Abstract
This study evaluates the effects of a green tea (Camellia sinensis) and hyaluronic acid gel on fibroblast activity and alveolar bone repair following third molar extractions. By examining the gene expression related to cell survival, proliferation, and angiogenesis, the study bridges in vitro findings with clinical outcomes in a split-mouth randomized trial. Human fibroblasts were exposed to the treatment gel, analysing gene expression through RT-qPCR. Twenty participants undergoing bilateral third molar extractions received the test gel on one side and a placebo on the other. Assessments included patient-reported outcomes, professional evaluations, and radiographic analyses at multiple postoperative intervals. The test gel significantly enhanced AKT, CDKs, and VEGF gene expressions, indicating a positive effect on angiogenesis and cell proliferation. Clinically, it resulted in reduced exudate, swelling, and secondary interventions, with radiographs showing improved alveolar bone density after 90 days. The green tea and hyaluronic acid gel significantly improves soft tissue and bone healing post-extraction, offering a promising adjunctive therapy for enhancing postoperative recovery. This gel represents a novel adjuvant treatment option for facilitating improved healing outcomes after third molar extractions, highlighting its potential utility in clinical dental practice.
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Introduction
The surgical extraction of third molars poses significant clinical challenges due to anatomical constraints, such as limited access, varying degrees of impaction, and proximity to critical structures like blood vessels and nerves1. These challenges often necessitate complex surgical techniques, leading to postoperative complications including pain, trismus, limited mouth opening, and alveolitis, thereby causing considerable patient discomfort2. In response, there has been a growing interest in exploring alternative therapeutic strategies aimed at enhancing tissue regeneration at post-extraction sites3,4,5. Among these, bioactive molecules that can expedite soft tissue healing and bone regeneration are increasingly recognized for their potential to improve the postoperative recovery of patients undergoing third molar extractions2,6,7.
Herbal medicines, known for their anti-inflammatory properties, have shown promising results in controlling postoperative inflammation and enhancing healing, with green tea-based compounds standing out for their preclinical efficacy in reducing bone resorption and inflammatory markers in periodontal disease models8,9,10,11,12,13,14,15. The antioxidant properties of green tea have also been clinically validated to improve periodontal health more effectively than traditional toothpaste formulations16, and systematic reviews have equated the anti-inflammatory efficacy of green tea mouthwashes with that of chlorhexidine17. Similarly, green tea's ability to downregulate pro-inflammatory cytokines has been demonstrated in keratinocyte cultures18.
Hyaluronic acid, another key molecule in tissue repair, has shown promising results in the management of oral inflammatory conditions and wound healing due to its significant role in connective tissue repair and anti-inflammatory effects19. Its application in mouthwashes and gels has been effective in reducing inflammation and preventing complications such as dry socket following dental extractions20,21,22,23,24.
Considering these findings, our randomized clinical trial aims to evaluate the effectiveness of a gel formulation combining green tea and hyaluronic acid in promoting mucosal healing after third molar extractions. This study seeks to determine whether such a gel can significantly enhance the healing process, thereby offering a beneficial adjuvant therapy for dental surgeries. We hypothesize that there will be a discernible difference in healing outcomes between sites treated with the green tea and hyaluronic acid gel and those treated with a placebo.
Material and methods
In vitro methodologies
Cell culture and compounds
Primary human gingival cells were purchased from ATCC (HGF, ATCC PCS-201-018). The cells were grown in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS, Vitrocel, Campinas, SP, Brazil) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin), in an environment maintained at 37 °C, 5% CO2, and 95% humidity. For experimental treatments, cells at a density of 1 × 105 cells/well in 6-well plates were exposed for 24 h to media supplemented with 10% concentrations of hyaluronic acid (Henrifarma, São Paulo, Brazil) and green tea extract (Camellia sinensis) (Chemspecs, São Paulo, Brazil), evaluating their synergistic effects on fibroblast activity without inducing cytotoxicity.
mRNA obtaining samples and gene expression
After 24 h of exposition, samples were harvested and mRNA extracted using Ambion TRIzol Reagent (Life Sciences—Fisher Scientific Inc, Waltham, MA, USA) and thereafter treated with DNase I (Invitrogen, Carlsbad, CA, USA). Quantification was performed using a plate reader (SYNERGY-HTX multimode reader, Biotek, USA). Then, cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kits from Applied Biosystems (2.0 μL 10 × RT Buffer, 0.8 μL 25 × dNTP Mix (100 mM), 2.0 μL 10 × RT Randon Primers, 1.0 μL MultiScribleTM Reverse Transcriptase, 4.2 μL Nuclease- Free H2O). The qPCR products were performed on the QuantStudio® 3 real-time PCR equipment (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a total of 10 μL, containing 2 × PowerUp™ SYBR™ Green Master Mix (5 μL) (Applied Biosystems, Foster City, CA), 0.4 μM of each primer, 50 ng of cDNA and nuclease-free H2O. Gene expression was analyzed using control cells by the ΔΔCT method, using β-ACT, GAPDH and 18S13 as housekeeping genes. The ΔΔCT method was used to calculate the values of expressions.
Clinical strategies
Study design
This study consisted of a double-blind, split-mouth, randomized clinical trial study that followed the CONSORT (Consolidated Standards of Reporting Trails) protocol. For statistical purposes, the patients were considered as individual cases and each one of them were submitted to the split-mouth model, in which the same was submitted to the test and control treatment. The sample consisted of 22 (twenty-two) participants from Inpes Odontologia (Uberlândia, Brazil) who underwent extraction procedures for the four third molars and a subsequent procedure to stimulate healing of the alveolar soft tissue after extraction by applying a gel containing green tea and hyaluronic acid over the mucosa of the surgical site. All the patients read and signed an informed consent agreeing to participate in this study. Furthermore, this study was approved by the Research Ethics Committee of the Federal University of Uberlândia with opinion number 48475621.0.0000.5152, and was conducted in accordance with resolution 466 of 12/12/2012 of the National Health Council of the Ministry of Health of Brazil and with the Declaration of Helsinki. The protocol of this study was registered in the REBEC (Brazilian Registry of Clinical Trials) under the number RBR-8z4ctxq (Approval date 12/06/2021—Protocol available at https://ensaiosclinicos.gov.br/rg/RBR-8z4ctxq).
Sample calculation
The sample calculation was performed based on the study by Heo et al. 200225, which carried out fractal analysis of bone in the regeneration phase after orthognathic surgery. It was found that a fixed mean difference of 0.10 points as being clinically relevant with an expected mean standard deviation of 0.05, a sample size of 20 patients would be sufficient to determine this level of difference with a β power of 0.90 and an α power of 0.05.
Inclusion and exclusion criteria
Inclusion criteria comprised individuals over 18 years of age, good oral hygiene (plaque index < 20%) and presence of four third molars with indication for tooth extraction. The tooth extraction was indicated if the 3rd molar was included into de bone and soft tissues, or if teeth are out of position with no occlusion with the antagonist teeth. Exclusion criteria included individuals with periodontal disease, use of drugs that alter bone metabolism, decompensated diabetics (glycated hemoglobin above 8%), heavy smokers (over 10 cigarettes a day), pregnant or lactating women.
Surgical procedure and study groups
All participants underwent an initial panoramic clinical and radiographic examination, in order to obtain the necessary data for surgical planning, in addition to subsequent classification of third molars according to Pell & Gregory26 and Winter27. The surgical procedure was performed under local anesthesia (neural block technique in the area of operation with Articaine 4% associated with epinephrine 1:100,000) and the four third molars were extracted at the same session, using sulcular flaps at the 3rd and 2nd molar associated with release incisions at the buccal-mesial surface of the 2nd molar, and instruments depending on the technical need. After removing the teeth, the flap was sutured with 4–0 nylon thread, and the gel corresponding to each surgical site was deposited on the surgical wound, in sufficient quantity to cover the surgical site (~ 3 ml per socket). The time of the surgical procedure was recorded from the time of application of the anesthetic until the completion of the suture.
The selection of treatment in each alveolar mucosa occurred prior to the procedure through an online randomization table (random.org) (GJO). The sides were distributed between the test and control groups according to the type of treatment that they would undergo after the extraction of the 4 third molars, in a split-mouth model. The surgical wound of the maxillary and mandibular molars on the test side was filled with hyaluronic acid gel and green tea, while the surgical wound of the maxillary and mandibular molars on the control side was filled with placebo gel, and the gel was held in position with a gauze (Fig. 1). The coding of the gel was known by only one researcher (GJO), so that the patient, the surgeons, and evaluators were blind to the treatment. The patient was instructed to continue using the gels for 7 days, and they should be used topically on the mucosa of the surgical socket 3 times a day, after oral hygiene (~ 3 ml per socket). At the 3-day period the patients were asked to use the gel in front of the examiners, and adjustments on the gel application were made if it is necessary. The patient was instructed not to consume liquids and solids after 30 min of using the gel. The gel container had only the identification of the side that should be used, so the patient was not aware of the test and control gel. The surgery were performed by two experience surgeons (DAP and MSB).
Example of the application of the gel. (a) Topic application of the gel on the upper region, (b) Topic application of the gel on the lower region. These maneuvers were performed by the patients. (c) Just after the surgery, a gauze was soaked with the gel, (d) the patients were instructed to bite it, (e) during the 3-day period of follow up the patients were reinstructed in the use of the gel, and (f) the gel was applied on the entire surgical site.
In all participants, the preoperative medication protocol with dexamethasone (8 mg) was performed in a single dose one hour before the surgical procedure. Postoperatively, diclofenac sodium (50 mg) was prescribed for 3 days every 8 h and dipyrone sodium (500 mg) for 3 days every 6 h, all for oral administration. The sutures were removed after 7 days. The patient and the evaluators did not know where the test and placebo gels were applied.
Gel composition and characteristics
The gel used in this study had the following composition (New Dental Care, Ribeirao Preto, Sao Paulo, Brazil): Carboxymethylcellulose 1.00%, Glycerin 5.00%, sorbitol 60.00%, sodium benzoate 0.30%, xylitol 0.50%, laurel glucoside 1.50%, sodium lauryl sulfate 0.50%, hyaluronic acid 0.05%, polyvinylpyrrolidone K 30 0.50%, dimethylsilanediol salicylate (DSBC) 0.50%, thixosil 43B 10.00%, thixosil 73 5.00%, tetrasodium pyrophosphate 0.5%, saccharin 0.05%, green tea extract (Camellia sinensis) 0.50%, oil of hydrogenated castor 2.00%, EDTA 0.05%, purified water 11.45%. The test gel was transparent and presented low-viscosity similar the control gel. The control gel had the same composition of the test gel except with no presence of the active compounds.
Clinical analysis
Participants were clinically analyzed at 3, 7, 14, 30 and 90 days after tooth extraction. For the analysis of tissue appearance, the examined area was gently dried with a triple syringe and well illuminated by a light from the dental equipment. Clinical parameters were evaluated through visual inspection in scores from 0 to 3, with bleeding (1—absent, 2—palpation-induced, 3—spontaneous), tissue color (1—100% pink gingiva, 2—< 50% of hyperemic and mobile gingiva; 3—> 50% of hyperemic and mobile gingiva), secretion aspect (1—absent, 2—absent with presence of pronounced plaque around the socket, 3—pronounced), and tissue consistency (1—close to normal, pink; 2—flaccid, red; 3—fragile, greenish/gray—3). All data were recorded at the four surgical sites of each patient.
To assess facial edema, two facial measurements were used, the vertical dimension, which consisted of a line from the lateral palpebral commissure to the gonion, and a horizontal dimension, a line from the lower border of the tragus to the labial commissure. These distances were measured on a malleable tape, transferred and quantified using a digital caliper. Mouth opening was measured through the interincisal distance. Regarding the presence of clinical complications, the participants were questioned and eventual complications were noted, and those that could be clinically observed were identified by visual inspection.
The presence of bone spicules, alveolitis and trismus were also noted, as well as the need for alveoli irrigation (pain associated with pronounced exudate secretion) throughout the postoperative period. All these parameters were evaluated by two blinded, trained and calibrated operators (SSS and PGJM).
As for the patient-centered analyses, each participant evaluated through a visual analogue scale (0 to 10, being: 0—none, 1 to 3—little, 4 to 6—fair, 7 to 9—average, 10—excessive) the parameters: pain, inflammation and swelling, hemorrhage and bleeding, difficulty in chewing, and limitation of mouth opening.
Radiographic analysis
Panoramic radiographs were used to assess bone tissue structure and density 3 months after tooth extraction. The fractal dimension is a mathematical technique that allows the quantification of complex structures and evaluates the level of irregularities of shapes and objects, its value being directly proportional to its complexity. For this analysis, a region of interest (ROI), in the area corresponding to the extracted tooth, was selected at each of the four sites in the image, thus corresponding to each of the third molars. The box counting method was used to perform the fractal analysis. First, an image of each ROI was taken from the original image. Then, these images underwent the following steps: duplication; applying a Gaussian filter at 35.00 to remove wide-scale variations such as overlaps; subtraction of the second image by the first; adding gray values to 128; image transformation into binary; erodization and dilation for noise removal; and skeletonization, step in which the trabecular bone delineation took place. The fractal dimension values were measured with the software's box-counting function. The image was overlaid with squares of 2, 3, 4, 6, 8, 12, 16 pixels in size. The number of squares that surrounded the trabeculae and the total square count were measured for each pixel of different size. The graph of the logarithmic scale of the values was drawn and the value of the fractal dimension was calculated by measuring the slope of the formed line aligned to the points plotted on the graph, providing a final D-value. To minimize the chances of error in this image treatment process to obtain the final D value, a macro was used that was programmed to execute all the steps, after the ROI cut was performed (PGJM).
Statistical analysis
The GraphPad Prism 8 software (San Diego, CA, USA) was used to perform the statistical analysis of this study. The results obtained in vitro were plotted as mean ± standard deviation and selected by one-way analysis of variance (ANOVA) with Tukey's post-test, to compare all pairs of groups, where p < 0.05 was considered statistically significant. Clinical outcomes were analyzed as follows: numerical data were evaluated in terms of their distribution using the Shapiro–Wilk normality test. The assessments of the VAS scale and of the healing analyzes were not distributed according to normality and due to this, the Wilcoxon non-parametric test was used to compare the different groups within each evaluation moment. Longitudinal data within each group were statistically compared using Friedman's test complemented by Dunn's test. The data from the analysis of facial measurements were analyzed by means of the parametric paired t-test for comparison between groups and by the Repeated Measurements ANOVA test complemented by Tukey's test for longitudinal evaluation within each group. All statistical tests were applied at a 5% significance level.
Ethics approval and consent to participate
This study was approved by the Research Ethics Committee of the Federal University of Uberlândia with opinion number 48475621.0.0000.5152,and was conducted in accordance with resolution 466 of 12/12/2012 of the National Health Council of the Ministry of Health of Brazil and with the Declaration of Helsinki. The protocol of this study was registered in the REBEC (Brazilian Registry of Clinical Trials) under the number U1111-1269–7482. All patients read and signed a consent form before the enrolled in this study.
Results
Synergistic enhancement of AKT gene expression by green tea and hyaluronic acid
Our investigation into the combined impact of green tea and hyaluronic acid on fibroblast gene expression revealed a significant modulation of cellular pathways crucial for survival and proliferation. Initial tests confirmed the absence of cytotoxic effects from both compounds, applied independently or in combination, at a 10% concentration (data not shown). Subsequent analyses focused on the expression of the AKT gene, a key indicator of cell survival28. The results demonstrated that both green tea and hyaluronic acid individually contributed to the up-regulation of the AKT gene. However, a remarkable synergistic effect was observed when these compounds were combined, leading to an approximately 100-fold increase in AKT expression compared to the control group (Fig. 2). This profound enhancement underscores the therapeutic potential of combining these natural compounds to activate cellular mechanisms that promote tissue healing and regeneration. The statistical significance of these findings is detailed in the accompanying analysis (Fig. 2).
AKT, an important cellular survival biomarker, was evaluated in response to the synergism between hyaluronic acid (10%) and green tea (10%) in human fibroblasts. Control cultures were considered when the cells were maintained under classical conditions, without any treatment. The data shows a significant higher expression of AKT in response to both compounds in synergism. It was considered differences when: *p = 0.0136, and ****p < 0.0001. The value of expression was normalized with 18S data (housekeeping gene).
Further investigation into the synergistic effects of green tea and hyaluronic acid revealed their influence on genes critical for cell cycle progression. Our findings indicate a significant upregulation of key genes associated with cell proliferation, including CDK2 (Fig. 3a), CDK4 (Fig. 3b), and CDK6 (Fig. 3c), when fibroblasts were treated with a combination of these compounds. This up-modulation points to an enhanced capacity for cell proliferation, underlining the therapeutic potential of this synergistic approach for tissue repair and regeneration.
Cell growth related genes were significantly affected by the synergism between hyaluronic acid (10%) and green tea (10%) in fibroblasts. Control cultures were considered when the cells were maintained under classical conditions, without any treatment. The value of expression was normalized with 18S data (housekeeping gene). CDK Cyclin Dependent Kinase. ****p < 0.0001.
Enhancement of angiogenesis through VEGF modulation
We have further explored the synergistic interaction between green tea and hyaluronic acid on angiogenesis, a critical process in tissue regeneration28. We specifically examined the expression of the Vascular Endothelial Growth Factor (VEGF), known for its pivotal role in angiogenesis29. The combined treatment significantly upregulated VEGF expression in fibroblasts, indicating a potent enhancement of the angiogenic potential (Fig. 4). This finding highlights the synergism's effectiveness in promoting angiogenesis, suggesting a promising avenue for improving tissue repair and healing processes through enhanced vascular formation.
VEGF, an important growth factor to drive angiogenesis, was higher in response to the synergism between hyaluronic acid (10%) and green tea (10%) in human fibroblasts. Control cultures were considered when the cells were maintained under classical conditions, without any treatment. The value of expression was normalized with 18S data (housekeeping gene). VEGF vascular endothelial growth factor. ****p < 0.0001.
Clinical outcomes
The patients were enrolled between Jan/2022 to dec/2022. At the end of the study, 20 patients completed the follow-up and two patients were excluded in this meantime—one of then lost the 3-day follow-up and discontinued the use of the gels, while another patient used antibiotics during the postoperative phase (Fig. 5).
Flow diagram of this study.
Green tea gel and hyaluronic acid reduced exudate secretion in post-extraction sockets
A gradual improvement in the clinical aspect of the lesion was verified in both groups with increasing follow-up time, however the test group had a lower alveoli secretion level at 7 days than the control group in lower molars (p < 0.05) (Tables 1 and 2).
Patients noted progressive improvement with increasing post-extraction follow-up time, but without differences between treatments.
Regarding the patient-centered analyses, there was a progressive improvement in all parameters tested with increasing post-surgery follow-up time, although there were statistically significant differences between the groups (Table 3).
Green tea gel and hyaluronic acid reduced edema up to the 14th postoperative day
A progressive improvement in edema and mouth opening was observed with the increase in the observation period after the extraction surgery. It was also verified that the test group had lower edema values measured by the horizontal dimension of the patients' face up to 14 days-period (p < 0.05) (Table 4).
Post-extraction sockets treated with green tea gel and hyaluronic acid showed higher bone density at 90 days
Radiographic analysis showed that there were no differences between groups and different experimental periods in relation to fractal dimension analysis. However, it was verified that in both groups there was an increase in bone density in the alveoli in the period of 90 days compared to the baseline period, and that the group where the gel based on green tea and hyaluronic acid was applied had higher bone density than the wells treated with the placebo gel within 90 days (p < 0.05) (Table 4).
Post-extraction socket irrigations were required only in sites treated with placebo gel
No differences were observed between groups regarding the complexity of extracted teeth (Tables 5, 6, 7). There was a need to perform osteotomies (19 in the test gel group and 18 in the control gel group) and sections (14 teeth in each group) in lower teeth with no differences between the groups in relation to these types of surgical maneuvers. The presence of trismus was observed in 5 patients after 3 days of the surgical procedure and in one patient after 7 days of the surgery. Irrigations were performed in one patient in a period of 3 days and in four patients in a period of 7 days in mandibular molar alveoli that was noticed fibrinolytic secretion associated with pain and the presence of biofilm. All these wells were previously treated with the control gel.
Discussion
Regenerative mechanism is related with original morphofunctional capacity of cells in renewing the loss/injured tissue. In dentistry this mechanism is widely observed during third molar extraction or implant-related procedures30,31,32. It is known that the wound healing in those cases is critical to prevent the invasion of microorganisms present in the oral microbiome into other tissues and further establishing a chronic infection33. Specifically, gingival wound healing comprises the growth of fibroblasts and consequent maturation and remodeling of extracellular matrix (ECM)31,34,35,36. In this way, to identify novel compounds to trigger this mechanism is very welcome for accelerating regenerative events and this study looked for understanding the synergism effect of green tea and hyaluronic acid on regeneration of soft tissue; it seems important to supplement fibroblasts by stimulating them to proliferate during the wound healing37. The workplan was based on the analysis of both compound in modulating specific gene expression of human fibroblast and later validate these findings in clinical strategies during third molar extraction related soft tissue healing.
Our experimental study conducted in vitro shows the effect of the compounds on fibroblast phenotype by promoting the overexpression of genes related with cell cycle progression, such as CDKs 2,4, and 638,39. In other words, the synergism of green tea and hyaluronic acid affect the growth of fibroblast and suggest a positive effect in regeneration of soft tissues, such as mucosa present on the oral environment. In fact, evaluating the effect on fibroblasts in vitro is an important starting-point strategy mainly looking for reducing the experimental animal’s number in pre-clinical stages40. Moreover, fibroblasts are the main connective tissue cells and responsible to release molecules related with ECM constitution such as collagen fibers, desired during the repair of injures sites41,42,43. Nowadays, based on the well-known function of these cells in remodeling tissue, and more credit has been deposited in fibroblast during pro-regenerative events, mainly by interfering on the regenerative related microenvironment by releasing bioactive molecules such as growth factor or mitogens44,45.
Complimentarily, this study has shown also that challenged fibroblast with green tea and hyaluronic acid significantly over-expresses VEGF (Vascular Endothelial Growth Factor; ~ 18 fold-changes). VEGF is one of the most significant factors during the entire angiogenesis46,47. From our point of view, challenged fibroblast creates a natural scaffold that is able to drive the regeneration steps, such as the angiogenesis. Angiogenesis is related with the success of regenerative mechanisms guaranteeing nutrients to metabolically active cells in response to increase in concentration of pro-angiogenic factors produced by the fibroblast. A summary of the main findings found in vitro is assembled in the Fig. 6.
Summary of the main findings found in this study. Note that the conjunction of signaling promoted by the synergism led to the activation of AKT as a hub to drive cellular events related with cell growth, survival, and stimulus for angiogenesis; Taken into account, this signaling pathway is related with connective tissue regeneration, and fibroblast is a pivotal cell in this scenario.
Importantly, this study brings a very interesting strategy to investigate whether green tea and hyaluronic acid are stimulators of soft tissue regeneration by associating 2 different approaches: a pre-clinical analysis by evaluating the biology of fibroblasts on this response, and the second looking for validating (or not) these findings in clinical outcomes. Now, considering the clinical outcomes, it is important to mention that the present study demonstrates that a gel-based on green tea and hyaluronic acid modifies the healing course in post-extraction sockets of third molars, reducing the secretion of inflammatory exudate in the lower sockets, less edema, lower frequency of complications and higher bone density. Furthermore, there were no report of side effects regarding the gel use, which showed the safety of the test gel in the treatment of oral surgical sites. Based on these data, the null hypothesis of this study was rejected.
The reduction in inflammatory exudate and in the extraoral edema associated with the use of the test gel can be explained by the previously described antibacterial and anti-inflammatory effects of green tea and hyaluronic acid. Previous studies have indicated that green tea is capable of inducing the secretion of human beta-defensin (hBD) by epithelial cells, protecting these cells from proteolytic degradation and increasing the barrier function of a gingival keratinocyte against the invasion of periodontopathogens48,49,50. Furthermore, green tea was previously shown to reduce the extent and intensity of the inflammatory response, as well as the pro-inflammatory cytokine pattern when used as adjunctive therapy to scaling and root planning12. The catechins contained in green tea are responsible for decreasing pro-inflammatory cytokines such as Il-1β and TNFα, in addition to inhibiting peptidase and collagenase activity8. Important, this organized microenvirnment promoved by the green tea and hyaluronic acid synergism led to driving the fibroblast performance.
On the other hand, the hyaluronic acid included in the composition of the gel may also have had an influence due to its range of beneficial biological effects. An in vitro anti-plaque effect of hyaluronate (0.025%) similar to that of chlorhexidine was demonstrated in inhibiting bacterial growth51. These results are also in agreement with a systematic review that demonstrated benefits in the use of hyaluronic acid in the treatment of periodontal disease, especially due to reductions in plaque index and bleeding on probing52, and these effects may be associated with reduced expression of pro-inflammatory cytokines as demonstrated in an in vitro study in fibroblast cultures stimulated by P.gingivalis in which hyaluronic acid suppressed the MAPK and NF-κB signaling pathways53.
The alveoli that presented pronounced secretion associated with pain were treated by irrigation with saline solution to wash the post-extract sockets and remove food debris, and inflammatory exudate. In fact, the need for this type of management was verified in 5 lower post-extraction wells, all of which had been treated with the control gel. The higher bone density in the group of alveoli treated with the test gel may have occurred due to the absence of mechanical manipulation of the lower alveoli with irrigation. However, hyaluronic acid has also been reported to increase bone formation in areas grafted with osteoconductive bone substitutes in humans54, as well as in post-extraction sockets of teeth with periodontal and endodontic damage in dogs55. In addition, the catechins present in green tea have also shown an osteoprotective effect by reducing osteoclastogenesis and stimulating differentiation of undifferentiated mesenchymal cells into osteoblasts56, and these effects on bone tissue have been indicated to be the reason for the success in the application of green tea in the treatment of periodontitis16, and in the prevention of osteoporosis57. However, the effect of the association of these two compounds on bone metabolism still needs to be better described.
Other local therapies have been applied to treat lesions in the oral cavity. It has been shown that hyaluronic acid gel enriched with amino acids58, chlorhexidine-based gels59,60, and the use of blood concentrates2,61 positively alter healing in sockets after extraction of third molars2,58,59, periodontal bone defects61, and soft tissue injuries58,60. Thus, the possibility of therapeutic choices to accelerate healing of wounds in the oral cavity is great and the comparison of different therapies is necessary in order to indicate the best therapy for each clinical situation.
The patient centered outcomes of pain, inflammation, bleeding, masticatory function and mouth opening, despite being better on the side that received the test gel, were not statistically different when compared to the control side. The analysis by the patient can often be subjective, making it difficult for the patient to compare certain parameters. This factor could partially contribute to the results found. Some proposed factors could explain these results. Furthermore, analgesic and anti-inflammatory medications prescribed pre and postoperatively directly influence the initial healing phase of the surgical wound, reducing or eliminating pain and mediating inflammation through the reduction of prostaglandins1. Thus, since the model of this study was split-mouth, the drug effects would act both on the test side and on the control side, not causing important differences between the sides in the patient's perception. The aforementioned aspects can be considered limitations of this study.
Another important limitation of this study is that the patients self-applied the gel, and this aspect was not controlled. It is worth mentioning that the ability of these patients to apply the gel in a region of difficult access in the oral cavity may have influenced the therapeutic potential of the gel. Furthermore, information regarding gel retention in the oral cavity is still needed in the future. However, the patients were blinded about the type of gel used, and any difficulties encountered in applying the gel must have influenced the results of this study with a similar impact at the control and test sides. Regarding the analysis performed in this study, the radiographic 3D analysis is more accurate method to evaluate the bone formation than the 2D analysis. Anyway, despite the limitations, the bone density and fractal dimension analysis are methods used before to analyze bone healing in panoramic radiographies. In addition, it is important to state that the split-mouth design reduce the effects of the methods errors on the outcomes. Finally, the exclusion criteria in this study impaired the participation of individuals with characteristics with delays the healing of the soft and hard tissues healing. Even soft smokers were not included since no patient with these characteristics were enrolled, despite the intention to include them if they where sorted. Anyway, the effects of the hyaluronic acid and green tea gel on the healing in patients with risk factors (eg. Smokers, diabetics) must be tested in future.
Conclusion
In conclusion, our study provides compelling evidence that the combination of green tea and hyaluronic acid gel significantly enhances both soft tissue and bone healing post-third molar extraction. The synergistic effect of these compounds led to notable improvements in angiogenesis, cell proliferation, and reduced inflammatory response, as evidenced by gene expression analyses and clinical outcomes. These findings suggest that the green tea and hyaluronic acid gel represents a promising adjunctive therapy for improving postoperative recovery in dental surgery. Future research should focus on further elucidating the molecular mechanisms underlying these beneficial effects and exploring the potential for broader applications in tissue regeneration and wound healing.
Data availability
The datasets generated and/or analysed during the current study are available in the Universidade Federal de Uberlândia repository https://repositorio.ufu.br/handle/123456789/35802, however if not accessible data is available from corresponding author on request.
References
Sreesha, S. et al. Postoperative pain, edema and trismus following third molar surgery—A comparative study between submucosal and intravenous dexamethasone. J. Family Med. Prim. Care 9(5), 2454–2459. https://doi.org/10.4103/jfmpc.jfmpc_188_20 (2020).
Daugela, P., Grimuta, V., Sakavicius, D., Jonaitis, J. & Juodzbalys, G. Influence of leukocyte- and platelet-rich fibrin (L-PRF) on the outcomes of impacted mandibular third molar removal surgery: A split-mouth randomized clinical trial. Quintessence Int. 49(5), 377–388 (2018).
Meng, L., Wei, Y., Liang, Y., Hu, Q. & Xie, H. Stem cell homing in periodontal tissue regeneration. Front. Bioeng. Biotechnol. 10, 1017613. https://doi.org/10.3389/fbioe.2022.1017613 (2022).
Wang, X., Chen, J. & Tian, W. Strategies of cell and cell-free therapies for periodontal regeneration: The state of the art. Stem Cell Res Ther. 13(1), 536. https://doi.org/10.1186/s13287-022-03225-z (2022).
Thalakiriyawa, D. S. & Dissanayaka, W. L. Advances in regenerative dentistry approaches: An update. Int. Dent. J. 74(1), 25–34. https://doi.org/10.1016/j.identj.2023.07.008 (2024).
Kahraman, S. A., Cetiner, S. & Strauss, R. A. The effects of transcutaneous and intraoral low-level laser therapy after extraction of lower third molars: A randomized single blind, placebo controlled dual-center study. Photomed. Laser Surg. 35(8), 401–407 (2017).
Szwed-Georgiou, A. et al. Bioactive materials for bone regeneration: Biomolecules and delivery systems. ACS Biomater. Sci. Eng. 9(9), 5222–5254. https://doi.org/10.1021/acsbiomaterials.3c00609 (2023).
Jenabian, N., Moghadamnia, A. A., Karami, E. & Mir, A. P. B. The effect of Camellia Sinensis (green tea) mouthwash on plate-induced gingivitis: A single-blinded randomized controlled clinical trial. Daru 20(1), 39. https://doi.org/10.1186/2008-2231-20-39 (2012).
Ledder, R. G., Latimer, J., Humphreys, G. J., Sreenivasan, P. K. & McBain, A. J. Bacteriological effects of dentifrices with and without active ingredients of natural origin. Appl. Environ. Microbiol. 80(20), 6490–6498. https://doi.org/10.1128/AEM.02315-14 (2014).
Abdulbaqi, H. R., Himratul-Aznita, W. H. & Baharuddin, N. A. Evaluation of Salvadora persica L. and green tea anti-plaque effect: A randomized controlled crossover clinical trial. BMC Complement Alternate Med. 16(1), 493. https://doi.org/10.1186/s12906-016-1487-0 (2016).
Piekarz, T. et al. The influence of toothpaste containing australian melaleuca alternifolia oil and ethanolic extract of polish propolis on oral hygiene and microbiome in patients requiring conservative procedures. Molecules 22(11), 1957. https://doi.org/10.3390/molecules22111957 (2017).
Almeida, J. M. et al. Influence of adjuvant therapy with green tea extract in the treatment of experimental periodontitis. Arch. Oral Biol. 102, 65–73 (2019).
Yoshinaga, Y. et al. Green tea extract inhibits the onset of periodontal destruction in rat experimental periodontitis. J. Periodont. Res. 49, 652–659 (2014).
Figueiredo, L. C. et al. Propolis, aloe vera, green tea, cranberry, calendula, myrrha and salvia properties against periodontal microorganisms. Microorganisms 10(11), 2172. https://doi.org/10.3390/microorganisms10112172 (2022).
López-Valverde, N. et al. Antioxidant, anti-inflammatory and antimicrobial activity of natural products in periodontal disease: A comprehensive review. Front. Bioeng. Biotechnol. 11, 1226907. https://doi.org/10.3389/fbioe.2023.1226907 (2023).
Hrishi, T. S. et al. Effect of adjunctive use of green tea dentifrice in periodontitis patients—A randomized controlled pilot study. Int. J. Dent. Hyg. 14(3), 178–183. https://doi.org/10.1111/idh.12131 (2016).
Mathur, A. et al. Efficacy of green tea-based mouthwashes on dental plaque and gingival inflammation: A systematic review and meta-analysis. Indian J. Dent. Res. 29(2), 225–232. https://doi.org/10.4103/ijdr.IJDR_493_17 (2018).
Hagiu, A., Attin, T., Schmidlin, P. R. & Ramenzoni, L. L. Dose-dependent green tea effect on decrease of inflammation in human oral gingival epithelial keratinocytes: In vitro study. Clin. Oral Investig. 24(7), 2375–2383. https://doi.org/10.1007/s00784-019-03096-4 (2020).
Miglani, A., Vishnani, R., Reche, A., Buldeo, J. & Wadher, B. Hyaluronic acid: Exploring its versatile applications in dentistry. Cureus. 15(10), e46349. https://doi.org/10.7759/cureus.46349 (2023).
Ni, J., Shu, R. & Li, C. Efficacy evaluation of hyaluronic acid gel for the restoration of gingival interdental papilla defects. J. Oral Maxillofac. Surg. 77(12), 2467–2474. https://doi.org/10.1016/j.joms.2019.06.190 (2019).
Sahayata, V. N., Bhavsar, N. V. & Brahmbhatt, N. A. An evaluation of 0.2% hyaluronic acid gel (Gengigel®) in the treatment of gingivitis: A clinical & microbiological study. Oral Health Dent. Manag. 13(3), 779–785 (2014).
Casale, M. et al. Hyaluronic acid: Perspectives in dentistry. The systematic review. Int. J. Immunopathol. Pharmacol. 29(4), 572–582. https://doi.org/10.1177/0394632016652906 (2016).
Abdulkareem, A. A., Al Marah, Z. A., Abdulbaqi, H. R., Alshaeli, A. J. & Milward, M. R. A randomized double-blind clinical trial to evaluate the efficacy of chlorhexidine, antioxidant, and hyaluronic acid mouthwashes in the management of biofilm-induced gingivitis. Int. J. Dent. Hyg. 18(3), 268–277. https://doi.org/10.1111/idh.12432 (2020).
Almushalbn, A., Albassal, A. & Harfouch, M. Comparative clinical study between chlorhexidine Gel (0.2%) and hyaluronic Gel (1%) in the prevention of a dry socket after tooth extraction for orthodontic treatment. Cureus 14(12), e32391. https://doi.org/10.7759/cureus.32391 (2022).
Heo, M. S. et al. Fractal analysis of mandibular bony healing after orthognathic surgery. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 94(6), 763–767. https://doi.org/10.1067/moe.2002.128972 (2002).
Pell, G. J. & Gregory, G. T. Impacted mandibular third molars: classifications and modified technique for removal. Dental Digest. 9, 330–338 (1933).
Winter, G. B. Principles of Exodontia as Applied to the Impacted Third Molar: A Complete Treatise on the Operative Technic with Clinical Diagnoses and Radiographic Interpretations St Louis (American Medical Books, 1926).
Manning, B. D. & Cantley, L. C. AKT/PKB signaling: Navigating downstream. Cell 129(7), 1261–1274. https://doi.org/10.1016/j.cell.2007.06.009 (2007).
Everts, P. A. et al. Angiogenesis and tissue repair depend on platelet dosing and bioformulation strategies following orthobiological platelet-rich plasma procedures: A narrative review. Biomedicines 11(7), 1922. https://doi.org/10.3390/biomedicines11071922 (2023).
Liu, Z. L., Chen, H. H., Zheng, L. L., Sun, L. P. & Shi, L. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct. Target. Ther. 8(1), 198. https://doi.org/10.1038/s41392-023-01460-1 (2023).
Smith, P. C., Cáceres, M., Martínez, C., Oyarzún, A. & Martínez, J. Gingival wound healing: An essential response disturbed by aging?. J. Dent. Res. 94(3), 395–402. https://doi.org/10.1177/0022034514563750 (2015).
Jeyaraj, P. E. & Chakranarayan, A. Soft tissue healing and bony regeneration of impacted mandibular third molar extraction sockets, following postoperative incorporation of platelet-rich fibrin. Ann. Maxillofac. Surg. 8(1), 10–18. https://doi.org/10.4103/ams.ams_185_17.PMID:29963419;PMCID:PMC6018276 (2018).
Toma, A. I., Fuller, J. M., Willett, N. J. & Goudy, S. L. Oral wound healing models and emerging regenerative therapies. Transl. Res. 236, 17–34. https://doi.org/10.1016/j.trsl.2021.06.003 (2021).
Cho, Y. D., Kim, K. H., Lee, Y. M., Ku, Y. & Seol, Y. J. Periodontal wound healing and tissue regeneration: A narrative review. Pharmaceuticals (Basel) 14(5), 456. https://doi.org/10.3390/ph14050456 (2021).
Moretti, L., Stalfort, J., Barker, T. H. & Abebayehu, D. The interplay of fibroblasts, the extracellular matrix, and inflammation in scar formation. J. Biol. Chem. 298(2), 101530. https://doi.org/10.1016/j.jbc.2021.101530 (2022).
Diller, R. B. & Tabor, A. J. The role of the extracellular matrix (ECM) in wound healing: A review. Biomimetics (Basel). 7(3), 87. https://doi.org/10.3390/biomimetics7030087 (2022).
Gomes, R. N., Manuel, F. & Nascimento, D. S. The bright side of fibroblasts: Molecular signature and regenerative cues in major organs. NPJ Regen. Med. 6(1), 43. https://doi.org/10.1038/s41536-021-00153-z (2021).
Diril, M. K. et al. Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration. Proc. Natl. Acad. Sci. U. S. A. 109(10), 3826–3831. https://doi.org/10.1073/pnas.1115201109 (2012).
Jirawatnotai, S., Dalton, S. & Wattanapanitch, M. Role of cyclins and cyclin-dependent kinases in pluripotent stem cells and their potential as a therapeutic target. Semin. Cell Dev Biol. 107, 63–71. https://doi.org/10.1016/j.semcdb.2020.05.001 (2020).
Martinez-Lopez, S. et al. The 3Rs in experimental liver disease. Animals (Basel) 13(14), 2357. https://doi.org/10.3390/ani13142357 (2023).
da Costa Fernandes, C. J., do Nascimento, A. S., da Silva, R. A. & Zambuzzi, W. F. Fibroblast contributes for osteoblastic phenotype in a MAPK-ERK and sonic hedgehog signaling-independent manner. Mol. Cell Biochem. 436(1–2), 111–117. https://doi.org/10.1007/s11010-017-3083-0 (2017).
Costa Fernandes, C. J. D. & Zambuzzi, W. F. Fibroblast-secreted trophic factors contribute with ECM remodeling stimulus and upmodulate osteocyte gene markers in osteoblasts. Biochimie 168, 92–99. https://doi.org/10.1016/j.biochi.2019.10.013 (2020).
Fadl, A. & Leask, A. Hiding in plain sight: Human gingival fibroblasts as an essential, yet overlooked, tool in regenerative medicine. Cells 12(16), 2021. https://doi.org/10.3390/cells12162021 (2023).
Castro-Silva, I. I., Zambuzzi, W. F., de Oliveira, C. L. & Granjeiro, J. M. Periosteal-derived cells for bone bioengineering: A promising candidate. Clin. Oral Implants Res. 23(10), 1238–1242. https://doi.org/10.1111/j.1600-0501.2011.02287.x (2012).
Yanagihara, M. et al. Fibroblasts are the most suitable cell source for regenerative medicine due to their high intracellular fibroblast growth factor 2 content. Biochem. Biophys. Rep. 35, 101510. https://doi.org/10.1016/j.bbrep.2023.101510 (2023).
Tatullo, M. et al. Promising scaffold-free approaches in translational dentistry. Int. J. Environ. Res. Public Health 17, 3001. https://doi.org/10.3390/ijerph17093001 (2020).
Bokhari, S. M. Z. & Hamar, P. Vascular endothelial growth factor-D (VEGF-D): An angiogenesis bypass in malignant tumors. Int. J. Mol. Sci. 24(17), 13317. https://doi.org/10.3390/ijms241713317 (2023).
Lombardo Bedran, T. B., Feghali, K., Zhao, L., Palomari Spolidorio, D. M. & Grenier, D. Green tea extract and its major constituent, epigallocatechin-3-gallate, induce epithelial secretion of beta-defensin and prevent beta-defensin degradation by Porphyromonas gingivalis. J. Period. Res. 49(5), 615–623 (2014).
Ben Lagha, A., Haas, B. & Grenier, D. Tea polyphenols inhibit the growth and virulence properties of Fusobacterium nucleatum. Sci. Rep. 7, 44815. https://doi.org/10.1038/srep44815 (2017).
Lagha, A. B., Groeger, S., Meyle, J. & Grenier, D. Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis. Pathog. Dis. https://doi.org/10.1093/femspd/fty030 (2018).
Rodrigues, S. V., Acharya, A. B., Bhadbhade, S. & Thakur, S. L. Hyaluronan-containing mouthwash as an adjunctive plaque-control agent. Oral Health Prev. Dent. 8(4), 389–394 (2010).
Eliezer, M., Imber, J. C., Sculean, A., Pandis, N. & Teich, S. Hyaluronic acid as adjunctive to non-surgical and surgical periodontal therapy: A systematic review and meta-analysis. Clin. Oral Investig. 23(9), 3423–3435. https://doi.org/10.1007/s00784-019-03012-w (2019).
Chen, M. et al. High molecular weight hyaluronic acid regulations P. gingivalis-induced inflammation and migration in human gingival fibroblasts via MAPK and NF-κB signaling pathway. Arch. Oral Biol. 98, 75–80. https://doi.org/10.1016/j.archoralbio.2018.10.027 (2019).
Lee, J. B. et al. Effects of hyaluronic acid and deproteinized bovine bone mineral with 10% collagen for ridge preservation in compromised extraction sockets. J. Periodontol. 92(11), 1564–1575. https://doi.org/10.1002/JPER.20- (2021).
Kim, J. J., Song, H. Y., Ben Amara, H., Kyung-Rim, K. & Koo, K. T. Hyaluronic acid improves bone formation in extraction sockets with chronic pathology: A pilot study in dogs. J. Periodontol. 87(7), 790–795. https://doi.org/10.1902/jop.2016.150707 (2016).
Huang, H. T. et al. Osteoprotective roles of green tea catechins. Antioxidants (Basel) 9(11), 1136. https://doi.org/10.3390/antiox9111136 (2020).
Li, X. et al. Tea consumption and bone health in Chinese adults: A population-based study. Osteoporos Int. 30(2), 333–341. https://doi.org/10.1007/s00198-018-4767-3 (2019).
Canciani, E. et al. Effects of vitamin and amino acid-enriched hyaluronic acid gel on the healing of oral mucosa: In vivo and in vitro study. Medicina (Kaunas) 57(3), 285. https://doi.org/10.3390/medicina57030285 (2021).
Teshome, A. The efficacy of chlorhexidine gel in the prevention of alveolar osteitis after mandibular third molar extraction: A systematic review and meta-analysis. BMC Oral Health 17(1), 82. https://doi.org/10.1186/s12903-017-0376-3 (2017).
Asbi, T. et al. A single application of chlorhexidine gel reduces gingival inflammation and interleukin 1-β following one-stage implant placement: A randomized controlled study. Clin. Implant Dent. Relat. Res. 23(5), 726–734. https://doi.org/10.1111/cid.13041 (2021).
Rexhepi, I. et al. Efficacy of inorganic bovine bone combined with leukocyte and platelet-rich fibrin or collagen membranes for treating unfavorable periodontal infrabony defects: Randomized non-inferiority trial. J. Periodontol. 92(11), 1576–1587. https://doi.org/10.1002/JPER.20-0305 (2021).
Funding
The authors like to Thanks the New Dental Care for the donation of the gel used in this study. This study was supported by grants from CNPq, National Council for Scientific and Technological Development (Grants INCT Saúde Oral e Odontologia 406840/2022-9 and Grants CNPq—Financial code 001; 314166/2021-1).
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Conceptualization, GJO, WZ, FJB, RSP; methodology, MSB, DAP, SSS, PJM, GSF.; formal analysis, WZ, GJO; investigation, MSB, DAP, SSS, PJM, GSF; data curation, WZ, GJO.; writing—original draft preparation, DAP, MSB, GSF, WZ, GJO.; writing—review and editing, GJO, WZ; visualization, all the authors; supervision, WZ, GJO.; project administration, WZ, GJO.; funding acquisition, GJO, WZ, FJB, RSP. All authors have read and agreed to the published version of the manuscript.
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FJBB has financial conflicts because he is a partner of the New Dental Care company. All the other authors have no conflict of interesting to declare.
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Bonatto, M.d.S., Feltran, G.d.S., Barbosa, T.P. et al. Green tea and hyaluronic acid gel enhance fibroblast activation and improves the gingival healing post-third molar extraction. Sci Rep 14, 7124 (2024). https://doi.org/10.1038/s41598-024-57821-5
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DOI: https://doi.org/10.1038/s41598-024-57821-5
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