1 Volume 23 2024 e243733 Original Research Braz J Oral Sci. 2024;23:e243733http://dx.doi.org/10.20396/bjos.v23i00.8673733 1 BDS., MSc. Assistant Professor. College of Dentistry, University of Mosul, Mosul, Iraq. 2 BDS., MSc., PhD. Assistant Professor. College of Dentistry, University of Mosul, Mosul, Iraq. Corresponding author: Rasha M. Al-Shamaa University of Mosul/College of Dentistry rasha.mozahim@uomosul.edu.iq rasha.mozahim79@gmail.com Phone:+9647738496492 Iraq/Duhok Editor: Dr. Altair A. Del Bel Cury Received: June 14, 2023 Accepted: April 19, 2024 Assessment the bioactivity of zinc oxid eugenol sealer after the addition of different concentrations of nano hydroxyapatite-tyrosine amino acid - an in vitro study Rasha M. Al-Shamaa1* , Raghad A. Al-Askary2 Aim: Zinc oxide eugenol sealer has been used till now in endodontic obturation. However, despite many improvements in its formula, it still does not have, the essential root canal sealer’s properties which is the apatite forming ability. The aim of the present study is to assess the effect of the incorporation of nano Hydroxyapatite- tyrosine amino acid at different concentrations in the zinc oxide eugenol sealer formula in terms of bioactivity analysis. Methods: The nano hydroxy apatite-tyrosine amino acid was incorporated into the original zinc oxide eugenol (endosell) at different concentrations starting from (10 – 20)%. The chemical changes in zinc oxide eugenol before and after addition were characterized using FTIR and XRD. The setting time test was done according to ADA specification no. 57. The bioactivity analysis for the zinc oxide eugenol before and after the addition was evaluated according to ISO/FDIS 23317:2007(E) by using 28 days of storage in phosphate buffer saline, and then the hydroxyapatite precipitation and Ca/P ratio was evaluated using FESEM/EDX. Results: The FTIR and XRD confirmed the setting reaction occurrence among the (original ZOE, nHAP, and Tyr). The XRD and FESEM/EDX analyses confirmed the HAP precipitation on the ZOE sample surfaces after the addition of (nHAP-Tyr a.a) and this precipitation was increased with increased concentrations of additions. Conclusion: Incorporated (20) % of equal amounts of “nHA-Tyr a.a” can convert the ZOE to bioactive sealer as confirmed by XRD and FESEM/EDX. However, other characteristic analyses like Nuclear magnetic resonance, atomic force microscopy, and in vivo animal study were needed to further confirm the results. Keywords: Root canal filling materials. Tyrosine. Nanocomposites. zinc oxide-eugenol cement. https://orcid.org/0000-0002-5244-3757 https://orcid.org/0009-0001-8365-4303 2 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 Introduction The main goal of root canal obturation is to provide an appropriate filling of anatomi- cal irregularities with minimal voids. To achieve this objective, gutta-percha cones are associated with endodontic sealers1,2. The sealers are mainly grouped according to their main chemical components into (zinc oxide eugenol, glass ionomer, silicone, resin, and bioceramics-based root canal sealers3,4. The zinc oxide eugenol (ZOE) sealers are the early root canal sealers based on Gross- man or Rickerts’s formula that was commonly used amongst clinicians. It has good antimicrobial activity. However, ZOE has a weak mechanical characteristic, its sealing property was inferior in comparison to other sealers due to its relatively high solubility in addition to the recorded cytotoxic effect of these types of sealers and it lacks the ability of bone formation5,6. Biomaterials are materials that possess some novel properties that make them appropriate to come in immediate contact with the living tissue without eliciting any adverse immune rejection reactions. There are many types of biomaterials: Bioinert material (material that once placed in the human body has minimal interaction with its surrounding tissue, examples of these are metals, ceramics, and zirconia); Bioac- tive material (a material that elicits a specific biological response at the interface of the material, which results in the formation of a bond between the tissue and that material, these materials are either non-resorbable like bioactive glasses, hydroxyap- atite, and ceramic) or Bioresorbable materials (material that upon placement within the human body starts to dissolve, slowly replaced by advancing tissue (bone) for example tricalcium phosphate7. A bioactive material is defined as a material that has an effect on or induces a response from living tissue, organisms, or cells to form nano-hydroxyapatite (nHAP). Bioactive endodontic sealers have been developed to improve the quality of root canal obtura- tion. However, the bioactivity, and remineralization potential, may provide additional benefits for root canal obturation materials by strengthening root dentin and promot- ing the formation of hard tissue8. The Hydroxyapatite with a chemical formula Ca10 (PO4)6(OH)2 is a calcium phosphate bioceramic regarded as the main inorganic biomineral constituent in enamel and den- tine with a Calcium/Phosphate (Ca/P) ratio of 1.67 which is a major component of hard tissues such as bone and dentin and Ca/P ratio of 2.17 in Enamel9,10. Nanotechnology is also used to produce a large number of dental materials. The advan- tages of nanoparticles, that have attracted attention in endodontics, are their better penetration into the dental tubules, profound antibacterial properties, and decreased microleakage. Because of these valuable properties, the utilization of nanoparticles in producing endodontic sealers has become favorable for many researchers11,12. Calcified human tissues consist of the collagen matrix as the organic phase and the apatite as the inorganic phase; deposition of the latter is regulated by noncollage- nous proteins (NCPs), which have highly anionic nature that enable them to cap- 3 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 ture calcium ions from the body solution and to provide bounded ions to collagen fibrils during the formation of bone and dentin. Thus, it has been assumed that bind- ing of NCPs/ collagen fibrils could be a means of promoting intrafibrillar nucleation of hydroxyapatite13,14. Tyrosine (Tyr) is a type of amino acid, which serves as the building block of proteins. It is a non-essential amino acid and is also classified as an aromatic amino acid, derived from phenylalanine15,16. Tyrosine (Tyr) has an aromatic ring that consists of a six-link carbon-hydrogen ring with three conjugated double bonds. This ring’s substituents regulate whether the side chain of amino acids employs polar or hydrophobic interactions. In the case of Tyr, a hydroxyl group on the phenyl ring participates in hydrogen bonds; that is, the side chain of Tyr is more polar and hydrophilic. In other words, Tyr is less hydrophobic and more reactive due to its aromatic ring with a hydroxyl group17,18. The purpose of the research was developed as a trying to induce the bioactivity of ZOE sealer by the addition of (nHAP – Tyr.) a.a. materials. Materials and Methods Addition of Materials The addition of (nHAp) - tyr powders was done equally (50% nHA, 50% tyr). The addi- tion starts from 10% before this concentration. There was apatite precipitation that could be seen on the surface of sealer specimens after 28 days of storage in PBS as confirmed by FESEM/EDX data. The selective concentrations used in the current study were (10, 12, 16, 20) %. However, the addition ended at 20 % since no further setting reaction for the original ZOE sealer had occurred. The addition of the equal parts of each (nHA and tyr) powder was done by adding the same amount of powder that would be withdrawn from the original ZOE sealer, as the following: Dispense one spoon of the original ZnO on the sensitive balance of 0.0001 g accuracy which weighs about (0.150) g, then remove from this mass what is equal to the per- cent of (nHAP and Tyr) powder that will be added. The additions occur as the following: • 0.150 / 100 × 2 = 0.003 g [amount of original ZOE powder that will be withdrawn]. • 0.003/ 2 = 0.0015 g [for each nHAP and Tyr] powder. • Final spoon mass = 0.150g (after addition). Chemical Setting Reaction The chemical setting reactions for nHAP alone, Tyr. a.a alone, original ZOE, and ZOE after addition at (10 and 20) % were done using FTIR and XRD analyses. Setting Time Test 4 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 Setting time Test for the ZOE after addition at (10-12-16-20) % was done according to ADA specification no. 57 using a Gilmore needle with a mass of (100 ± 0.5) g and a flat end with a diameter of (2.0 ± 0.1) mm positioned vertically at right angles against the sealer sample. Five samples for each freshly mixed original ZOE sealer and the experimental sealer at different concentrations were prepared using a stainless-steel ring mold with a diam- eter of (10 mm and height of 2 mm). The time from the end of mixing until the set of the material was recorded as the setting time. Bioactivity (Assessment of apatite forming ability) The bioactivity of the sealer was assessed according to ISO 23317 (Implants for sur- gery - In vitro evaluation for apatite-forming ability of implant materials) using the FESEM/EDX analysis to confirm the apatite deposits on the sealers’ surface after immersion in (PBS) for 28 days. Phosphate-buffered saline is a buffer solution (pH ~ 7.4) commonly used in biological research. One of the characteristics of a bioactive material is its ability to form an apa- tite-like layer on its surface when it comes in contact with physiological fluids in vivo or with simulated body fluids such as (PBS) in vitro16,17. Three samples for each of the original ZOE (control positive group) and selective con- centrations of experimental sealer (10%, 12%, 16%, and 20%) were prepared mea- suring (2.0 ± 0.1) mm in thickness and (20 ± 0.1) mm in diameter, and used for the assessment of apatite forming ability. Then all the prepared samples were placed in an incubator at 37 °C and 95% relative humidity for 1 week to ensure the com- plete setting of sealer. Then in such a way, the sealer samples were soaked in (10) ml PBS-containing container, which was refreshed every 3 days and then incubated at 37 °C and 95% relative humidity for 28 days. After incubation, the apatite precipitate on the surface of the sealer specimens was confirmed using an X-ray diffractometer, with 2θ = (10˚-80˚) and radiation source with Cu Kα (λ = 1.5406 ˚A) at 30 kV and 30 mA. The surface morphology of the specimens was analyzed using FESEM adjusted at 20 kV accelerating voltage and 10 mA and the Ca/P ratio was assessed using EDX analysis. Results Chemical-Setting Reaction Using Fourier Transform Infra Reds (FTIR) The FTIR was used to detect the functional groups changed before and after the addi- tion to identify the chemical bonds and to produce the sample profile19,20. The FTIR Spectra of Tyrosine Amino Acid The functional groups of Tyr a.a were illustrated in Figure 1 as: At (3399.52) which represented the (OH) phenolic part of Tyr. At (3198.52) is represented the amine part (NH2). At (1266.47-1213.22) are represented the phenolic, C–O “aromatic” 5 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 At (1014.91-876.95) are represented the carboxylic acid part (COOH) The FTIR spectra of nano-hydroxyapatite The two major functional groups of nHAP are illustrated in Figure 1: At (1024.84) which represented the (P=O) functional part of nHAP. At (599.99) which represented (C–Ca) functional part of nHAP. The FTIR spectra of the original ZOE sealer The functional groups of ZOE are illustrated in Figure 1: At (636.89) is represented (C–H) bend of the eugenol group. At (1637.87) is represented (C=O) of the zinc oxide. At (2928.73-2866.63) are represented the (C–H) “aliphatic” At (3076.74) is represented the (C–H) “aromatic”. The FTIR of experimental sealer (10% and 20 %) nHA+Tyr The FTIR spectrum confirmed the occurrence of chemical reactions among the com- ponents of ZOE sealer and nHAP and Tyr. a.a. and this was confirmed as the func- tional groups of (amine and phenol) of Tyr. disappeared at (3500-3000). However, the following representative functional groups were identified, in Figure 1: At (2926.65-2866.27) are represented the aliphatic (C–H), from Tyr. At (1719.77) is represented the carbonyl group (C=O), from Tyr. At (1359.95-1260.93) are represented the (P=O), from nHAP. At (1030.33-982.68) are represented the aliphatic phosphates (P-O-C) from nHAP. At (1226.81- 1180.87) are represented the aromatic phosphates (P-O-C) from nHAP. The possibility of the formation of the (COO–Ca) complex that results from the acid- base reaction between the calcium oxide (base) from nHAP and Tyr a.a. (acid) in the presence of water, in which the carboxylic groups of Tyr act as a proton donator (H+) that replaced by the Ca++ ions to form (COO–Ca) complex with its characteristic peaks at (1610-1550/1420-1300). The FTIR spectral pattern of 10% and 20% experimental sealer confirmed the dis- appearance of some functional groups that are present in the original ZOE sealer, these are (1655.54, 1578.89, 1560.60, 1450.10, 1430.43, 1388.31, 993.87, 795.97, 722.36) and (2928.73, 2866.63, 1655.54, 1578.89, 1450.10, 1430.43, 1388.31, 722.36, 993.87) respectively 6 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 Figure 1. Show FTIR spectra for tyrosine amino acid (Tyr), nano hydroxyapatite (nHAP), original ZOE, 10% ZOE, and 20% ZOE. The X-Ray Diffraction Analysis (XRD) The XRD was done for nHAP alone, Tyr. a.a alone, original ZOE, and ZOE after addition at (10 and 20) % as shown in Figure 2. Figure 2. Show the XRD spectra pattern for tyrosine amino acid (Tyr), nano hydroxyapatite (nHAP), original ZOE, 10% ZOE, and 20% ZOE. The XRD of Tyrosine amino acid Tyrosine amino acid has significant characteristics peaks at the following 2 theta values (15.18°, 18.00°, 20.30°, 25.70°), these peaks disappeared or decreased in XRD spectra results in data of the experimental ZOE sealer (after the addition of 10 and 20 % of nHA + Tyr), Figure 2. 7 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 The characteristic peaks of Tyr were in good agreement with the ICDD data (00-031-1970) that represented the high purity of Tyr that was used in this study21,22. The XRD of nHA The XRD pattern represents the significant characteristics peaks at the following 2 theta values (25.93°, 33.10°, 34.12°, 34.63°) that were in good agreement with the ICDD data (01-082-1429) indicating that the nHAP was in hexagonal phase20. However, these specific peaks were decreased after mixing (10 and 20)% of (Tyr and nHAP), indicating the occurrence of a chemical reaction, Figure 2. The XRD of the original ZOE The original ZOE has significant characteristics peaks at the following 2 theta values (24.10°, 26.04°, 31.96°, 32.96°, 36.43°, 35.49°, 36.50°,43.15°, 47.13°, 56.73°, 62.98°, 68.05°) according to JCPDS card No. 79-22053 3a, and ICDD data (01-079-0205)23,24. These peaks either increased or decreased after mixing (10 and 20) % of (Tyr and nHAP), indicating the occurrence of setting chemical reactions among the composi- tions of the original ZOE root canal sealer with the nHAP and Tyr, Figure 2. The Setting Time Table 1 demonstrates the descriptive statistics of setting a time that includes the mean and standard deviation (SD) of the control (original ZOE) and the experimental groups, at different concentrations (10, 12, 16, and 20) %. However, with more than 20 % (Tyr + nHAP) addition; the original ZOE no further underwent setting. Table 1. Descriptive statistics (mean, standard deviation) for the experimental groups of four different concentrations and original ZOE. Sealer types/ Concentration N Control (ZOE) M ± SD Experimental (ZOE) M ± SD Control 10 0.310±0.000 10% 10 0.5902±0.0016 12% 10 1.0200±0.01581 16% 10 1.0340±0.01140 20% 10 1.0700±0.01000 Total 0.8048±0.30972 N = Number of specimens, M = Mean, SD = Standard deviation ZOE = zinc oxide eugenol. One-way analysis of variance “ANOVA test”, (Table 2) was performed to compare the four experimental concentrations. The results revealed that there was a significant difference at (P≤0.01) between four concentrations of setting time. 8 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 Table 2. Show One Way ANOVA tests among The Experimental Groups. ANOVA Sum of Squares df Mean Square Fc (Sig.) Between Groups 2.300 4 0.575 5.990 0.000Within Groups 0.002 20 .000 Total 2.302 24 Post Hoc Tests (Duncan’s Multiple Range tests) were performed to evaluate the sys- tem that gives the best value of experimental concentrations (Table 3) Table 3. Post Hoc Duncan ‘s Multiple Range tests of experimental groups at P<0.01. Duncan’s Group N Subset for alpha = .05 Control 10% 12% 16% 20% Control 10 0.3100 0.5902 1.0200 1.0340 1.0700 10% 10 12% 10 16% 10 20% 10 Bioactivity Analysis After immersion of the experimental sealer in (PBS) for about 28 days, HAP precipita- tion on the surface of the experimental groups was shown visibly. However, the HAP precipitation was confirmed using FESEM/EDX analyses. The EDX spectra for the control (original) and experimental ZOE sealer groups were represented in Figure 3.A. The spectra do not reveal the presence of phosphorus and calcium elements in the control ZOE group. While the EDX for experimental groups at (10, 12, 16, and 20) % revealed the presence of phosphorus and calcium elements, Figure 3 (B-E). The Ca/P ratio of the natural HAP is (1.67), the ratio for the experimental groups was calculated from EDX analysis and it indicates the formation of HAP. It was (1.60), (1.68), (1.77), and (1.83) for (10, 12, 16, 20) % respectively. 9 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 A B C D E Figure 3. EDX data of the control group after immersion in PBS for 28 days (A). Compositional changes in the experimental group after immersion in PBS for 28 days showed the Ca/P ratio at 10% (B), at 12% (C), at 16% (D), and at 20% concentrations respectively (E). The FESEM images showed that there was no HAP precipitate on the outer surface of the sample in the control group, Figure 4 (A). The FESEM images of the experimental sealer groups revealed an irregular and amor- phous HAP precipitate on the outer surfaces figure 4 (B-E) in comparison with the control group (original ZOE) which showed no HAP precipitation. However, the precip- itation has appeared to be increased with increasing the percentage of addition with the higher at 20 %. 10 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 Figure 4. Show FESEM image of experimental ZOE group after 28 days of immersion in PBS, at different degree of magnification (2 µm, 1 µm and 200 nm) that registered HAP precipitate on the outer surface of the sample. At 200nm degree of magnification, the deposits represented the HAP precipitation which is usually taken plate-like morphology due to the presence of the amino acid, (A: Original ZOE, B:at 10 %, C: At 12%, D: At 16% and E:At 20%) Discussion Endodontic root canal sealer is used to achieve a fluid-tight or hermetic seal through- out the canal including the canal irregularities, the apical foramen, and minor discrep- ancies between the dentinal wall of the root and the core filling material. That is why, sealers help to prevent leakage, decrease the possibility of bacterial entrance from the canal to invade the periapical tissues, and resolve the lesion25. Biomimetic dentistry is the newest branch of minimally invasive dentistry that aids in mimicking the natural tooth structure utilizing bioactive materials that resemble the materials that teeth are composed of one of the most important materials that 11 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 biomimetic dentistry concerns is HAP. HAP is a type of mineral that is naturally a constituent of teeth and bones and can be used to repair and rebuild tooth structure26. The HAP is used in a variety of fields with many advantages including biocompatibil- ity, bioactivity, and the absence of cytotoxicity effect, enhances the osteoconductive properties since it permits the attachment, proliferation, and migration of bone cells, thus actively promoting the growth and regeneration of new bone26,27. The presence of amino acid in the sealer formula has been supposed to form a chem- ical bond with calcium and phosphate ions of dentin hydroxyapatite through both carboxyl and amino functional groups in addition to the chemical bond that might occur between amino functional groups of amino acid and the collagen fibers of den- tin. Therefore, this will aid in the improvement of the bond between the sealer and tooth dentin26,28. For the interaction of Tyr a.a. with uncharged polar side groups, It has been suggested that their adsorption on the surface of HAP according to the number of the adsorp- tion sites. HAP surface has centers of positively and negatively charged ions and therefore the solute molecules may be accommodated on the surface of the crystals. It appears that these molecules adsorb onto specific sites of the surface where adsorption could take place through hydrogen bonding. Such interaction is expected in the case of Tyr and nHAP which all have a free hydroxyl side group. It has been proposed that the carboxyl and amino groups of the amino acid have a minor contri- bution to the adsorption process on the surface of HAP12. The finding of current study outcomes showed that the incorporation of (20 % nHAP-Tyr a.a.) to the original ZOE sealer produced high clinical results i:e better apatite formation on the surface of the original material among all other experimen- tal concentrations, these results because of the use of bioactive remineralization can offer several advantages over traditional treatments in conservative dentistry such as: 1)Preservation of healthy tooth structure: the utilization bioactive materials can promote the remineralization of the defective tooth structure. 2) Decrease the need for invasive procedures such as drilling, filling, or crowns.3) Decrease sensitiv- ity: through obstruction of the defect present in tooth tissue.4) Improved esthetics: Biomaterials remineralization can enhance the quality and appearance of the miner- alized tooth tissues18,22,29,30. Also, the methodology of the current study included that the incorporation of syn- thetic nano Hydroxyapatite particles (nHAp) - tyrosine amino acid at different concen- trations to the original composition of zinc oxide eugenol-containing sealer will have a positive effect on the original ZOE sealer. However, the ZOE sealer is used now in many private dental clinics although many sealers developed. However, it is not con- sidered as a bioactive sealer. Therefore, the idea of the research was developed as an trying to induce the bioactivity and improve the biocompatibility of ZOE sealer by the addition of nano-hydroxyapatite - tyrosine amino acid materials. In conclusion and according to the outcomes of FTIR, XRD, FESEM/EDX analyses in the current study, the addition of 20% nHAP-Tyr a.a equally (50% nHA, 50% tyr) to the original ZOE could be induced a reaction that produced the bioactive type of ZOE. 12 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 However, other characteristic analyses like NMR and AFM analyses were needed to further confirm the results. Acknowledgments The authors acknowledge the University of Mosul/College of Dentistry. Special thanks to the University of Mosul/College of Chemistry. Data availability statement The findings of the manuscript were discussed in-depth by all authors, who also con- tributed to its revision and gave their approval to the final edition. Author Contribution Rasha Mozahem Al-Shamaa: Study conception and design, data collection, analy- sis and interpretation of results and author; draft manuscript preparation. Raghad Adnan Rashid: Study conception and design, analysis and interpretation of results and author; draft manuscript preparation. Both authors actively participated in the dis- cussion of the manuscript’s findings, and the Ethical policy and institutional review board statement. References 1. Yanpiset K, Banomyong D, Chotvorrarak K, Srisatjaluk RL. Bacterial leakage and micro- computed tomography evaluation in round-shaped canals obturated with bioceramic cone and sealer using matched single cone technique. Restor Dent Endod. 2018 Jul;43(3):e30. doi: 10.5395/rde.2018.43.e30. 2. Tanomaru-Filho M, Torres FFE, Pinto JC, Santos-Junior AO, Tavares KIMC, Guerreiro-Tanomaru JM. Micro-computed tomographic evaluation of a new system for root canal filling using calcium silicate-based root canal sealers. Restor Dent Endod. 2020 Jun;45(3):e34. doi: 10.5395/rde.2020.45.e34. 3. Al-Haddad A, Che Ab Aziz ZA. Bioceramic-Based Root Canal Sealers: A Review. Int J Biomater. 2016;2016:9753210. doi: 10.1155/2016/9753210. 4. Jeong JW, DeGraft-Johnson A, Dorn SO, Di Fiore PM. dentinal tubule penetration of a calcium silicate-based root canal sealer with different obturation methods. J Endod. 2017 Apr;43(4):633-7. doi: 10.1016/j.joen.2016.11.023. 5. Alaenazi MS, Al-Qahtani SS, Algarn HA, AL- Mutairi SF. Contemporary endodontic sealers. J Healt Med Nurs. 2018;46:42-52. 6. Bezerra CP, Campos CF, Leite J, Fernandes MS, Coury Saraceni CH, Rodrigues FP, Dutra-Correa M. On the understanding of zinc-oxide eugenol cement use prior to etch-rinse bonding strategies. Indian J Dent Res. 2019 May-Jun;30(3):424-7. doi: 10.4103/ijdr.IJDR_302_16. 7. Prasada K, Bukhari SMUH. Biomaterials in restorative dentistry and endodontics. Int J Cur Adv Res. 2018 Feb;7(2G):10065-70. doi: 10.24327/ijcar.2018.10070.1690. 8. 8.Hegde MN, Attavar S, Sreenath N, Bioactive materials–a review. Int J Adv Sci Technol Res. 2017;6(7):1-7. doi: 10.26808/rs.st.i7v6.01. 13 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 9. 9.Calabrese L, Fabiano F, Currò M, Borsellino C, Bonaccorsi LM, Fabiano V, et al. hydroxyapatite whiskers based resin composite versus commercial dental composites: mechanical and biocompatibility characterization. Adv Mater Sci Eng. 2016 Feb;21(7):1-9. doi: 10.1155/2016/2172365. 10. Alagarsamy K, Vishwakarma V, Kaliaraj GS, Viswanathan K, Chavali M. Implant application of bioactive nano-hydroxyapatite powders- a comparative study. Mater Resh Express. 2018;5(1):123-9. doi: 10.1088/2053-1591/aaa6e7. 11. Shayegan A, Atash R, Petein M, Abbeele AV. Nanohydroxyapatite used as a pulpotomy and direct pulp capping agent in primary pig teeth. J Dent Child (Chic). 2010 May-Aug;77(2):77-83. 12. Omidi S, Javidi M, Zarei M, Mushakhian S, Jafarian A. Subcutaneous connective tissue reaction to a new nano zinc-oxide eugenol sealer in rat model. Iran Endod J. 2017 Winter;12(1):64-9. doi: 10.22037/iej.2017.13. 13. Li Z, Ren Q, Cui J, Hu D, Tian T, He T, et al. Comparing the efficacy of hydroxyapatite nucleation regulated by amino acids, poly-amino acids and an amelogenin-derived peptide. Cryst Eng Comm. 2020;22(22): 3814-23. 14. Pullishery F, Alhejoury HA, Turkistani M, Souror YR. Is zinc oxide eugenol cement still impeding the use of resin-based restoration? A systematic review. Dent Med Res. 2021;9(2):59-67. 15. Tsuchiya H. anesthetic agents of plant origin: a review of phytochemicals with anesthetic activity. Molecules. 2017 Aug;22(8):1369. doi: 10.3390/molecules22081369.  16. Uyaver, S. Tyrosine, phenylalanine, and tryptophan undergo self-aggregation in similar and different manners. Atmosphere. 2022;13(9):1448. doi: 10.3390/atmos13091448. 17. Donnermeyer D, Bürklein S, Dammaschke T, Schäfer E. Endodontic sealers based on calcium silicates: a systematic review. Odontology. 2019 Oct;107(4):421-36. doi: 10.1007/s10266-018-0400-3. Epub 2018 Dec 15. 18. Wang Y, Chang Y, Yin L, Xue Y, Li Z, Xue C. A novel technological process of extracting l-tyrosine with low fluorine content from defatted antarctic krill (Euphausia superba) by-product by enzymatic hydrolysis. Food Bioprocess Technol. 2016;9:621-7. doi: 10.1007/s11947-015-1658-x. 19. Lim M, Jung C, Shin DH, Cho YB, Song M. Calcium silicate-based root canal sealers: a literature review. Restor Dent Endod. 2020 Jun 9;45(3):e35. doi: 10.5395/rde.2020.45.e35. 20. Abu Zeid ST, Saleh AAM, Khafagi MGED, Abou Neel EA. Setting reaction of new bioceramic root canal sealers. Spectrosc Lett. 2018;51(8):426-30. doi: 10.1080/00387010.2018.1485703. 21. Kumar A, Biswas K, Basu B. On the toughness enhancement in hydroxyapatite-based composites. Acta Mater. 2013;61(14):5198-215. doi: 10.1016/j.actamat.2013.05.013. 22. Kottapalli PK, Madu GP, Ambati NR, Bolla D, Rayala VS, Narra R. Clinical and radiographic evaluation of mixture of zinc oxide powder and nanohydroxyapatite as an obturating material in primary molars. Braz Dent Sci. 2019;22(1):63-9. doi: 10.14295/bds.2019.v22i1.1651. 23. Yuvakkumar R, Suresh J, Hong I. Green synthesis of zinc oxide nanoparticles. Adv Mater Res. 2014;952:137-40. 24. Javidi M, Zarei M, Naghavi N, Mortazavi M, Nejat AH. Zinc oxide nano-particles as sealer in endodontics and its sealing ability. Contemp Clin Dent. 2014 Jan;5(1):20-4. doi: 10.4103/0976-237X.128656. 25. Zhou HM, Shen Y, Zheng W, Li L, Zheng YF, Haapasalo M. Physical properties of 5 root canal sealers. J Endod. 2013 Oct;39(10):1281-6. doi: 10.1016/j.joen.2013.06.012. 26. Almulhim KS, Syed MR, Alqahtani N, Alamoudi M, Khan M, Ahmed SZ, et al. Bioactive inorganic materials for dental applications: a narrative review. Materials (Basel). 2022 Oct;15(19):6864. doi: 10.3390/ma15196864. https://www.researchgate.net/journal/Materials-Research-Express-2053-1591 http://dx.doi.org/10.1088/2053-1591/aaa6e7 14 Al-Shamaa et al. Braz J Oral Sci. 2024;23:e243733 27. De Carvalho B, Rompen E, Lecloux G, Schupbach P, Dory E, Art JF, et al. Effect of sintering on in vivo biological performance of chemically deproteinized bovine hydroxyapatite. Materials (Basel). 2019 Nov;12(23):3946. doi: 10.3390/ma12233946. 28. Fiume E, Magnaterra G, Rahdar A Verné E, Baino F. Hydroxyapatite for biomedical applications: a short overview. Ceramics. 2021;4(4):542-63. doi: 10.3390/ceramics4040039. 29. Moskovitz M, Tickotsky N, Dassa M, Fux-Noy A, Shmueli A, Halperson E, et al. Zinc oxide zinc sulfate versus zinc oxide eugenol as pulp chamber filling materials in primary molar pulpotomies. Children (Basel). 2021 Sep;8(9):776. doi: 10.3390/children8090776. 30. Al-Shamaa RM, Chakmakchi M, Thiab KA. An in vitro comparative study to evaluate the apical seal of root canals prepared by rotary versus reciprocating wave-one nickel titanium system. Al-Rafidain Dent J. 2014;14(2):288-93. doi: 10.33899/rden.2014.160895. https://doi.org/10.3390/ceramics4040039