Effect of spirulina platensis algae purified bioactive peptides on wound healing after periodontal flap surgery: a randomized clinical trial

Abstract

Spirulina platensis, a blue-green alga rich in nutritional value and the beneficial pigment phycocyanin, is a source of bioactive peptides with antioxidant properties. This study aimed to evaluate the effects of bioactive peptides extracted from spirulina on wound healing and inflammation after periodontal surgery. In this double-blind, split-mouth randomized controlled trial study, twenty patients with periodontitis were assigned to either an experimental group (receiving spirulina peptide gel) or a control group (receiving placebo). Before surgery, all patients underwent scaling and root planning to eliminate pre-existing plaque. The surgical procedure involved a modified Widman flap technique. Wound healing was assessed at 1, 4, and 8 weeks post-surgery using parameters including soft tissue swelling, gingival color, probing depth, bleeding index, and plaque index. followed by a saline wash. Statistical analysis using SPSS 20 software employing non-parametric tests (Wilcoxon and Kruskal-Wallis) were carried out. The spirulina group showed a significant reduction in plaque and bleeding indices at weeks 4 and 8, as well as less gingival redness and lower pain scores at week 1, compared to controls. In addition, the need for pain medication was significantly lower in the spirulina group. These findings indicate that spirulina bioactive peptide gel can significantly improve wound healing, reduce inflammation, and alleviate post-surgical pain in periodontal procedures. These findings suggest its potential as a valuable adjunct therapy in periodontal surgery, enhancing patient recovery and improving overall periodontal health. However considering the limitation of the study, further studies are warranted to confirm and generalize the results.

Introduction

Surgical wounds in the oral cavity often heal more slowly than similar wounds on the skin and are particularly prone to inflammation and infection—complications that can negatively affect patient outcomes and quality of life. Approaches based on tissue regeneration are promising strategies for oral wounds healing. These approaches improve tissue re-epithelialization and extracellular matrix regeneration¹. Despite advancements in surgical techniques, optimal management of oral soft-tissue healing remains a clinical challenge. In the last few decades, the search for drug and food products to find peptides with bioactive compounds have attracted great attention^2,3. Bioactive peptides usually contain 2 to 20 amino acids per molecule, and in some cases may contain more than 20 amino acids. According to the sequence of amino acids in the structure of these peptides, the desired peptide can show positive effects e.g. improving the immune system, antimicrobial, antioxidant and anti-inflammatory activities^4–6.

Current literature has focused primarily on wound healing and antioxidant effects in skin wounds or fibroblast cultures, with far less emphasis on intraoral wounds and especially on human periodontal conditions. To date, there is a limited body of research specifically addressing interventions that enhance healing after periodontal flap surgery, which underscores an important gap in our knowledge.

In this study, spirulina algae was used to extract bioactive peptides in order to investigate their possible effects on wound healing and tissue regeneration. Spirulina platensis is a blue-green algae and one of the most promising microalgae that has been declared as “the best food” and “the best solution for tomorrow” by World Health Organization^7,8. The presence of phycocyanin pigment indicates it as a valuable resource. Phycocyanin has antioxidant, anti-cancer, anti-inflammatory and other positive systemic effects; and its safety and non-toxicity has been evaluated⁹.

Various factors including different cell types, complex cell signaling networks, growth factors and inflammatory mediators play role in wound healing process. Inadequate attention to wound healing may affect healing stages and thus lead to pathological inflammation¹⁰. On the other hand, excessive response of the immune system is the main reason for slow wound healing; hence, researchers have been exploring to regulate immune responses focusing on free oxygen radicals (ROS) as a major factor. These factors are produced during cellular respiration under normal conditions, but their overexpression at the wound site usually delays tissue regeneration. The use of spirulina compounds with antioxidant capabilities can provide the possibility of speeding up the repair of damaged tissues by inhibiting the effects of these radicals^11,12.

Several studies have been conducted to assess the anti-inflammatory, antioxidant and regenerative effects of spirulina. In a study by Syarina et al. investigating wound healing effects of spirulina focusing on skin fibroblasts, it was concluded that it may have potential use in treating various chronic wounds¹³. However, few studies have evaluated their effects on oral or periodontal wound healing; among those, evidence remains scarce and largely preclinical. If no robust clinical periodontal evidence exists, this gap is acknowledged as a focus of the present work.

In the studies on living tissue, no cases of complications or toxicity by Spirulina platensis have been reported. In a study investigating the wound healing activity of spirulina, no complications were reported during the study, and its LD50 was estimated to be more than 5000 mg/kg¹⁴. In another study that investigated the effect of spirulina on human fibroblast cells, no interactions or toxicity was reported in normal doses¹³.

The aim of the present study was to investigate the effect of Spirulina platensis purified bioactive peptides on healing of soft-tissue wounds following periodontal flap surgery. In this way, the patients in whom periodontal surgical treatment was indicated in at least 2 areas of the mouth were selected for the split-mouth study. The gels containing Spirulina bioactive compounds were applied under mucoperiosteal flaps and on exposed root surface in one of the two surgical sites, and its effects were compared with the control group on the opposite side. We hypothesized that surgical sites treated with Spirulina bioactive compounds will exhibit improved healing and reduced inflammation compared to untreated control sites.

Methodology

This double-blind split-mouth clinical trial study was conducted on patients with periodontitis who were candidates for periodontal flap surgery and referred to the Department of periodontics of Tabriz University of Medical Sciences. This study was a double-blind parallel randomized clinical trial with an allocation ratio of 1:1. Twenty patients that were candidates for periodontal flap surgery in at least 2 separate areas who met the inclusion criteria were randomly selected.

This study employed a double-blind design. Participants were not explicitly informed about the specific contents of the gel applied to each surgical site, but given the split-mouth design, complete blinding of participants was not possible. The treating surgeon could not be blinded due to the nature of the intervention. However, the outcome assessor, who performed all measurements and data collection, was blinded to the treatment allocation. The blinding of the outcome assessor was maintained by ensuring they were not present during the surgical procedure and had no knowledge of which treatment was applied to each site. Data were coded to prevent unblinding during analysis.

The research protocol was approved by Ethics Committee of Tabriz University of Medical Sciences with ethical code of IR.TBZMED.REC.1401.654 at 10/10/2022 and IRCT code of IRCT20221021056257N1 at 09/11/2022. Informed consents were obtained from all patients. and all methods were performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki.

The sample size was determined based on previous studies with similar designs and interventions, considering the primary outcome of periodontal probing depth (PD) reduction. Assuming a clinically significant difference of 1 mm in PD reduction between the spirulina and control groups, a standard deviation of 1.2 mm, a power of 80%, and a significance level of 5% (α = 0.05), a minimum sample size of 18 sites per group was required. To account for potential dropouts and ensure adequate statistical power, a total of 20 patients were included in the study, with each patient contributing data from two sites in a split-mouth design. This approach allowed for paired comparisons and reduced inter-individual variability¹⁵.

The inclusion criteria were the age range of 18 to 75 years; no systemic diseases; non-smokers; available during the 12-week study period; diagnosis of periodontitis; at least two non-contiguous interproximal pockets with periodontal pocket depths (PPDs) greater than 4 mm or other radiographic evidence indicating for periodontitis e.g. alveolar bone loss.

The exclusion criteria were pregnancy; using antibiotics and anti-inflammatory drugs within a month before surgery; patients who were using orthodontic appliances; presence of removable restorations; intraoral pathological conditions; presence of five or more decayed teeth; patients who participated in another study in the past one month; allergic reaction to health care products; patients who were forbidden to eat or drink liquids due to their medical conditions for four hours.

The sample selection method for allocating the type of treatment was randomized controlled sampling. In this method, the list of qualified people was entered into the Excel software and people were randomly selected from the statistical population. Regarding the type of treatment, a sealed envelope was given to the doctor by the patient. In other words, in this study, the wound was selected randomly and after opening the envelope, the doctor used a gel containing spirulina bioactive compounds for the wound on one side, and the wound on the other side was considered as control and no special substance was used. This study was double-blind in a way that the evaluating researcher and the patient did not know about ​​the case and the control areas.

No interim analyses were conducted during the study, and no stopping guidelines were established. The trial was designed to proceed to completion with the planned sample size. This decision was based on the relatively low risk associated with the intervention (topical application of spirulina gel) and the lack of expected adverse events that would warrant early termination of the study.

Preparation of bioactive spirulina gel

The Spirulina algae protein extraction, purification, hydrolysis and characterization were carried out according to Ebrahimi et la¹⁶. Briefly, algae powder was dissolved in water. The pH of the solution was adjusted to 10. Then, the solution was centrifuged and the resulting supernatant containing soluble protein was collected. The pH of supernatant was adjusted at 3 by HCl to precipitate the proteins. Then, the precipitate was collected by centrifugation and was neutralized using NaOH. After that, the protein sample was lyophilized by freeze-dryer. Finally, the amount of protein was measured by Bradford assay using an UV spectrophotometer. To hydrolyze the protein, Spirulina protein was dissolved at a concentration of 5%w/v in 0.2 M phosphate buffer (pH of 7.4) and allowed to be completely hydrated while stirring. Then, the enzymes stabilized in the buffer was added in the solution containing Spirulina protein. The enzymes of pepsin, pancreatin, trypsin, and alcalase were used. The reaction medium was placed in a water bath with 85 ◦C for 15 min to inactivate the reaction and enzyme activity. Then, the solution was cooled, centrifuged and the obtained supernatant was separated and pulverized by the freezer for further work. Protein content was also determined by Bradford method. Hydrolyzed protein was characterized by the mean of degree of hydrolysis, amino acid analysis, Sodium dodecyl sulphate–poly acrylamide gel electrophoresis (SDS-PAGE), and antioxidant activity via DPPH, ABTS, and total phenol assessments,. The results have been reported by our research group previously^16,17.

Application of purified gel of bioactive spirulina peptides in periodontal flap surgery

In this study, the effect of bioactive peptides obtained from spirulina algae was investigated on patients referred to the Department of Periodontics of Tabriz University of Medical Sciences with periodontitis diagnosis who were candidates for periodontal surgery in at least 2 areas. Scaling and leveling of the root surface was done for the participants before the surgical procedure, so no supra or subgingival plaque and mass were seen^18,19. Then the patients were evaluated after 8 weeks, and if there were areas with pocket depth of more than 5 mm, they underwent surgery²⁰. During surgery, a modified Widman flap was applied and the buccal and lingual tissues were separated mucoperiosteally. Any sub-gingival plaque was removed with manual and ultrasonic equipment, the soft tissue wall of the pocket was removed with a curt to remove the epithelium of the pocket and the germ tissue, and the area was washed with normal saline. Using the envelopes previously prepared for the random implementation of the process, the left or right quadrants were randomly selected to place the gel containing spirulina compounds and no compositions were places in the control site²¹.

All measurements were performed by a single experienced examiner using a standardized protocol and Williams periodontal probe (Hu-Friedy, Michigan, LLC, USA) probe for consistency.

The wound healing outcomes were evaluated after 1, 4 and 8 weeks post-surgery. The evaluation criteria included the following items: soft tissue swelling, gingival color, periodontal probing depth, bleeding index and plaque index.

All measurements and data collection were performed by a third person who was not aware of the surgical procedures. Seven days after surgery, sutures were removed and only visible signs measured. After 4 and 8 weeks, all items including soft tissue swelling, gingival color, periodontal probing depth, bleeding index and plaque index were measured. In order to achieve the desired level of plaque control after surgery, patients used 0.2% chlorhexidine gluconate mouthwash twice a day for 1 min until the 14th day. Furthermore, mechanical plaque control was avoided in the first week²⁰.

In order to check the amount of swelling, number 0 was used for no swelling, 1 for moderate and 2 for significant swelling. In order to evaluate the gingival color, the number 0 for no redness, 1 for moderate and 2 for significant redness were used. To measure the depth of the pocket and CAL, the reference of these measurements was from the CEJ location. Bleeding index and Plaque index were measured in 6 levels of the involved teeth and finally numerical criteria were presented as an average. The presence or absence of bleeding during probing was recorded using two scales: 0 for no bleeding and 1 for bleeding. The presence or absence of plaque was also measured with the same scale along the gingival margin²⁰.

During the first 7 days after surgery, patients completed a questionnaire to declare postoperative complaints, which included items such as the amount of pain experienced, type of pain, the number of analgesics used, swelling of the mucous membrane, swelling of the face, gingival swelling and bleeding of the.

The VAS scale, which is a visual analogue index with a pain level from 0 to 10 was used to determine the pain level. The degree of swelling of the mucosa and face was asked using two options: no swelling and high swelling, and the presence or absence of bleeding in surgical area was asked. The patients were asked not to refer to the previous days’ information when completing the questionnaire daily.

No changes were made to the trial outcomes after the trial commenced. The primary outcome of this study was periodontal probing depth (PD) reduction. Secondary outcomes included bleeding index, plaque index, soft tissue swelling, gingival color, pain severity, and analgesic consumption. All outcomes were assessed at baseline and during the follow-up visits as described.

Statistical analysis

The data were analyzed using SPSS 20 statistical software. The results of the study were reported using descriptive statistics methods. Wilcoxon signed-rank test was used to compare quantitative variables in two groups, Kruskal-Wallis test to compare follow-ups, and Chi-square and Fisher tests for qualitative variables. The significance level was p < 0.05. No subgroup analyses or adjusted analyses were pre-planned or conducted in this study due to the limited sample size and the split-mouth design of the study.”

Results

In order to investigate the healing effect of the purified compounds of spirulina bioactive peptides in periodontal flap surgery, bleeding index, plaque index, pain severity, soft tissue swelling, gingival color and periodontal pocket depth were compared in two sites of case and control in periodontal flap surgery candidates.

All 20 patients completed the study protocol and were included in the final analysis. No patients were lost to follow-up or excluded from the analysis. There were no losses to follow-up or exclusions after randomization. All patients adhered to the study protocol and completed all follow-up visits at 1, 4, and 8 weeks post-surgery.

The mean plaque index was 0.29 and 0.40 for test- and control groups at the 4 week after surgery. Also, the average of this value was 0.2 and 0.3 for the test and control groups at 8 weeks after surgery. At the results, PI values ​​in the experimental group at 4 weeks and 8 weeks after surgery were significantly lower than the control group (p value = 0.022, 0.011 respectively). (Table 1; Fig. 1)

Table 1.

The comparison of PD, BI, PI values in two groups of case and control.

Variable		Case		Control		p-value**
		Mean	Standard deviation	Mean	Standard deviation
CAL	Pre-op	5.8500	1.13671	5.4500	0.82558	0.055
PD	Pre-op	6.5500	1.27630	6.3500	0.74516	0.360
	Follow-up1	2.3470	0.34876	2.5050	0.33594	0.010
	Follow-up2	2.2635	0.24489	2.3460	0.22846	0.056
	p-value*	0.114		0.068
BI
	Follow-up1	0.4500	0.51042	0.8500	0.36635	0.005
	Follow-up2	0.1660	0.36274	0.3830	0.47484	0.023
	P value*	0.113		0.002
PI
	Follow-up1	0.2965	0.10312	0.4050	0.15800	0.022
	Follow-up2	0.2365	0.17452	0.3205	0.15830	0.011
	P value*	0.280		0.79

*p-value: Kruskal Wallis Test. ≠: To compare follow-up times (significant by Mann-Whitney U Test). **p-value: Wilcoxon Test (To compare two groups).

Fig. 1.

Comparison of BI and PI in two case and control groups.

The mean bleeding index at the 4-week examination was 0.85 and 0.38 for test- and control groups. Also, the average of this criterion was 0.45 and 0.16 for the test and control groups at 8 weeks after surgery. At the results, BI values ​​in the experimental group at 4 weeks and 8 weeks after surgery were significantly lower than the control group (p value = 0.005, 0.023 respectively) (Table 1; Fig. 1).

At the time of surgery, the PD value was on average 6.55 and 6.35 mm for the test and the control group respectively. At the 4-week examination the reduction in PD was 4.21 and 3.85 mm, respectively, which was a significant change from baseline in both groups. (p value = 0.010) The average of this value was 2.26 and 2.34 for the test and control groups at 8 weeks after surgery. this value although was reduced in comparison with the control group at 8 weeks after surgery, but it was not statistically significant (p value = 0.056). (Table 1) This reduction in probing depth from approximately 6.5 mm to 2.3 mm represents a gain of more than 4 mm, indicating a marked improvement in periodontal status that is considered clinically significant for patient outcomes.

At the time of surgery, the distance from bone to marginal gingiva was on average 6.55 and 6.35 mm for the test and the control group respectively. The distance from CEJ to the bone level was 5.85 and 5.45 mm for the test and control groups, respectively. The results show that there was no significant difference between two groups in CAL and PD values before surgery.

The comparison of gum redness in the experimental and control groups during the investigated times is shown in Fig. 2. The gum redness in the first follow-up (1 week) in the Exp group was significantly lower than the control group. At 4 weeks and 8 weeks after surgery, the frequency of absence of gum redness was higher in the case group, but no significant difference can be seen in the two groups.

Fig. 2.

Comparison of gum redness in case and control groups

At the first to the fourth days after surgery, facial swelling, mucosal swelling and post-surgical bleeding, although in the experimental group was less than the control group, but this difference was not statistically significant (Figs. 3, 4 and 5). Figure 6 shows the mean number of analgesics used in each group per day after surgery. Drug consumption was significantly higher in the control group. Pain severity in the experimental group during 7 days after surgery was significantly lower than the control group (Fig. 7).

Fig. 3.

Comparison of facial swelling in experimental and control groups.

Fig. 4.

Comparison of mucosal swelling in experimental and control groups.

Fig. 5.

Comparison of post-surgical bleeding in experimental and control groups.

Fig. 6.

Comparison of drug consumption in two Experimental and control groups.

Fig. 7.

Comparison of post-surgical pain in two Experimental and control groups.

The trial was completed as planned, with all patients completing the 8-week follow-up period. No interim analyses were conducted, and the trial was not stopped early for any reason.

Safety and Adverse Events.

No adverse events or unintended effects were reported or observed in either the spirulina or control groups throughout the study period. All patients tolerated the application of spirulina bioactive peptide gel well, with no reports of allergic reactions, irritation, or other local or systemic adverse effects.

Discussion

Surgical approach is considered as one of the conventional treatment methods for periodontal diseases in many cases e.g. the ineffectiveness of mechanical treatments to remove plaque. One of the side effects of any surgery is symptoms e.g. inflammation and pain during the process of tissue regeneration.

According to the results of the present study, there was no significant difference between experimental and control groups in the amount of CAL and PD before surgery in patients who were candidate for periodontal flap surgery. Spirulina bioactive peptides caused a statistically significant decrease in PD, BI and PI values at 4 weeks after surgery in comparison with the control group. 8 weeks after surgery, BI and PI values were lower in experimental group compared to the control group, but the differences were not significant. Furthermore, the amount of gingival redness in one week after surgery in the experimental group was significantly lower than the control group at one week after surgery, but this difference was not significant at 4 and 8 weeks after surgery. The reduction in PD from approximately 6.5 mm at baseline to around 2.3 mm at 8 weeks post-surgery in the spirulina groupsuch a considerable reduction in PD reflects a significant gain in periodontal health, which can be associated with improved long-term tooth prognosis and reduced risk of disease progression for these patients.

The results of the present study are in line with the findings of the study by Kang et al. (2021). They reported that spirulina reduce periodontitis caused by Porphyromonas Gingivalis through its anti-inflammatory effect and thus reduced bone loss, indicating that spirulina may be a potential agent for treatment of periodontitis²². Mahendra et al.‘s in a clinical trial study (2013) on the subgingival use of spirulina gel in chronic periodontitis showed a significant improvement in pocket depth and clinical attachment loss. According to their the results, topical application of spirulina gel combined with scaling and root leveling had beneficial effects and the effectiveness of this product as a local drug delivery system in the non-surgical treatment of periodontitis without any side effects was observed¹⁵.

Dranseikienė et al. (2022) showed that the purity of cyano-phycocyanin has significant effect on its antimicrobial and antioxidant properties. Cyano-phycocyanin significantly contributes to wound healing and tissue regeneration by inducing fibroblast proliferation and increasing cell migration²². According to the study by Agustina et al. (2021), the aqueous extract of spirulina is a potential source of antioxidant activity with wound healing capacity without toxic and irritating effects²³.

Effective pain control after oral surgeries such as periodontal flap surgery is one of the problems in periodontology surgeries. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to reduce pain after periodontal flap surgery, but these drugs can be associated with some side effects, such as platelet dysfunction and gastrointestinal problems²⁴. In the present study, the VAS pain scale was used. According to the patients’ opinion, the results indicated that the effectiveness of the bioactive peptides of spirulina significantly reduced the amount of pain within 7 days after surgery, as well as the consumption of analgesics was significantly decreased compared to the control group. Based on the current data, Spirulina bioactive peptides may be considered a promising adjunct to conventional periodontal therapy. However, given the limitations of our study, it is recommended to carry out larger, long-term studies before definitive recommendations regarding its routine clinical application.

In a study by Zamani et al. (2020), topical cream produced from Spirulina platensis phycocyanin improved wound healing in mice infected with Candida Albicans²⁵. Also, in an in vitro study, Panigrahi et al. observed a significant improvement in wound healing using spirulina²⁶. These studies, like the present study, investigated the wound healing and anti-inflammatory effects of spirulina and its compounds and proved its cellular and tissue mechanisms. According to the results of the above studies, spirulina improves wound healing and reduces inflammatory complications, which is in line with the results of the study.

It should be noted that while the beneficial effects of Spirulina on wound healing and inflammation are well-supported in preclinical in vitro and animal studies, there remains a significant translational gap. The clinical evidence, particularly within periodontology, is still limited. Therefore, future human trials with larger sample sizes are warranted to validate the clinical applicability of Spirulina bioactive peptides as adjuncts to periodontal therapy.

To our knowledge, this is one of the first clinical trials to assess the direct application of purified Spirulina platensis bioactive peptides in the context of human periodontal flap surgery. Given the paucity of clinical studies targeting intraoral wound healing using microalgae-derived peptides, our results provide new insight and initial clinical evidence supporting their therapeutic potential. This research thus represents an important step forward in translating promising preclinical findings into the domain of oral and periodontal regenerative therapies.

While this study supports the therapeutic potential of spirulina bioactive peptide gel as an adjunct in periodontal surgery, limitations should be considered. The relatively small sample size limits statistical power and may increase the risk of Type II errors. Furthermore, the follow-up period was restricted to 8 weeks, preventing evaluation of the long-term stability of wound healing. The study was conducted at a single academic center in Tabriz, using only the modified Widman flap technique. These factors may restrict the generalizability of the findings to other populations with different demographic characteristics, disease severities, surgical techniques. Therefore, caution is warranted when extrapolating these results to broader or more diverse clinical settings. In light of these promising findings, future studies should include larger sample sizes, longer-term follow-up periods, and multi-center participation to confirm efficacy and safety across diverse populations. Additionally, exploring the effects of spirulina gel in other types of periodontal surgeries or in medically compromised patient populations could further elucidate its clinical applications. More in-depth analysis and identification of specific peptide components may also optimize therapeutic protocols.

Conclusion

Our study demonstrated that wound healing following periodontal flap surgery was notably improved in sites treated with Spirulina bioactive peptides compared to control sites. Specifically, both Bleeding Index (BI) and Plaque Index (PI) values were significantly lower in the Spirulina group at 4 and 8 weeks post-surgery. Clinical examinations showed that Spirulina also reduced redness of the gums at one week. Additionally, patients in the Spirulina group reported significantly lower pain severity and required fewer analgesics within the first 7 days after surgery. Spirulina bioactive peptides led to a significant reduction in Probing Depth (PD) at 4 weeks; although PD values remained lower than controls at 8 weeks, this difference was not statistically significant at that time point. Patient feedback indicated no significant difference in post-surgical facial or surgical area swelling and bleeding between the groups. These findings suggest that Spirulina bioactive peptides may be a promising adjunct for improving early healing phases, reducing discomfort, and improving periodontal indices after flap surgeries. However considering the limitation of the study, further studies are warranted to confirm and generalize the results.

Author contributions

P.J., data analysis and interpretation, manuscript drafting and critical revision. P.A., study design, data acquisition, analysis and interpretation, manuscript drafting. M.F., data acquisition, analysis and interpretation, manuscript drafting. H.H conception, data analysis and interpretation, critical revision of the manuscript. M.K., conception, study design, data analysis and interpretation, manuscript drafting and critical revision. All authors gave their final approval and agree to be accountable for all aspects of the work.

Funding

This manuscript was financially supported by research council of Tabriz University of Medical Sciences (thesis No. 70294).

Data availability

The used and analyzed datasets available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The research protocol was approved by Ethics Committee of Tabriz University of Medical Sciences with ethics code of IR.TBZMED.REC.1401.654. All of the participants were informed verbally about the clinical process and their written informed consents were obtained.

Competing interests

The authors declare no competing interests.

The full trial protocol is available from the corresponding author upon reasonable request.