups [Table 1]. Thereafter, the data were processed by multivariate comparison analysis by using the post hoc LSD test, whose results showed significant differences among the treatment groups [Table 4]. This indicates that the concentration of propolis solution used for immersion affects the formation of inhibition zones.
Figure 4: Inhibition zone against P. gingivalis and A.actinomycetemcomitans

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Table 3: Within-group comparison between treatment group in antibacterial A. actinomycetemcomitans testing by Mann–Whitney U test

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Table 4: Within-group comparison between treatment group in antibacterial P. gingivalis testing by least significant differences

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Viability assay

[Figure 5] shows the mean viability of fibroblasts. The control group, which was only treated with CHA material, had the lowest fibroblast viability as compared to the treatment groups. The treatment group, which was administered with CHA incorporated with 10% propolis, had the highest fibroblast viability. The statistical tests resulted in a value of P = 0.00 (P < 0.05), thereby confirming the presence of significant differences among the treatment groups [Table 1]. Thereafter, the data were processed by multivariate comparison analysis by using the post hoc LSD test, whose results showed significant differences among the treatment groups [Table 5]. This indicates that the concentration of propolis solution used for immersion affects the viability of fibroblast.
Figure 5: Mean viability of fibroblasts

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Table 5: Within-group comparison between treatment group in viability fibroblast cell line testing by least significant differences

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  Discussion Top


Loading

Embedding technique was used as the incorporation technique in this study. An embedding process followed by incubation for a certain time is one of the easiest techniques of connecting antibacterial material with the bone graft material.[11] In fact, by embedding techniques, the porosity and surface area of bone graft materials affect the absorption of antibacterial material.[12] The bond that occurs between CHA and propolis is due to the use of gelatin in the bone graft material. The hydrophilic group in gelatin, which is a group of polar amino acids, causes dried gelatin to absorb liquid.[13]

This study showed that the use of low-concentration propolis solution caused the dissolving of immersed CHA bone graft material, which means that there was a reduction in the surface area of the bone graft material. This, then, decreased the amount of propolis solution absorbed into the CHA. This result is in line with a theory stating that the surface area of bone graft material affects the absorption of antibacterial material, in this case, of propolis.[12]

Release

Propolis release assay was performed at the specific intervals of 1, 3, 6, 24, 48, and 72h. The highest release was found in the first 1h. High propolis release in the first 1h is consistent with the results of other studies, thereby showing that the highest degradation of CHA bone graft material into the PBS solution occurs in the first 1h.[10]

A high release in the first 1h indicates a high concentration of propolis in the in vitro environment. A high initial release, also known as burst release, is useful for the eradication of local bacteria after the surgery.[14]

Propolis release was observed up to 72h, thereby indicating a gradual propolis release. In a static environment, such as PBS solution, most drugs are released within 24h. The percentage of propolis release in the first 6h was more than 40% in all the groups. This is in line with the results of previous studies on controlled drug delivery models using polymer bone graft materials. This way, this study model has followed the principles of a controlled delivery.[12]

Antibacterial power

The results of this study showed that CHA is able to carry the antibacterial properties of propolis, which is evident from the formation of a clear zone around the well. Clear zones around the wells of CHA, which was immersed in 5%, 7.5%, and 10% solutions, indicated that CHA could carry propolis as an antibacterial agent against P. gingivalis. On the contrary, as a negative control, CHA formed no clear zone. The antibacterial activity of propolis is affected by the concentration of solution used for immersion. The propolis solution with a concentration of 10%–30% is an optimal concentration that has a good antibacterial activity against the gram-positive and gram-negative bacteria.[15] CHA immersed in a solution with a concentration of 5% formed a zone of inhibition against A. actinomycetemcomitans with a diameter of 8.90 mm, and a zone of inhibition against P. gingivalis with a diameter of 9.59 mm. Immersion in a solution with a concentration of 10% created a greater inhibition zone, that is 12.7 mm, against A. actinomycetemcomitans, and 13.86 mm against P. gingivalis. CHA immersed in a solution with a concentration of 10% showed a greater antibacterial activity as compared to that immersed in solutions whose concentrations were 5% and 7.5%.

The group treated with CHA and 10% propolis resulted in the largest diameter. Propolis has various active substances, such as CAPE, which act as an antibacterial agent. CAPE could damage the bacterial cytoplasmic membrane, inhibit nucleic acid synthesis, and increase the permeability of cell membrane, thus causing bacterial lysis.[16] The antibacterial activity is affected by the concentration of propolis solution used for immersion. Lower concentration of propolis suggests lower antibacterial activity.[15]

Viability

Periodontal ligaments comprise fibroblasts, which maintain the periodontal tissue homeostasis under physiological conditions. Whenever there is an injury or damage in the periodontal tissues, fibroblasts are activated and they turn into myofibroblasts, which produce extracellular matrix that can stimulate healing.[17] CHA incorporated with propolis can improve the ability of tissues to repair themselves.[18]

The results of this study showed that propolis incorporation in the concentrations of 5%, 7.5%, and 10% could stimulate the growth of fibroblasts as compared to the control group treated with CHA. This is in line with the results of another study,[9] which revealed that 10% of propolis decreases the number of polymorphonuclear cells, yet increases the number of fibroblasts and leads to the formation of new blood vessels. A study,[19] which used nanohydroxyapatite immersed in propolis solution for 24h, showed a low toxicity effect on fibroblasts.

Propolis has an active substance in the form of CAPE and flavonoids. Flavonoids can stimulate transforming growth factor beta (TGF β), which mediates chemotaxis and the proliferation of fibroblasts into the healing area. The presence of large number of fibroblast in the healing area will induce to synthesis of more collagen so that wound healing will be more faster and better.[20]

Conclusion

The results from this study suggested that propolis could be loaded into CHA and then released in a controlled manner, with 10% propolis having the optimum ability in the loading process, antibacterial activity against P. gingivalis, and fibroblasts cell viability. However, another study is still needed to investigate longer release time, antibacterial assay against other periodontal pathogenic bacteria, and longer time in viability assay.

Acknowledgement

This research was accomplished by the help of Integrated Research Laboratory, Faculty of Dentistry, Universitas Gadjah Mada.

Financial support and sponsorship

This study was supported by a grant Hibah Penelitian Dana Masyarakat Berbasis Luaran (Outcome based), Faculty of Dentistry, Universitas Gadjah Mada, Republic of Indonesia, in the fiscal year 2019 under contract No. 4349/UN1/FKG1/SetKG1/PT/2019.

Conflicts of interest

There are no conflicts of interest.


  Authors contributions Top


All of the authors contributed equally in the preparation of the manuscript and finally approved by all authors.


  Ethical policy and Institutional Review board statement Top


The ethical aspect of this study was reviewed and approved by Ethic Committee of Faculty of Dentistry Universutas Gadjah Mada No. 0031/KKEP/FKG-UGM/EC/2019 on February 19th, 2019.


  Data availability statement Top


The data that supports the findings of this study are available upon reasonable request at the corresponding author.



 
  References Top

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Zohery AA, Nour ZM, El Rehim SS, Mady MI Histomorphometric analysis of bone regeneration after use of propolis versus nanobone graft materials for the management of class II furcation defect in dogs. J. Alexandria Dental 2017;42:198-203.  Back to cited text no. 2
    
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Titsinides S, Agrogiannis G, Karatzas T Bone grafting materials in dentoalveolar reconstruction: A comprehensive review. Jpn Dent Sci Rev 2019;55:26-32.  Back to cited text no. 3
    
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Bashutski JD, Wang HL Periodontal and endodontic regeneration. J Endod 2009;35:321-8.  Back to cited text no. 4
    
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Filho HN, Pinto TF, De Freitas CP, Ribeiro PD Jr, Dos Santos PL, Matsumoto MA Autogenous bone grafts contamination after exposure to the oral cavity. J Craniofac Surg 2014;25:412-4.  Back to cited text no. 5
    
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Mooduto L, Fredline C, Sosiawan A, Wahjuningrum DA Cytotoxicity of sodium hypochlorite, chlorhexidine, and propolis on human periodontal ligament fibroblast cell. J Int Dent 2019;12:476-80.  Back to cited text no. 6
    
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Habibovic P, Juhl MV, Clyens S, Martinetti R, Dolcini L, Theilgaard N, et al. Comparison of two carbonated apatite ceramics in vivo. Acta Biomater 2010;6:2219-26.  Back to cited text no. 7
    
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Freires IA, Santaella GM, de Cássia Orlandi Sardi J, Rosalen PL The alveolar bone protective effects of natural products: A systematic review. Arch Oral Biol 2018;87:196-203.  Back to cited text no. 8
    
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Suryono  , Hasmy NS, Pertiwi TL, Benyamin B, Ismail A Propolis 10%-gel as a topical drug candidate on gingivitis. Int J Med Pharm 2017;1:12-7.  Back to cited text no. 9
    
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Ardhani R, Setyaningsih S, Hafiyyah OA, Ana ID Preparation of carbonate apatite membrane as metronidazole delivery system for periodontal application. Key Eng Mater 2016;696:250-8.  Back to cited text no. 10
    
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Anagnostakos K, Schröder K Antibiotic-impregnated bone grafts in orthopaedic and trauma surgery: a systematic review of the literature. Int J Biomater 2012;2012:538061.  Back to cited text no. 11
    
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Dashti A, Ready D, Salih V, Knowles JC, Barralet JE, Wison M, et al. In vitro antibacterial efficacy of tetracycline hydrochloride adsorbed onto Bio-Oss VR bone graft. J Biomed Mater Res B 2010;93B2:394-400.  Back to cited text no. 12
    
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Peña C, de la Caba K, Eceiza A, Ruseckaite R, Mondragon I Enhancing water repellence and mechanical properties of gelatin films by tannin addition. Bioresour Technol 2010;101: 6836-42.  Back to cited text no. 13
    
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Phaechamud T, Darunkaisorn W Drug release behavior of polymeric matrix filled in capsule. Saudi Pharm J 2016;24: 627-34.  Back to cited text no. 14
    
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Ghasemi FS, Eshraghi SS, Andalibi F, Hooshyar H, Kalantar-Neyestanaki D, Samadi A, et al. Anti-bacterial effect of propolis extract in oil against different bacteria. Zahedan J Res Med Sci 2017;19:e7225.  Back to cited text no. 15
    
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Al-waili N Mixing two different propolis samples potentiates their antimicrobial activity and wound healing property : A novel approach in wound healing and infection. Vet World 2018;11:1188-95.  Back to cited text no. 16
    
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Li B, Wang JH Fibroblasts and myofibroblasts in wound healing: Force generation and measurement. J Tissue Viability 2011;20:108-20.  Back to cited text no. 17
    
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Mai R, Reinstorf A, Pilling E, Hlawitschka M, Jung R, Gelinsky M, et al. Histologic study of incorporation and resorption of a bone cement-collagen composite: An in vivo study in the minipig. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e9-14.  Back to cited text no. 18
    
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Grenho L, Barros J, Ferreira C, Santos VR, Monteiro FJ, Ferraz MP, et al. In vitro antimicrobial activity and biocompatibility of propolis containing nanohydroxyapatite. Biomed Mater 2015; 10:1-8.  Back to cited text no. 19
    
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Oryan A, Alemzadeh E, Moshiri A Potential role of propolis in wound healing: Biological properties and therapeutic activities. Biomed Pharmacother 2018;98:469-83.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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