1http://dx.doi.org/10.20396/bjos.v20i00.8661512 Volume 20 2021 e211512 Original Article 1 Postgraduate Program in Health Sciences, University of Brasília, Brasília, Distrito Federal, Brazil. 2 Proteomic and biochemical analysis center, Postgraduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brasília, Distrito Federal, Brazil. 3 Dentistry Course, Catholic University of Brasília, Brasília, Federal District, Brazil. 4 Postgraduate Program in Biotechnology and Biodivesity, Brasilia University, Brasilia, Distrito Federal, Brazil. 5 Graduate Program in Health and Development in the Midwest Region. Faculty of Medicine. Federal University of Mato Grosso do Sul. Campo Grande. Brazil. 6 S-Inova Biotech, Graduate Program in Biotechnology, Dom Bosco Catholic University, Campo Grande, Mato Grosso do Sul, Brazil. Corresponding author: Name: Taia Maria Berto Rezende Full postal address: Universidade Católica de Brasília Pós-graduação em Ciências Genômicas e Biotecnologia SGAN 916N – Av. W5 – Campus II – Módulo C, room C-221 Brasília-DF, Brazil Fone: + 55-61-98134-9001 Fax: + 55-61-3347-4797 e-mail: taiambr@gmail.com Secondary e-mail: taia@ucb.br Received: October 06, 2020 Accepted: January 21, 2021 Host defense peptides clavanins A and MO reduce in vitro osteoclastogenesis Ingrid Aquino Amorim1,2 , Stella Maris de Freitas Lima2,3 , Ana Paula de Castro Cantuária1,2 , Mirna de Souza Freire2,4, Jeeser Alves de Almeida5 , Octávio Luiz Franco2,6 , Taia Maria Berto Rezende1,2,3,* Aim: Several systemic diseases, such as periodontitis and apical periodontitis, can cause extensive bone resorption. Host defense peptides may have the potential for the development of novel therapies for the bone resorption process. This study evaluated the potential of host defense peptides clavanins A, MO, and LL-37 in in vitro osteoclastogenesis. Methods: RAW 264.7 cultures were stimulated with recombinant of receptor activator of nuclear factor kappa B ligand in the presence of different tested concentrations of host defense peptides, besides calcium hydroxide and doxycycline. Cellular viability, nitric oxide production, and a number of differentiated osteoclast-like cells were also evaluated. Results: Results showed that none of the substances were cytotoxic, except for 128 μg.mL-1 of doxycycline after 3 days. Host defense peptides, calcium hydroxide, and doxycycline did not interfere in nitric oxide production or downregulated it. An exception was observed in the presence of 2 μg.mL-1 of doxycycline, in which nitric oxide production was up-regulated. All host defense peptides were capable of reducing osteoclast-like cell differentiation. Conclusion: Host defense peptides clavanins A and MO demonstrated to be potential suppressors of osteoclastogenesis in vitro without interfering in cellular viability and nitric oxide production. These promising results need to be further analyzed in in vivo models of bone resorption. Keywords: Bone resorption. Antimicrobial cationic peptides. Nitric oxide. Osteogenesis. mailto:taia@ucb.br https://orcid.org/0000-0002-1340-7091 https://orcid.org/0000-0003-1359-8761 https://orcid.org/0000-0001-5828-450X https://orcid.org/0000-0002-3409-8005 https://orcid.org/0000-0001-9546-0525 http://orcid.org/0000-0002-4148-0659 2 Amorim et al. Introduction Bone remodeling is a process balanced between osteoblast-mediated bone deposi- tion and osteoclast-developed bone resorption. Many oral diseases are mediated by an inflammatory process, increasing the recruitment of osteoclasts and enhancing bone erosion1. Periodontal disease and apical periodontitis present bone resorption with high osteoclast formation or hyperactivation, overcoming bone formation, and decreasing osteoblast activity2. Inflammatory conditions, such as local osteolysis, can be associated with induc- ible nitric oxide synthase (iNOS) activation3. NO can also promote cytokine pro- duction and bone turnover besides indirect induction of bone resorption3. In this regard, periodontitis results in higher production of NO compared to healthy gin- giva4. Periodontal treatment may involve the use of several systemic antibiotics such as tetracycline (minocycline and doxycycline) as adjuvants due to its local distribution. It was demonstrated that doxycycline hyclate gel (local therapy) could aid in scaling and root planning in patients with moderate to severe chronic peri- odontitis, but the benefit is still uncertain5. For the endodontic treatment, the use of calcium hydroxide (Ca(OH)2) as a local antimicrobial is accepted worldwide as intracanal dressing6. Despite the high success rate in existing periodontal and endodontic therapies, there are some limitations, mainly in tissue repair activity6. To improve bone repair in these diseases, it is essential to develop new substances. New therapies involv- ing a direct effect on the bone can prolong the maintenance of the tooth in the oral cavity due to tissue support health. Host defense peptides (HDPs) are biomol- ecules from many organisms released in early defense response to infection and invasion by bacteria and other microorganisms7. HDPs may possess antimicrobial and immunomodulatory properties besides tissue repair induction8. In this context, human cells can be potential sources of HDPs7. Clavanin A is a promising HDP due to its known antibacterial, immunomodulatory, antitumor, and antiviral activities9. Besides, clavanin A was used as a model to create clavanin MO. Five hydrophobic amino acid residues (FLPII) were added to the N-terminus, being selected based on a computational search of the conserved region of other peptides with higher immu- nomodulatory activities10. It has been shown that different HDPs could improve ther- apies in the dental field. Some peptides have been reported as having the potential to inhibit osteoclasto- genesis such as LL-3711, human beta-defensin-3 with C-terminal end contains a 15-amino acid polypeptide (HBD3 - C15)12, synoeca-MP,13 and HHC-1013. A previous study demonstrated that LL-37 and clavanins A and MO can modulate the inflam- matory response of active cytokines presented in the osteoclastogenesis process, such as TNF-α, while Ca(OH)2 up-regulated the IL-6 and IL-1α production 14. This fact leads us to believe that clavanins A and MO may have the potential to inhibit osteoclastogenesis, a fact that has not yet been evaluated. Thus, this study aims to evaluate the biotechnological potential of HDPs clavanin A and MO in the oral oste- oimmunological context and their capability to reduce in vitro osteoclastogenesis, 3 Amorim et al. compared to LL-37 (HDP control), Ca(OH)2 (used in the endodontic treatment) and doxycycline (used in the periodontal treatment). Material and Methods Peptide synthesis Clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH2), clavanin MO (FLPIIVFQFLGKI- IHHVGNFVHGFSHVF-NH2), and LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFL- RNLVPRTES-NH2) were synthesized and purified (>95% purity) by Peptide 2.0 Inc. (USA). Molecular mass and purity of all peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry on an Auto-Flex III Speed instrument (Bruker Daltonics, Billerica, MA). Peptides were diluted in ultra- pure water and quantified by UV absorption at 205, 215, and 225 nm, according to Murphy and Kies15. Doxycycline and calcium hydroxide preparation Ca(OH)2 (Iodontosul, Porto Alegre, Brazil) was weighed and diluted in ultrapure water before each experiment. Doxycycline (Pharmac, Brasilia, Brazil) was handled in cap- sules (100 mg in each unit). The capsules were opened, and doxycycline was weighed and diluted in ultrapure water before each experiment. Cell culture, experimental groups and osteoclasts Osteoclast precursor RAW 264.7 cell line (RAW; BCRJ code 0212; RRID CVCL_0493 – Rio de Janeiro, Brazil) is composed of monocytes derived from tumors induced in male BALB/c mice (Mus musculus), infected with murine leu- kemia Abelson virus16. RAW cells were grown in high glucose Dulbecco’s modi- fied Eagle’s medium (DMEM; Gibco, California, USA) supplemented with 10% fetal bovine serum (Gibco, California, USA), 1% penicillin/streptomycin (1000 U.mL-1) (Gibco, California, USA), 1% nonessential amino acid solution (Gibco, California, USA), 1% L-glutamine (Gibco, California, USA) and 0.1% gentamicin (Gibco, Cali- fornia, USA). Cell cultures were maintained in an incubator containing 5% CO2 at 37°C and 95% humidity. Experiments were conducted with 2.5x103 cells per wells in 96-well plates (Kasvi, China), stimulated with or without rRANKL 100 ng.mL-1 (Peprotech, New Jersey, USA) and HDPs clavanin A, clavanin MO, and LL-37 (2, 8, 32, and 128 μg.mL-1). Peptide stimulated cultures were compared to doxycycline and Ca(OH)2 (2, 8, 32, and 128 μg.mL -1). The concentrations were based on a previ- ously published result14. Cell viability assay and NO production were analyzed after 3 and 7 days of cell culture. Half of the culture medium and stimuli were changed every 3 days. After 7 days, TRAP staining was performed, and the number of dif- ferentiated osteoclast-like cells was determined. Cytotoxicity analyses Peptides, Ca(OH)2, and doxycycline cytotoxicity were analyzed by MTT colorimetric assay (Sigma-Aldrich, St. Louis, USA), read in a microplate reader (Bio-Tek Power Wave HT, USA) at 570 nm17. Cell viability was determined after 3 and 7 days of cul- 4 Amorim et al. ture. All samples were compared to a positive control group (RAW culture), consid- ered as 100% cell viability. Nitric oxide production analysis Nitrite production was evaluated in supernatants of cell cultures by Griess reaction, with adaptations18. Briefly, 100 μL of cell culture supernatant was transferred to a new 96-well plate (Kasvi, China). Then, 100 μL of 1% sulfanilamide phosphoric acid solu- tion and 2.5% of 1% naphthyl ethylenediamine phosphoric acid (1:1) was added. After 10 min, reading was performed in a microplate reader (Bio-Tek PowerWave HT, USA) at 490 nm. The amount of nitrite was calculated based on a standard curve of sodium nitrite (1.5625 µM to 200 µM)18. Tartrate-resistant acid phosphatase (TRAP) staining TRAP staining was performed after 7 days of incubation for the quantification of dif- ferentiated osteoclast-like cells. The tartrate-resistant acid phosphatase (TRAP) kit (Sigma-Aldrich, St. Louis, USA) was used according to the manufacturer’s specifica- tions. Osteoclast-like cells were considered as TRAP-positive cells (with red/orange TRAP staining) with more than three nuclei. Statistical analysis Data obtained was analyzed by the standard error of the mean for each experiment. The normality was evaluated (Kolmogorov-Smirnov test), and subsequent parametric statistical analysis was carried out by two-way analysis of variance (two-way ANOVA) for the data from MTT and NO production and one-way ANOVA for TRAP analyses. Tukey’s posthoc test was applied to identify statistical differences. Analyses were considered at the 95% significance level, and statistical differences were considered when p<0.05. Statistical analysis was performed using GraphPad Prism 6.0 software (Instat California, USA). Results HDP cytotoxicity The cytotoxicity of substances was determined by cell viability assays after 3 and 7 days of cell culture in the presence of HDPs clavanin A, clavanin MO, LL-37, Ca(OH)2 and doxycycline. HDPs and Ca(OH)2 were not cytotoxic to pre-osteoclasts (data not shown). However, doxycycline, at the high concentration (128 μg.mL- 1) reduced cell viability by 48% (p<0.05), after 3 days of incubation, compared to the control group (data not shown). Similar viability results were observed in osteoclast-like cells (RAW cells with rRANKL), and substances after 3 and 7 days incubation. HDPs, Ca(OH)2 and doxycycline were not cytotoxic to rRANKL-stimu- lated cells (Figure 1). However, 128 μg.mL-1 of doxycycline reduced cell viability by 42% after 3 days, compared to the control group (p<0.05). Indeed, HDPs were not cytotoxic and only doxycycline at 128 μg.mL-1 demonstrated a cytotoxic effect on osteoclast-like cells (rRANKL-stimulated and RAW cells). 5 Amorim et al. Nitric oxide production Cell cultures with HDPs, Ca(OH)2, and doxycycline produced basal levels of NO, com- pared to the control group (data not shown). The rRANKL increased NO production in Figure 1. Clavanin A, clavanin MO, LL-37, Ca(OH)2 and doxycycline cytotoxicity at 2, 8, 32 and 128 μg.mL -1 on 2.5x103 RAW cells, after 3 and 7 days, by MTT assay. Cultures were stimulated with 100 ng.mL-1 of rRANKL. Cell viability was represented by percentage. Control group was represented by 2.5x103 RAW cells stimulated with 100ng.mL-1 of rRANKL and considered 100% of cell viability. All experiments were done in technical and biological triplicates. Statistical differences by two-way ANOVA test and Tukey’s post hoc were represented by *p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001 compared to each concentration and time tested conditions; Dark green bars represent statistical differences observed on day 3; Light green bars represent statistical differences observed on day 7. C el l v ia bi lit y (% ) Clavanin A ** 250 200 150 100 50 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 C el l v ia bi lit y (% ) Clavanin MO ** *** * 250 200 150 100 50 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 C el l v ia bi lit y (% ) LL-37 250 200 150 100 50 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 C el l v ia bi lit y (% ) Doxycycline 3 days 7 days 250 200 150 100 50 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 C el l v ia bi lit y (% ) Ca(OH)2 250 200 150 100 50 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 * ** ** **** *** **** *** * ** *** * ** ** **** ** **** **** * ** **** * ** 6 Amorim et al. RAW 264.7 cell cultures (Figure 2). After 3 days, rRANKL-stimulated cells with clavanin A downregulated NO levels at all concentrations, compared to the control group, while on the seventh day, NO levels were similar to the control group (p<0.0001). The down- regulation of NO production was also observed in cultures stimulated with clavanin Figure 2. Nitric oxide production in the presence of clavanin A, clavanin MO, LL-37, Ca(OH)2 and doxycycline at 2, 8, 32 and 128 μg.mL-1 on rRANKL-stimulated-RAW cells, after 3 and 7 days, as described in the method of Green et al., with adaptations. Cultures were stimulated with 100 ng.mL-1 of rRANKL. Control group consisted of 2.5x103 RAW cells stimulated with 100 ng.mL-1 of rRANKL. Bars represent the standard error of the mean of nitrite oxide production. All experiments were done in technical and biological triplicates. Statistical differences by two-way ANOVA test and Tukey’s post hoc were represented by *p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001 compared to each concentration and time-tested conditions; Dark green bars represent statistical differences observed on day 3; Light green bars represent statistical differences observed on day 7. N itr ite (μ M ) Clavanin A **** 5 4 3 2 1 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 N itr ite (μ M ) Clavanin MO5 4 3 2 1 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 N itr ite (μ M ) LL-37 5 4 3 2 1 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 N itr ite (μ M ) Doxycycline 3 days 7 days 5 4 3 2 1 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 N itr ite (μ M ) Ca(OH)2 5 4 3 2 1 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 ************ **** ** *** ** *** * **** **** *** * ** ** **** **** ******** **** **** ** * * **** **** **** **** **** 7 Amorim et al. MO at 128 μg.mL-1, after 7 days (p <0.05). However, the presence of 128 μg.mL-1 of LL-37 after 7 days, increased the NO levels compared to the control group (p<0.0001), while the others concentrations at 3 and 7 days were similar to baseline levels. Ca(OH)2 was able to upregulate NO production at 8 (p<0.005), 32 (p<0.05) and 128 μg.mL-1 (p<0.005) after 7 days of cell incubation. NO levels in the presence of Ca(OH)2 at other different concentrations were similar to the control group (p<0.05), after 3 and 7 days. The lower concentration of doxycycline up-regulated NO production after 7 days com- pared to all concentrations, including the control group (p<0.0001). Overall, reduced levels of NO were observed in some concentrations of all substances, except for dox- ycycline and Ca(OH)2 7 days after the test. Number of differentiated osteoclast-like cells RAW cell cultures stimulated with HDPs, Ca(OH)2, doxycycline and rRANKL were submitted to TRAP staining after 7 days of incubation (Figure 3A-B), for quantifi- cation of differentiated osteoclast-like cells. All HDPs, Ca(OH)2, and doxycycline, at all tested concentrations, were capable of reducing the differentiation of osteo- clast-like cells (Figure 3B). Indeed, clavanin A reduced osteoclastogenesis in an inverse dose-dependent concentration. Clavanin A at 128 μg.mL-1 demonstrated the lowest number of differentiated osteoclast-like cells (p<0.05). Osteoclastogen- esis was similarly reduced by clavanin MO at all tested concentrations (p<0.05). Likewise, LL-37 also downregulated osteoclastogenesis at 2, 4, 8 and 128 μg.mL-1, with the lowest number of osteoclast-like cells in 2 and 4 μg.mL-1 (p<0.05). Ca(OH)2 was most effective in reducing osteoclast-like cells at 8 μg.mL-1 (p<0.05). However, 2 μg.mL-1 of Ca(OH)2 showed the highest number of differentiated osteoclast-like cells (p <0.05). Meanwhile, doxycycline exhibited a gradual reduction in osteoclas- togenesis, and 128 μg.mL-1 stimulated cells demonstrated the lowest number of differentiated osteoclast-like cells. Based on the number of osteoclast-like cells differentiated by rRANKL-stimulated RAW cell culture, the concentration of 8 μg.mL-1 was the lowest common concentration for HDPs, Ca(OH)2, and doxycycline, capable of reducing differentiation in osteoclast-like cells. Therefore, the best results were exhibited by LL-37 and Ca(OH)2. LL-37 showed approximately 67% fewer osteoclasts than clavanin A, clavanin MO and doxycycline, while Ca(OH)2 showed 59% fewer osteoclasts compared to clavanins and doxycy- cline. Ca(OH)2 and LL-37 demonstrated better osteoclastogenesis downregulation, compared to the same concentration of clavanin A, clavanin MO and doxycycline (p<0.0001). Therefore, among the tested HDPs, LL-37 presented the best ability to reduce the number of osteoclasts in vitro (p<0.05). 8 Amorim et al. Figure 3. Representative photos of TRAP positive stained cells (A) and number of multinucleated osteoclast- like cells (B) induced by 2.5x103 RAW cell and 2.5x103 RAW cells stimulated with 100 ng.mL-1 rRANKL (Control). Cultures were rRANKL-stimulated and tested with 2, 8, 32, and 128 μg.mL-1 of clavanin A, clavanin MO, LL-37, Ca(OH)2 and doxycycline, after 7 days. Scale bar: 50 μM. Each well was completely checked, and osteoclasts were counted at 20x magnification. Black arrows show osteoclast-like cells with more than 3 nuclei. Statistical differences by one-way ANOVA test and Tukey’s post hoc were represented by *p<0.001 compared to control in each tested condition. Statistical differences of comparative analysis of the lowest common concentration, with greater reduction in osteoclast-like cell differentiation, of all tested materials: 8μg.mL-1 of clavanin A, clavanin MO, LL-37, Ca(OH)2 and doxycycline (samples at 8μg.mL -1) were represented by *p<0.0001. Number of osteoclast-like cells was represented as the standard error of the mean. All experiments were done in technical and biological triplicates. O st eo cl as ts Clavanin AB A 50 40 30 20 10 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 * * * * O st eo cl as ts Clavanin MO 50 40 30 20 10 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 * * * * O st eo cl as ts LL-37 Clavanin AControl 2 μg .m L- 1 8 μg .m L- 1 R A W c el ls R A W c el ls + rR A N K L 32 μ g. m L- 1 12 8 μg .m L- 1 Clavanin MO LL-37 50 40 30 20 10 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 * * * * O st eo cl as ts Ca(OH)2 50 40 30 20 10 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 * * * * O st eo cl as ts Doxycycline 50 40 30 20 10 0 Co nt ro l 2 μ g.m L -1 8 μ g.m L -1 32 μg .m L -1 12 8 μ g.m L -1 * * * * O st eo cl as ts Samples at 8 μg.mL-1 50 40 30 20 10 0 Cl av an in A Cl av an in M O LL -37 Ca (O H) 2 Do xy cy cli ne ** * * * * * Ca(OH)2 Doxycycline 9 Amorim et al. Discussion Although periodontal and endodontic therapies are highly effective, new substances can improve outcome expectations. Antimicrobial, immunomodulatory, and repar- ative activity could be better achieved by new therapies and biologic substances19. Indeed, antimicrobial resistance is also a current limitation for both therapies6. The present study demonstrated the in vitro potential of HDPs clavanins A and MO in an osteoclastogenesis model. Results of cellular NO production, cytotoxicity, and the effects of HDPs on rRANKL-mediated osteoclastogenesis were compared to Ca(OH)2 and doxycycline, widely used medications in endodontic 6 and periodontal5 areas, respectively. Study related to clavanin A has demonstrated different activities regarding this pep- tide, including important points for dentistry, such as antibiofilm and antimicrobial activity. HDP clavanin A showed antibiofilm activity against fungal biofilms when used to coat an amniotic membrane, which is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction20. HDPs clavanin A, clavanin MO and LL-37 did not show any degree of cytotoxicity to RAW cells. A previous study using clavanin A also showed no cytotoxicity against mammalian cells (L929) with low concen- trations9. Moreover, another study showed that 128 µg.mL-1 of clavanin MO did not demonstrate cytotoxicity compared to the other antimicrobial agents, with or without additional stimulation. LL-37 increased cell viability on RAW cells, and Ca(OH)2 did not interfere with cell viability at the same concentration. Besides, after 6 h of incubation, clavanins alone reduced cell viability14. NO regulates bone resorption through the regulation of the synthesis of OPG/ RANKL in bone marrow cells21, although other factors, including cytokines, are also involved. Our results demonstrated that HDPs downregulated NO production with or without the stimulation of rRANKL. Accordingly, clavanin A demonstrated a significant reduction in the number of osteoclast-like cells in a dose-dependent manner. Clavanin MO also reduced the number of differentiated osteoclast-like cells. Similarly, LL-37 at 8 and 128 μg.mL-1 demonstrated the best inhibition activ- ity. According to previous results, HDPs can also modulate inflammatory medi- ators that contribute to the bone resorption activation process, such as TNF-α, IL-6 and IL-1α, and NO production14. Different substances already used in clinical practice were also evaluated in this study. Ca(OH)2 did not show cytotoxicity, and doxycycline demonstrated a toxic effect on cells at high concentration and increased cell viability at low concentration. Accord- ingly, a study evaluated the effects of a sub-antimicrobial dose of doxycycline (SDD) on ligature-induced periodontitis in spontaneously hypertensive rats. It concluded that SDD therapy exerted a systemic modulating effect on inflammation, with reduced periodontal tissue destruction in hypertensive rats22. Ca(OH)2 presented similar NO results compared to HDPs, and doxycycline presented an increase in NO levels, especially at 2 µg.mL-1. In the osteoclastogenesis process, both tested drugs decreased the number of osteoclast-like cells in the presence of all concentrations tested. These facts suggest that although NO is strongly associated with osteoclast differentiation, this is not the only factor involved in the osteoclasto- 10 Amorim et al. genesis process23. Also, doxycycline might have another mechanism for downregulat- ing the osteoclastogenesis pathway23. However, these results are in agreement with previous results that suggest the inhi- bition of osteoclastogenesis in RAW cells in the presence of Ca(OH)2 23. Other studies suggest that the alkaline pH of Ca(OH)2 can neutralize the lactic acid secreted by osteoclasts and may help prevent the destruction of mineralized tissue24. In summary, this study aims to initiate the assessment of the biotechnological potential of HDPs clavanin A and MO in the oral osteoimmunological context and their capability to reduce in vitro osteoclastogenesis, compared to LL-37 (HDP con- trol), Ca(OH)2 (used in the endodontic treatment) and doxycycline (used in the peri- odontal treatment). We highlighted the results observed in the presence of 8 μg.mL-1 of LL-37 and Ca(OH)2, thus considering the use of these peptides as a possible product for endodontic and periodontal applications, in order to reduce the osteo- clastogenesis process. On the other hand, Ca(OH)2 shows low production costs when compared to LL-37. This HDP presents a relatively long sequence of amino acids, which raises its cost for synthesis, and it would probably only be indicated for restricted cases. Indeed, because of its immunomodulatory benefits and its bio- compatibility, by being a peptide present in the oral cavity, LL-37 presents itself as a good candidate for dentistry use. When LL-37 results were compared to the doxy- cycline, the HDP demonstrated better efficiency in osteoclastogenesis downregula- tion at low concentrations, thus showing an even greater potential in the context of periodontal bone loss. Despite the benefits highlighted in these data, in vitro results should be interpreted with caution and other in vivo studies are necessary to evaluate the potential of this biomolecule for clinical use. Other important points for future investigations should be focused on the large-scale expression of this peptide (lowering its cost), and the analysis of its integrity through various oral conditions, such as temperature changes, pH, and presence of lytic enzymes. In addition, other parameters should be evalu- ated, such as the peptides’ mechanism of action in the osteoclastogenesis process, in order to enhance the knowledge on these potential products indicated for bone resorption processes, present in the periradicular area and periodontitis. Acknowledgments This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Grant: 409196/2018-5, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisa do Distrito Fed- eral (FAPDF) Grant: 0193.001702/2017 and Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT). 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