1 Volume 23 2024 e244720 Original Research Braz J Oral Sci. 2024;23:e244720http://dx.doi.org/10.20396/bjos.v23i00.8674720 1 Periodontics Division, Department of Prosthodontics and Periodontics, Faculdade de Odontologia de Piracicaba (FOP), Universidade Estadual de Campinas (UNICAMP), Piracicaba, São Paulo, Brazil. 2 Microbiology and Immunology Division, Department of Biosciences and Oral Biopathology, São José dos Campos School of Dentistry, São Paulo State University, São José dos Campos, Brazil. 3 Periodontics Division, Department of Diagnosis and Surgery, São José dos Campos School of Dentistry, São Paulo State University, São José dos Campos, Brazil. 4 Department of Oral Health Practice and Center of Oral Health Research, University of Kentucky College of Dentistry, Lexington, Kentucky, USA Corresponding author: Renato Corrêa Viana Casarin Division of Periodontology, Department of Prosthodontic and Periodontics Piracicaba Dental School, University of Campinas - UNICAMP Avenida Limeira, 901 – Areião, Piracicaba, SP, Brazil. ZC 13414-903 Phone: (19) 2106-5301 email: rcasarin@unicamp.br Editor: Dr. Altair A. Del Bel Cury Received: October 3, 2023 Accepted: January 29, 2024 Impact of Aggregatibacter actinomycetemcomitans on epithelial repair: in vitro study in wound model Bianca Carvalho Mendes¹, Camila Schmidt Stolf1 , Hélvis Enri de Sousa Paz1 , Letícia Sandoli Arroteia1 , Lucas de Paula Ramos2 , Mauro Pedrine Santamaria3,4 , Karina Gonzales Silvério Ruiz1 , Mabelle de Freitas Monteiro1 , Renato Corrêa Viana Casarin1* ABSTRACT: Normal wound healing occurs in four overlapping stages - hemostasis, inflammation, proliferation, and remodeling. In the oral cavity, these processes occur in an infectious environment. Among the pathogens found in the oral community, Aggregatibacter actinomycetemcomitans (Aa) constitutes a well-recognized periodontal pathogen responsible for expressing several virulence factors, which activate a host response. Aim: This study investigated whether Aa’s presence can interfere with oral keratinocyte tissue healing in an in vitro wound healing model. Methods: Two groups were defined: Group KO (n=5) and Group KO+Aa (n=5). The Aa (JP2 strains) were cultivated in anaerobiosis, and the total protein extract was obtained. The keratinocytes were cultivated with the medium of standard culture until their confluence. After confluence, plates were allocated to each group. With the pipette’s tip, a “scratch” was made in the middle of each well of the plate, and the cells were cultured at 37°C in a humidified atmosphere with 5% CO2. The cells received the stimulus according to groups, and, at times 0, 5, 10, 24, and 48 hours, the wound areas were visualized and standardly recorded using an inverted microscope. Results: When analyzing the timeframe, differences in wound measurements indicate a faster closure in the control group compared to the KO+Aa group, although not statistically significant. However, upon examining the wound closure measures, it was observed that the Aa protein extract significantly reduced wound closure at 10 and 48 hours (p<0.05), negatively impacting the keratinocyte’s behavior. Conclusion: In summary, it was demonstrated that the pathogen Aa can interfere with the re-epithelization in vitro. Keywords: Wound healing. Microbiota. Keratinocytes. HaCaT cells. http://dx.doi.org/10.20396/bjos.v23i00.8674720 mailto:rcasarin@unicamp.br https://orcid.org/0000-0002-5125-2326 https://orcid.org/0000-0002-0619-2547 https://orcid.org/0000-0003-2675-9675 https://orcid.org/0000-0002-2682-2796 https://orcid.org/0000-0001-9468-0729 https://orcid.org/0000-0001-5879-9095 https://orcid.org/0000-0001-9333-4349 https://orcid.org/0000-0003-1743-5855 2 Mendes et al. Braz J Oral Sci. 2024;23:e244720 Introduction Normal wound healing involves four overlapping stages: hemostasis, inflammation, proliferation, and remodeling1,2,3. Hemostasis initiates healing by forming a blood clot that initially seals the wound. Afterward, platelets and inflammatory cells arrive at the site primarily. They provide essential functions and signals for the influx of import- ant cells, such as fibroblasts and endothelial cells, to the injury site1. For healing to occur satisfactorily, restoring the intact epidermal barrier through re-epithelialization is necessary. For that, it is important to have the directed migration of keratinocytes and their proliferation and survival, as they are critical for the re-epithelialization of the wound4. Several diseases in the oral cavity and their treatments led to wounds that should heal under an infective condition. The oral community harbors more than 500 cultured and not-yet-cultivated species5. Some presented cytotoxic factors that could affect healing processes.  Among these species, some of them are well-known pathogens, presenting diverse vir- ulence factors, as Aggregatibacter actinomycetemcomitan (Aa), Fusobacterium nuclea- tum (Fn), Tannerella forsythia (Tf), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Treponema denticola (Td)6. Aa is a well-recognized Gram-negative anaerobic periodontal pathogen, responsible for expressing class II major histocompatibility complex molecules, the cell adhesion molecules, intercellular adhesion molecule-1, and cytokines and chemokines during inflammation4, which activates a host response that may be associated with the pathogenesis of periodontitis7. However, once Aa can trigger a host-inflammatory response, its presence could affect healing processes. Nevertheless, little is known about the impact of pathogens on oral wound healing. In a study investigating palate wounds for connective tissue harvesting, temporal changes in the wound area during healing were demonstrated, with alterations depen- dent on the surgical technique employed. Following the rupture of the mucosal barrier, oral bacteria were found to potentially impact the healing process, as observed in sig- nificant effects on wound re-epithelialization caused by Pg and Fn, identified as major contributors to the inhibition of healing8, being the impact of Aa still not explored. How- ever, a proliferative behavior of oral keratinocytes when stimulated by Aa-hsp60 (Heat shock protein 60) has been reported9. On the other hand, cytolethal distending toxin (CDT), a virulence factor of Aa, exhibited the potential for cell cycle arrest, cellular dis- tension, and death10. Aa leukotoxin may also play a major role in destroying the peri- odontal tissues11. However, its impact on the wound-healing model remains unknown. Under healthy conditions, oral polymicrobial communities still contain periodon- tal pathogens, although in lower proportions. However, when oral dysbiosis occurs, successive alterations in bacterial proportions and changes in the expression pro- file are observed, rendering them more pathogenic5,6. Recognizing that epithelial cells undergo alterations when challenged by periodontal pathogens, this virulent profile may potentially interfere with and delay the healing process8, especially concerning Aa, one of the bacteria most strongly associated with severe periodontal disease. Therefore, this study investigated how oral keratinocytes can modify their healing capacity when exposed to a protein extract of Aa serotype JP2 in an in vitro wound healing model. 3 Mendes et al. Braz J Oral Sci. 2024;23:e244720 Materials and methods: Study design To fulfill the objectives, a laboratory study was designed involving the culture of lin- eage cells. Cells were cultured, stimulated, and analyzed as described below. Groups The following groups were defined: • KO group (n=5): Oral keratinocyte cells derived from lineage submitted to the scratching test and cultivated in Standard medium. • KO+Aa  group (n=5): Oral keratinocyte cells derived from lineage submitted to the scratching test and cultivated in Standard medium addition by total Aa protein extract. Aa total protein extract obtention Aa total protein extract was obtained as described previously12. Briefly, Aa strains (JP2) were cultivated on the surface of TSBYE agar (trypticase soy agar) (Oxoid Ltd, Bas- ingstoke, Hampshire, England) and incubated in an atmosphere of 10% CO2 (Shel Lab, Oregon, USA), at 37°C between 2 to 3 days. Grown cells as planktonic cultures were centrifuged (15min, 2°C, ≈6300g), and the culture medium was discarded. For collec- tion and washing, 1 ml of ice-cold saline solution (NaCl 0.9%) was added to the pel- let, which was resuspended. The cells were transferred to 2ml microtubes with screw caps and an o-ring (Axygen Inc., Corning Life Sciences, Union City, CA, USA). The tubes were centrifuged (4 min, 2°C, ≈13000g), and the saline solution was discarded. The cell pellet was immediately stored at -80°C until protein extraction. 700μl of ultra-pure water and ≈0.16g of 0.1mm diameter zirconia beads (BioSpec Products, Inc., Bartles- ville, OK, USA) were added for total protein extraction. The microtubes were placed in a Mini-BeadBeater device (Biospec) at maximum power (3 cycles of 60 seconds with 1 min of rest on ice). Samples were centrifuged twice (8min, 4°C, ≈13000g). The bead-free supernatant was transferred to another microtube, vortexed (10s), and had the protein concentration measured using the Bradford reagent (Sigma). The homog- enized extract was divided into single-use aliquots and stored at -80°C. Collections were performed on at least 3 independent cultures. The quality and integrity of the extractions were evaluated by separating 8μg of total proteins on 8.5% SDS-PAGE gels, with the aid of the Mini-Protean III system (Bio-Rad, Hercules, CA, USA), in buffer run (TBS -100mM Tris-HCl buffer, 200mM Tris, 1.37M NaCl), at 26-36mA, for 2h. Quality gels were made for all protein extracts and evaluated by Coomassie Blue. The pres- ence of intact bands of different sizes indicated the integrity of the samples. Cell Viability Cell viability was performed by the MTT method (3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyl tetrazolium bromide). Cells were plated in 96-well plates at a 5.0x103 cells/ well concentration in standard medium. After 24  hours, the medium was replaced with medium containing 0,15, 0,30, 0,45, 0,60, 0,75, and 0,90 μg/ml of total Aa protein extract. On days 3 and 7, MTT reagent was added to each well and incubated for 4 Mendes et al. Braz J Oral Sci. 2024;23:e244720 4 hours at 37°C in a humidified 5% CO2 incubator. At the end of the incubation period, the medium was removed, and the converted dye was solubilized with 100% ethanol. Absorbance was measured at a wavelength of 570  nm. It was decided to use the 0.45 μg/ml concentration because it was the lowest concentration that showed via- bility greater than 80% until day 7. The results are shown in Figure 1. Cell culture and Scratching test This study used lineage human cells (HaCAT oral keratinocytes) from Periocells biobank - Periodontics Area, FOP-UNICAMP. The keratinocytes were first grown in a standard culture medium at 37°C in a humidified atmosphere with 5% CO2 until confluence. Then, the cells were sown in a concentration of 1x105 cells/well on 24 well plates with standard culture medium for 24 hours. After this period, the culture medium was replaced by medium containing or not the total Aa extract in a concen- tration of 0.45 μg/ml, and with the pipette’s tip, a “scratch” was made in the middle of each well of the plate. Then, the cells were kept at 37°C in a humidified atmosphere with 5% CO2 for up to 48 hours. Only cells between the fourth and sixth passage were used, and the experiment was replicated six times. Wound closure evaluation At time 0 (immediately after the “scratch”), wound areas were visualized and recorded using an inverted microscope. The images were analyzed, and each image had its area evaluated by the ImageJ software by a calibrated single examiner (BCM, Intra-class Correlation Coefficient (ICC) > 85%). For calibration, the BCM examiner had to perform two measurements of the same scratch with an interval of 24 hours between them and obtain an ICC greater than 85%. Only wounds of similar sizes (between 900 and 120% 100% 80% 60% 40% 20% 0% DAY 01 NC 0.15 µg/ml 0.3 µg/ml 0.45 µg/ml 0.6 µg/ml 0.75 µg/ml 0.9 µg/ml DAY 03 DAY 07 Figure 1. Effect of AaPE on cell viability. HGFs were challenged with 0 (control) to 0,9 μg/ml of AaPE (Aggregatibacter actinomycetencomitans protein extract), and the MTT assay for assessing cell metabolism and viability was performed on days 1, 3, and 7. The control represents 100% of viability. 5 Mendes et al. Braz J Oral Sci. 2024;23:e244720 1000 μm2/field) were included in the study. The heterogeneous ones were excluded. The scratch measurement was done at 0, 5, 10, 24, and 48 hours in the same plate position and region to standardize the wound healing analysis. Times were also stan- dardized according to a previous protocol13. In the methodology used, wound healing or wound closure refers to the capability of epithelial cells (keratinocytes - HaCaT) to migrate to the scratch region and close the created space (wound). Data Analysis Repeated measures ANOVA followed by the Tukey test was used to compare groups/ times of wound measurements since it obtained a normal distribution in the Shap- iro-Wilk test. A one-tailed Student’s t-test was used to compare groups for the Wound closure parameter. All analyses considered 5% of significance and were done using Sigmaplot software. Results As seen in Figure 2, at time 0, the wounds in the control group (NC) and the group with Aa were similar in size (p>0.05) and were maintained for up to 48 hours. How- ever, when analyzing the timeframe and difference between time assessments, a dif- ference in how the wound measures behave could indicate a faster closure in con- trol than in the KO+Aa group. This phenomenon could be confirmed when analyzing wound closure (Figure 3). Standard measurements allow the determination of wound closure, considering time 0 as a reference. It is clear in Figure 3 that Aa protein extract reduced wound closure at 10 and 48 hours (p<0.05), negatively impacting the kerati- nocyte’s behavior. 600 500 a a b b c d c b a a400 300 200 100 0 0 hours 5 hours KO KO+Aa 10 hours 24 hours 48 hours Figure 2. Wound measures (μm±SD) in each period. Different letter in the same color indicates statistical differences among times within the group; No difference between groups was seen (Two-way ANOVA/Tukey, p<0.05). 6 Mendes et al. Braz J Oral Sci. 2024;23:e244720 Discussion A wide variety of microorganisms inhabit the oral cavity: more than 1000 different bac- terial species can colonize the mouth, and some studies carried out from the genetic sequencing of the biofilm show that approximately 500 species are similar between healthy individuals and those with periodontal diseases14,15. It is acceptable and cred- ible that there is symbiotic and protective colonization of oral tissues by commensal microorganisms, which can, through different ways, act as “guardians” of periodontal health. At the same time, while simultaneously inhabiting the same niches, pathobiont microorganisms can, modulated by local and systemic conditions, participate in the installation of diseases. Oral wounds caused by disease or their treatment will heal in this environment. Thus, it is likely, although still unexplored, that bacterial agents can alter cellular events during healing. In this community, pathogens that express cytotoxic virulence factors and, thus, modulate the cellular processes involved in healing stand out. The present study showed that the presence of the protein extract of Aa could alter wound closure induced in keratinocyte cultures. The healing process of oral wounds involves several cellular profiles. Keratinocytes and fibroblasts are the main constitutive cells of oral tissues and actively participate in this process8. Macrophages, monocytes, and neutrophils also act in the wound closure process16 by removing necrotic tissue, cell debris, bacteria and curbing infection17,18. Moreover, studies confirmed that fibroblasts can be affected by different stimuli, such as hydrogen-rich water16, hemp extract and cannabidiol19, and L-ascorbic acid15. Cells involved in the hematopoietic response can also alter their response when in contact with oral pathogens, modifying cytokine production or even inducing a disease-asso- ciated pro-inflammatory response16,17. Figure 3. Wound closure (μm±SD) in each period. * Indicates a statistical difference between groups (Student’s t-test, p<0.05) 450 400 350 300 250 200 150 100 50 0 5 hours KO KO+Aa 10 hours 24 hours * * 48 hours 7 Mendes et al. Braz J Oral Sci. 2024;23:e244720 Studies in vitro assessing the role of fibroblasts in wound closure16,18,19 observed that they present a high migration speed and proliferation during healing. However, KO is even more essential in wound healing once it is responsible for wound re-epithe- lialization18 and for protecting the internal face of the wound from external stimu- lus while the connective tissue heals. It has been reported that keratinocytes can re-epithelialize the wound area very quickly, usually closing a wound in approximately 18  hours. This  re-epithelization phenomenon enrolls keratinocytes moving into the defect 24  hours after the injury20. During this migration, keratinocytes from wound borders have their hemidesmosome connections dissolved from residual epithelial structures. They detach from the base of the membrane and move quickly across the wound defect. Later, as the re-epithelialization proceeds, the keratinocytes proliferate to the wound area, supporting the advancing epithelial edge. However, which cells are the first to arrive in the wound21 is still unclear. Meanwhile, as previously discussed, in oral wounds, the healing process will occur under an infective environment, and the type of bacteria could alter cell behavior – positive or negative. Some probiotic species, such as Lactobacillus rhamnosus GG, could improve wound healing, acting as a healing accelerator20. These results corrob- orate subsequent findings13, whose two types of probiotics (Lactobacillus casei 324 and  Bifidobacterium pseudolongum) accelerated the re-epithelialization of gingival epithelial cells compared to the control group. However, pathobionts could negatively alter this process. Studies showed that when these cells are exposed to Pg, their proliferation capacity drops considerably. Pg can inhibit tissue growth8,18 and display a destructive effect13. When OBA-9 (immortalized human gingival epithelial cells) are infected with Pg W83, the wound becomes larger instead of lessening by reducing the cell viability compared to the control group13. Besides, keratinocytes can present healing delayed by the influence of two oral patho- gens (Pg and Fn), and it was noticed that the long-term effect (especially in days 4 and 8) caused a major delay in the healing in at least 70%8. In our study, we evaluate the effect of total protein extract from  Aa  serotype JP2, which also has a destructive effect.  Aa-JP2 is the most cytotoxic serotype and a highly pathogenic species because its highly leukotoxic strains can produce 10 to 20-fold more toxin than the others22. It is well known that Aa-JP2 interferes with the host’s innate immune defense and response11,12. Studies with oral cells showed that this could lead to an imbalanced inflammatory response12, which could affect healing, as observed in the study. All this can disrupt normal periodontal tissue remodeling processes, ultimately promoting collateral tissue damage, which explains the delay in healing22. However, although potentially cytotoxic, up to now, there has been no study assessing the impact of Aa on wound healing. The present study shows that it neg- atively affected the healing process since lower wound closure could be seen when keratinocytes were cultivated with total Aa protein.  One factor that may explain why  Aa  decreases healing is that it disrupts epithelial integrity, facilitates bacterial invasion into deeper tissues, and subsequently destroys periodontal connective tissue23. Disturbances caused by the cell-cell junction’s sta- bility and dynamics can affect the epithelial layers’ barrier properties, harm tissue remodeling throughout development, and impair the healing compared to healthy 8 Mendes et al. Braz J Oral Sci. 2024;23:e244720 tissue23. Aa can act on tissue destruction and delay healing. A study demonstrated that Aa secretes a cytolethal distending toxin that likely permeates the widened inter- stitial spaces of the junctional epithelium and in non-keratinized forms of gingival epi- thelium to affect their basal cell layers24. The distension of human gingival epithelial cells and the dissolution of cell junctions may promote broader penetration of the toxin throughout the tissue, which may signal the recruitment of inflammatory cells, consequently increasing the extent of tissue damage due to the production of inflam- matory mediators. This study showed a significant difference at two follow-up times (10 and 48 hours). On the other hand, no statistical difference could be identified after 5 and 24 hours of exposure. Epithelial cells are the first to arrive at the wound site, and the epithelial healing process is expected to occur rapidly20. There is a response time for keratinocytes to the Aa protein extract (AaPE) exposure. After exposure to AaPE, the cell recognizes the pathogenic protein through TLRs. Its metabolic activity begins to be modulated, generating a lack of production or defective production of proteins responsible for cell junctions23. This entire process can take hours to occur. This is the plausibility of the absence of difference in the cell response after 5 hours in this study. After this process, cellular activity is damped by Aa toxins, slowing down the epithelial repair, which explains the later closure response for the test group (48 hours). The results achieved in this study elucidate a slight portion of the etiopathogenic aspects of periodontal disease. In periodontitis, the dysbiotic presence of periodon- topathogens such as Aa and Pg at subgingival biofilm harms gingival tissue through the bacterial adherence to the epithelial cells, which colonizes the connective tissue, stimulating the cells to secrete cytokines responsible for recruiting inflammatory cells by inducing changes in signaling pathways and gene expression25. This methodol- ogy used AaPE for keratinocyte stimulation because of its proximity to periodontitis. Although there are six serotypes of Aa (a–g), serotype b (JP2 genotype) is the one that is associated with a greater risk of periodontal insertion loss22. In our study, the total protein of this more cytotoxic strain was used, and several virulent aspects were given to cells. Given the results obtained, in addition to being a trigger for the onset of the disease, the presence of Aa can also alter tissue healing after initial damage or periodontal instrumentation. This way, clinicians must focus more on controlling subgingival plaque and dysbiosis after periodontal treatment and other procedures involving periodontal tissues and re-epithelization, e.g., aesthetic crown lengthening. In this study, we assessed that wound healing could indeed be influenced by a bac- terial pathogen such as Aa; however, this study presented some limitations: The lack of an  in vivo study to see how the subgingival microbiota and a complete biofilm with a larger number of pathogens, as in periodontitis, can affect oral tissue healing, and the lack of another cell type, such as fibroblasts, for greater comparison effect in the results. Therefore, it would be necessary to include at least one more patho- gen and cell type for a better in vitro study and to evaluate the possibility of carrying out additional  in vivo  studies to determine the impact of oral bacteria on wound healing in vivo. In conclusion, the pathogen  Aggregatibacter actinomycetemcomitans  can interfere with wound closure by keratinocytes. This may be due to disturbances caused by the stability and dynamics of the cell-cell junction caused by Aa.  9 Mendes et al. Braz J Oral Sci. 2024;23:e244720 Acknowledgments This study was financed by the National Council for Scientific and Technological Development – Brazil (CAPES) – Finance Code 001. The authors also declare no con- flicts of interest in this study. Funding sources This study did not receive any specific grant from funding agencies in the public, com- mercial, or not-for-profit sectors. Conflict of interest The authors have no conflict of interest to disclose. Data availability Datasets related to this article will be available to the corresponding author upon request. Author contribution Bianca Carvalho Mendes: Data collection, Writing the manuscript. Camila Schmidt Stolf: Conception and design of the study, Data analysis, Writing the manuscript. Hél- vis Enri de Sousa Paz: Review and Editing, Writing the manuscript. Letícia Sandoli Arroteia: Data analysis, Interpretation and Statistics. Lucas de Paula Ramos: Data collection, Interpretation and Statistics. Mauro Pedrine Santamaria: Data collection, Interpretation and Statistics. Karina Gonzales Silvério Ruiz: Conception and design of the study, Review and Editing. Mabelle de Freitas Monteiro: Writing the manu- script, Review and Editing. Renato Corrêa Viana Casarin: Conception and design of the study, Writing the manuscript, Review and Editing. We declare that all authors actively participated in the manuscript findings, revised and approved the final version of the manuscript. References 1. Mohammed BM, Fisher BJ, Kraskauskas D, Ward S, Wayne JS, Brophy DF, et al. Vitamin C promotes wound healing through novel pleiotropic mechanisms. Int Wound J. 2016 Aug;13(4):572-84. doi: 10.1111/iwj.12484. Epub 2015 Aug 20. 2. Diegelmann RF, Evans MC. 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