Content area
Abstract
Pulp exposure cases are often found in daily dental practice. This cases are commonly caused by caries, trauma or other iatrogenic factors. Direct pulp capping using calcium hydroxide is one of the treatment option for these cases. Calcium hydroxide is currently the gold standard material for pulp capping because of its anti-bacterial properties, high pH content, and it's ability to stimulates the formation of reparative dentin. However, calcium hydroxide also has some disadvantages such as necrosis liquefaction and formation of tunnel defect on dentinal bridge formation. Calcium Phosphate in nano extract of anchovy (Stolephorus insularis) is expected to make calcium hydroxide that is applied as a pulp capping material more effective.
The purpose of this research is to examine the combination of calcium hydroxide with Stolephorus insularis nano extract effect on HSP 70 and NF-κB expression in exposed pulp.
This study used 54 rats, that were grouped into three groups. A cavity with perforated pulp chamber was made on the maxillay first molars: For the control group, calcium hydroxide and aquadest combination was given, the test group was given calcium hydroxide combined with Stolephorus insularis nano extract 1:1, and calcium hydroxide combined with Stolephorus insularis nano extract 1:2. All cavities were filled with RMGIC. On days 1, 3, and 7, experimental animals from each treatment group were terminated by peritoneal injection, and then HSP 70 and NF-κB expression were seen using immunohistochemical staining.
The Tukey HSD Multiple Comparison test, which was used to analyse the data, revealed a significant difference between the test group and the control group, but The expression of NF-κB and HSP 70 did not significantly differ between the first and second test groups.
Application of calcium hydroxide combined with anchovy (Stolephorus insularis) nano extract can reduce HSP 70 and NF-κB expression in exposed pulp.
Experimental article (J Int Dent Med Res 2025; 18(1): 7-13)
Keywords: Calcium hydroxide, Stolephorus insularis, HSP 70, NF-κB, immunology.
Received date: 14 November 2024 Accept date: 12 December 2024
Introduction
Dental caries is an infectious disease that still has a very high prevalence in Indonesia. The national dental caries prevalence rate based on Riskesdas 2018 is 88.8% and the adult dental caries index is 7.11. In dental caries cases that are located near the dental pulp, the application of a specific material is needed, this procedure is known as pulp capping. There are two types of pulp capping, direct and indirect pulp capping. Indirect pulp capping is pulp coating which is carried out in the cavity at a depth close to the pulp, but has not been yet exposed the pulp. Meanwhile, for vital pulp that is exposed either by iatrogenic factors, caries or trauma, direct pulp capping is carried out2. Direct pulp capping is a dental treatment that is indicated for exposed vital pulp. Direct pulp capping is done with application of dental material over the exposed cavity to induce the formation of barrier and also maintain the vitality of the pulp3.
Calcium hydroxide has been marked as the gold standard of pulp capping treatment procedures due to it's ability to break down into calcium, hydroxyl ions, and it's high pH content, antibacterial characteristic, also the odontoblasts pulp cells stimulation ability for reparative dentin forming2. However, the success rate of this material usage for pulp capping decreases over time4.
Currently, the government is promoting the use of natural materials as an alternative in health services. One example of a natural material that is an alternative to medicinal materials is the anchovy (Stolephorus insularis). Anchovies are a high quality food. All of the anchovies body parts can be consumed. Anchovy bones contain high protein and calcium percentage. Every 100 grams of fresh anchovies have 77 kcal of energy; protein 16 gr; fat 1.0 gr; calcium 500 mg; phosphorus 500 mg; iron 1.0 mg; Vitamin A 47; and Vitamin B 0.1 mg. The nutritional content of anchovies, both fresh and dried, is higher than other fish5.
Nanoparticies are particles that are less than 100 nm in size which are used as biomaterials in the fields of medicine and dentistry. Nanoparticles are designed to enhance the solubility of poorly soluble active substances, improve bioavailability, refine drug delivery systems for targeted localisation, increase the stability of active compounds = against environmental degradation (including enzymatic decomposition, oxidation, and hydrolysis), facilitate the absorption of macromolecular entities, and mitigate the irritating effects of active substances on the gastrointestinal tract6.
Heat shock protein 70 (HSP 70) is an endogenous danger signal carrier in the body which is an intracellular molecule but is released into the extracellular environment when there is necrosis or damage to cells and tissues. HSP 70 interacts strongly with the immune system in the extracellular environment and acts as a ligand for TLR2 and TLR47. Extracellular HSP 70 acts as a DAMP (Damage-Associated Molecular Pattern) to mediate inflammatory, in a pathological conditions, immune and metabolic responses by stimulate the activation of Nuclear Factor Kappa B (NF-κB), and pro-inflammatory cytokines secretion such as TNF- α, IL-1α, IL-6, IL-1b, and IL-8 by macrophages8.
Pulp inflammation can occur due to stimulation or injury to the pulp, one important indicator of inflammation is NF-κB. NF-κB is a protein in the cytoplasm that is activated by pro-inflammatory cytokines. One of the mediators of the inflammatory response is cytokines. Cytokines are intimately linked to inflamed pulp. In reaction to environmental stimuli, cytokines work as intracellular messengers that control inflammation and immune response activity both locally and systemically. A transcription factor, Nuclear factor kappa B (NF-κB) has the ability to trigger gene transcription factors for a number of pro-inflammatory cytokines that are linked to signal activation in cells caused by infections or damage. The primary targets of pulp inflammation are pro-inflammatory cytokines, which are produced by the transcription factor NF-κb9.
Materials and methods
Research Samples
The study had a total sample size of 54 male Rattus norvegicus, aged 12 to 16 weeks, with a body weight ranging from 250 to 300 grammes. The samples were randomly allocated into 3 groups, each including 18 male rats. The concentration of Stolephorus insularis nano extract used in this research was 0.1 mg (ratio 1:1) and 0.2 mg (ratio 1:2).
Research Methods
Calcium hydroxide and distilled water were administered in a 1:1 ratio to the positive control group (Group 1), while the Stolephorus insularis nano extract was administered to the subsequent group mixed with distilled water at 1:1:1 ratio (Group 2), the last group was given calcium hydroxide and Stolephorus insularis nano ekstract mixed with distilled water at 1:2:2 ratio (Group 3). The cavity was prepared, and the substance was positioned on the pulp surface. After that, the mice were put back in their cages.
On the first day, 18 mice were sacrificed in order to examine the activation of NF-κB and HSP 70. The remaining 18 mice were sacrificed on the 3rd day, followed by the last 18 on the 7th day,. Six mice were assigned to each treatment group each day. To perform the histopathological procedure, the jaw bones from the right maxillary first molar's interdental region were removed. Histological preparations were made parallel to the tooth's long axis and had a thickness of ± 4µm. Two slides were made by each specimen for observation.
To concentrate on protein and antibody binding, primary antibodies (monoclonal mouse) (Santa Cruzz B) were used for immunohistochemical (IHC) observations on HSP 70 and NF-θB activation. After reacting with the chromogen substrate, the enzyme was examined under Olympus BX51 light microscope, which has a 1000x magnification and a 20x field of view calculation.
Statistical Methods
The mean and standard deviation were used to display the data from this study. The Kolmogorov-Smirnov Lilliefors test was performed to assess the homogeneity and data distribution, followed by the homogeneity Levene test. To assess the differences, the Tukey HSD test was employed, with a significance threshold of 0.05, and the ANOVA test was utilised to determine if there were significant differences in the treatment group.
Results
The IHC result of HSP 70 on days 1, 3, and 7 is shown in Figures 1-3. The arrows indicate that HSP 70 expression was present on macrophage cells in all groups: A. Group 1; B. Group 2; and C. Group 3. Graph 1 displays the average HSP 70 expression for each treatment group, with Group 1 having the highest mean value, 9,89; Group 2 had 3,89; and Group 3 had 2,44.
The IHC detection of NF-κB on days 1, 3, and 7 is illustrated in Figures 4-6. The arrows indicate NF-κB expression in macrophage cells across each group. Graph 2 displays the expression of NF-κB in eachtreatment group. Group 1 has the greatest mean value of NF-κB expression. The average of Group 1, Group 2, and Group 3 sequentially was 9.5; 6.6, and 2.56.
The data were homogenous and normally distributed based on the normality and homogeneity tests on NF-κB expression and HSP 70. The effect of HSP 70 and NF-κB expression on the treatment groups was also verified using the ANOVA test.
The F value between groups in this study showed a significant value of 0.000 (p < 0.05), according to the ANOVA result on the expression of NF-κB and HSP 70 (Tables 1 and 2). This indicates that a significant effect was suggested by the combination of the treatment groups on the expression of NF-κB and HSP 70.
Significant differences between each group were observed on days 1, 3, and 7 based on the expression of HSP 70 (p < 0.05). On days 1, 3, and 7, no significant difference was found between Groups 2 and 3 (p > 0.05) (Table 3).
Significant differences between each group were discovered on days 1, 3, and 7 based on the expression of NF-κB (p < 0.05). On days 1, 3, and 7, a significant differences were found between Groups 2 and 3 (p > 0.05) (Table 4).
Discussion
In this study, a mechanical injury was given to the test animals (Rattus novergicus)'s maxillary first molar until it reached the dental pulp, and direct pulp capping was applied using calcium hydroxide mixed with distilled water as a control, Stolephorus insularis in a ratio of 1:1 and 1:2. The pulp responds to tissue injury by inflammation. Odontoblasts are the cells located most peripherally in the pulp10. With pulp capping, it is hoped that bioactive molecules such as Transforming Growth Factors (TGFs) and Bone Morphogenic Proteins (BMPs) released by the organic dentin matrix due to the action of the pulp capping material will cause odontoblast differentiation and regulation10. In the exposed pulp it is hoped that reparative tertiary dentin will form.
This study used calcium hydroxide combined with nano extract Stolephorus insularis as a direct pulp capping material on Wistar rat molars to determine HSP 70 and NF-κB expression in exposed pulp. Observations of HSP 70 and NF-κB were carried out on days 1, 3, and 7 because a study in rat indicated an increase in HSP 70 levels in the odontoblast process within 3 days after dentin cavity preparation (tooth preparation). In research on tooth separation in rat, it was found that HSP 70 expression increased after 24 hours and decreased gradually over a period of 1 week11.
NF-κB increased within 2 hours after trauma during the CPIP procedure which continued to be expressed on the first day, 7, 14, and even 21 days12. Research on HSP 70 levels in acute pancreatitis (AP) patients measured on days 0, 3 and 7, found that HSP 70 levels increased on days 3 and 77. By knowing the increase in HSP 70 and NF-κB levels, the conclusion was drawn to examine HSP 70 and NF-κB on days 1, 3 and 7, to see the differences in levels that occurred on the three days after calcium hydroxide with nano extract Stolephorus insularis combonation. HSP 70 and NF-κB Expression in the Ca(OH)2 with Aquadest Group (Group 1)
In Figure 4 and Figure 8, it can see the average value of HSP 70 and NF-κB expression for the group combining Ca(OH)2 with distilled water, which is the highest value compared to the other groups. In these two control groups, the HSP 70 and NF-κB expressionincreased on observation days 1, 3 and 7. The mean value of the control group Ca(OH)2 combined with distilled water was higher than the mean of the treatment group. Due to the alkaline pH of Ca(OH)2, a necrotic area is formed which is called the cauterization zone, as a consequence, inflammation occurs in the surrounding tissue after Ca(OH)2 application. There is no antiinflammatory effect of Ca(OH)2, so it cannot suppress NF- кВ translocation to the nucleus which ultimately causes the degree of inflammation to continue to increase13. HSP 70 Expression in Combination of Ca(OH)2 with Stolephorus insularis Nano Extract in Ratio 1:1 (Group 2) and Ratio 1:2 (Group 3)
The second group showed a mean value of HSP 70 expression which tended to increase on days 1, 3 and 7, spesifically 2.33, 2.33 and 7.00. Meanwhile, the mean value of HSP 70 expression in the third group tended to be stable on observation days 1, 3 and 7 with values of 2.50, 2.33 and 2.50. From table 3 above, there is a significant difference in HSP 70 expression, shown by a p value <0.05 in the 1st group and the 2nd group (p=0.000), as well as in the first group and the third group (p=0.000) on day 1, 3, and 7 of observation. Meanwhile, there was a nonsignificant difference in HSP 70 expression as indicated by a p value > 0.05 in the 2nd group and the 3rd group on days 1 (p=0.974), 3 (p=1.000), and 7 (0.882). The mean HSP 70 expression in the 3rd group was lower than the 2nd group on observation day 1, 3, and 7. NF-κB Expression in Combination of Ca(OH)2 with Stolephorus insularis Nano Extract in Ratio 1:1 (Group 2) and Ratio 1:2 (Group 3)
The second group showed a mean NF-κB expression value of 5.00 for day 1; 6.50 for day 3; and 6.50 for day 7. Meanwhile the mean value of NF-κB expression in the third group was 2.83 for day 1; 2.17 for day 3; and 2.67 for day 7.
The Tukey HSD statistical test was used to determine the different treatment groups. Table 4 shows that the expression of NF-κB has changed significantly. indicated by a p value <0.05 in the comparison of the first and the second group (p=0.014) and with the third group (p=0.000) on day 1, and on the first group with the second (p=0.002) and with the third group (p=0.000) on the 3rd day of observation, and the first group with the second group (p=0.002) and with the third group (p=0.000) on the 3rd day of observation 7. Meanwhile, there was also a significant difference in NF-κB expression, shown by a p value <0.05 in the second and the third group on days 1 (p=0.032), 3 (p=0.001), and 7 (0.005). The mean expression of NF-κB in the third group was lower than the second group on observation days 1, 3 and 7.
The hypothesis of this research is that there will be a decrease in HSP 70 and NF-κB expression after administering a Ca(OH)2 with Stolephorus insularis nano extract combination. A significant difference was found from the control group (Ca(OH)2 and distilled water) and the combination group of Ca(OH)2 and Stolephorus insularis nano extract 1:1, and also from the Ca(OH)2 and Stolephorus insularis nano extract 1:2 combination. This hypothesis is in line with the research results, where a significant decrease in the expression of HSP 70 and NF-κB after administering Ca(OH)2 with Stolephorus insularis nano extract 1:1 combination and Ca(OH)2 with Stolephorus insularis nano extract 1:2 combination. This is because the protein found in anchovies includes a number of necessary amino acids, including lysine, leucine, and isoleucine, as well as nonessential amino acids that are involved in cell regeneration, such glutamic acid and methionine. Protein is hard to digest because of its large molecular size. Particle size reduction will enhance the particles' surface area, which will improve the active compound's absorption and bioavailability14.
Nanotechnology applied in direct pulp capping treatment optimizes the distribution of active molecules so that it is important to inhibit infection and inflammation as well as trigger colonization and differentiation of pulp cells15. The calcium and phosphate ions found in anchovies have an important role in the mineralization of the reparative dentin matrix. Stolephorus insularis extract contains 76% calcium phosphate compounds in the form of hydroxyapatite16. The important function of hydroxyapatite is able to preserve the vitality of perforated pulp by playing a role in the formation of reparative dentin.
In another study, It was shown that a group of foetuses whose mothers received intraperitoneal Adriamycin prior to pregnancy were administered L-arginine for two weeks of gestation, a NO substrate, which decreased the expression of HSP 70. Comparatively speaking to control foetuses, its expression was decreased. It proves that NO can lessen cellular stress in this group17. In the case of this study, Arginine can reduce oxidative stress caused by Adriamycin and avoid unwanted apoptosis. HSP 70 serum levels were considerably lowered in hypoxic rats when carnosine and/or arginine were administered. According to this study, carnosine and arginine can prevent the adverse effects by lowering the endogenous cell leakage of HSP 70 into the circulation. This implies that these substances have a positive cytoprotective effect against renal tissue damage caused by hypoxia. Carnosine and arginine have been shown to control intra cellular HSP-70 expression, that is crucial to prevent organ failure18.
Conclusions
Application of calcium hydroxide combined with anchovy (Stolephorus insularis) nano extract can reduce HSP 70 and NF-κB expression in exposed pulp.
References
1. Kementerian Kesehatan Republik Indonesia. Laporan Riskesdas 2018. Badan Penelitian dan Pengembangan Kesehatan, Kementerian Kasehatan Republik Indonesia. Jakarta. 2019.
2. Alex G. Direct and Indirect Pulp Capping: A Brief History, Material Innovations, and Clinical Case Report. Compendium. 2018;39(3):182-189.
3. Versiani M. A., Basrani B. & Sousa-Neto M. D. The Root Canal Anatomy in Permanent Dentition. Springer International Publishing AG. 2019. https://doi.org/10.1007/978-3-319-734446_2.
4. Cushley, S., Duncan, H. F., Lappin, M. J., Chua, P., Elamin, A. D., Clarke, M., El-Karim, 1. A. Efficacy of Direct Pulp Capping for Management of Cariously Exposed Pulps in Permanent Teeth: A Systematic Review and Meta-Analysis. International Endodontic Journal. 2020;54(4):556-571. DOI: https://doi.org/10.1111/iej.13449.
5. Aryati, E., Wulan, A.S.D.. Manfaat Kar Teri Segar (Stolephorus Sp.) Terhadap Pertumbuhan Tulang dan Gigi. Odonto Dental Journal. 2014;1(2):52-56.
6. Abdassah, M. Nanopartikel Dengan Gelasi lonik. Farmaka. 2017;15(1): 45-52. DOI: https://doi.org/10.24198/jf.v15i1.12138 https://jurnal.unpad.ac.id/farmaka/article/view/12138/pdf
7. Pizano, L. A., Gutiérrez, C. R., González, С. M., Sanchez, J. A., Vazquez, J. L., Valverde, R., Pérez, J. L., Jiménez, M., Ceballos, A., Álvarez, M., Avila, J., & Gómez, A. High Serum Levels of High-Mobility Group Box 1 (HMGB1) and Low Levels of Heat Shock Protein 70 (HSP70) are Associated with Poor Prognosis in Patients with Acute Pancreatitis. Archives of Medical Research. 2018;49:504-511. DOI:https://doi.org/10.1016/j.arcmed.2018.08.004.
8. Nugraha, A. P. Wijaya, D.I. Amalia, S. Syahputra, R. High Mobility Group Box 1 and Heat Shock Protein 70 Expression Post (-)-Epigallocathechin-3-Gallate in East Java Green tea Methanolic Extract Administration During Orthodontic Tooth Movement in Wistar Rats. Pesquisa Brasileira en Odontopediatria e Clinica Integrada, 2020;20(1):1-12. DOI: https://doi.org/10.1590/pboci.2020.024.
9. Pribadi, N., Budiarti, D., Kurniawan, H.J, Widjiastuti, I. The NFkB and Collagen Type 1 Expression in Dental Pulp after Treated Calcium Hydroxide Combined with Propolis. Eur J Dent. 2021;15(1):122-126. DOI: 10.1055/s-0040-1716319.
10. Luo, X., Feng, W., Huang, S. et al. Odontoblasts release exosomes to regulate the odontoblastic differentiation of dental pulp stem cells. Stem Cell Res Ther. 2023;14:176. DOI: https://doi.org/10.1186/s13287-023-03401-9.
11. Nakano, K., Mori, T., Tominari, T., & Sato, Y. HSP 70 Expression in the Mouse Dental Pulp after Immediate Teeth Separation. Journal of Hard Tissue Biology. 2013;22(1):7-12.
12. Nahm, S. S., Han, W. K., Gil, H. Y., Coi, E. Increased cerebral nuclear factor kappa B in a complex regional pain syndrome rat model: possible relationship between peripheral injury and the brain. Journal of Pain Research. 2019;12:909-914. DOI: https://doi.org/10.2147/JPR.S 166270.
13. Nirwana, 1., Agustantina, T. H. & Soekartono, K. H. NF-κB Expressions in Rat Dental Pulp Mechanically Exposured after Pomegranate Fruit Extract Administration. Journal of International Dental and Medical Research, 2017;10(1):123127.
14. Sapiun, Z., Imran, A. K., Wicita, P. S., Kamba, V., Slamet, N. S. Preparation and Characterization of Anchovy (Stolephorus Sp) Protein Concentrate Nanoparticle Using lonic Gelation Method. International Journal of Applied Pharmaceutics. 2020;12(2): 109-111. DOI: http://dx.doi.org/10.22 159/ijap. 2020.12i2.36570.
15. Keller, L., Offner, D., Schwinté, P., Morand, D., Wagner, Q. Active Nanomaterials to Meet the Challenge of Dental Pulp Regeneration. Multidisciplinary Digital Publishing Institute. 2015;8(11):7461-7471. DOI: 10.3390/ma8115387.
16. Kumala, Y. R., Rachmawati, D., Hersanto, K. Stimulasi Dentin Reparatif Direct Pulp Capping Menggunakan Ekstrak Ikan Teri (Stolephorus sp). FKG Universitas Brawijaya. Malang. EProdenta Journal of Dentistry. 2017;1(2):45-53. DOI: https://doi.org/10.21776/ub.eprodenta.2017.001.02.1.
17. Pedrycz, A. Brzeski, Z. L-Arginine Decreases Heat Shock Protein 70 (Marker of Environmental Stress) Expression in Kidney Cells of Rat Fetuses During Apoptosis - Late Effect of Adriamycin Action. Ann Agric Environ Med. 2006;13(1): 129132. PMID: 16841884.
18. Rasheed, N. M, Fadda, L., Mohamed, A. M., Attia, H. A. Regulating Effect Of Carnosine And /Or L- Arginine On The Expression Of Inflammatory Molecules Induced Nephropathy In The Hypoxic Rat Model. Journal Brazilian Archives of Biology and Technology. 2016; 59(1):1-15. DOI: http://dx.doi.org/10.1590/1678-4324-2016150622. https://www.scielo.br/j/babt/a/NJqRGCXVNXqvMYNd9CZzNyLM/ ?lang=en&format=pdf
Copyright Ectodermal Dysplasia Group - Turkey 2025