Levan Bazlı Hidrojellerin Dental Pulpa Kaynaklı Mezenkimal Kök Hücreler Üzerinde Canlılık Testlerinin Değerlendirilmesi
Year 2025,
Volume: 9 Issue: 3, 173 - 178, 31.12.2025
Ezgi Altuntaş
,
Sabriye Senem Kılıç
,
Selay Tornacı
,
Pınar Yılmaz Atalı
,
Safa Tuncer
,
Tunç Akkoç
,
Ebru Öner
,
Cafer Türkmen
Abstract
Amaç: Bu in vitro çalışmada, anti-enflamatuar özelliğe sahip olan levan bazlı hidrojellerin ve pulpa kuafajı tedavilerinde yaygın olarak kullanılan iki materyal olan mineral trioksit agregat (MTA) ve trikalsiyum silikat bazlı Biodentin (Septodont)'in, dental pulpa kaynaklı mezenkimal kök hücrelerin (DPSCs) canlılıkları üzerindeki etkilerini değerlendirmeyi amaçlamaktadır.
Gereç ve Yöntemler: Levansükraz enzimi kullanılarak enzimatik levan (EL) sentezlenmiş, ardından EL’ye 1:1 oranında nanohidroksiapatit (nHAP) eklenerek ikinci bir hidrojel formülasyonu elde edilmiştir. MTA, Biodentin ve hidrojeller kompleks Dublecco’ s Modified Eagle Medium (DMEM) solüsyonu içerisinde 3 gün inkübe edilerek özütleri hazırlanmıştır. Dental pulpa kaynaklı mezenkimal kök hücreler (DPSC’ler), her bir kuyucuğa 100.000 hücre olacak şekilde 96 kuyucuklu plaklara ekilmiştir. Deney gruplarına ait steril ajanlarla hazırlanan özütler hücrelere uygulanmış ve hücre canlılığı 24., 48. ve 120. saatlerde değerlendirilmiştir. Hücre canlılığı ve mitokondriyal aktivite MTT analizi ile ölçülmüş; elde edilen veriler One-way ANOVA testi ve Tukey testi ile analiz edilmiştir (p<0.05).
Bulgular: 48. saatte, levan bazlı hidrojeller hücre canlılığında istatistiksel olarak anlamlı bir artış göstermiştir (başlangıç ortalaması: 164,2; 48. saat ortalaması: 123,4); ancak 120. saate gelindiğinde belirgin bir düşüş gözlenmiştir (başlangıç ortalaması: 73,4; 120. saat ortalaması: 22,0). Buna karşılık, MTA grubunda 120 saatlik süre boyunca hücre canlılığında sürekli bir artış gözlenmiş ve ortalama 219,8’e ulaşmıştır.
Sonuç: Bu çalışmanın limitasyonları dâhilinde elde edilen bulgular, levan bazlı hidrojellerin kısa vadede hücre canlılığı üzerinde olumlu etkiler gösterdiğini ortaya koymaktadır; ancak mineral trioksit agregatın daha uzun süreli ve kalıcı bir etki sağlayabileceği düşünülmektedir.
References
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Akkulah CY, Erginer M, Cumbul A, Kirtel O, Bayram F, Oner ET. Enhanced effects of levan hydrogels and bovine grafts on guided bone regeneration: In-vitro and in-vivo analysis. International Journal of Biological Macromolecules. 2025;292, 139129.
-
Avsar G, Agirbasli D, Agirbasli MA, Gunduz O, Oner ET. Levan based fibrous scaffolds electrospun via co-axial and single-needle techniques for tissue engineering applications. Carbohydrate polymers. 2018;193, 316-325.
-
Bai X, Gao M, Syed S, Zhuang J, Xu X, Zhang XQ. Bioactive hydrogels for bone regeneration. Bioactive materials. 2018;3(4), 401-417.
-
Combie J. Öner ET. From healing wounds to resorbable electronics, levan can fill bioadhesive roles in scores of markets. Bioinspiration & biomimetics. 2018;14(1), 011001.
-
Derakhshi M, Naseri M, Vafaeipour Z, Malaekeh-Nikouei B, Jafarian AH, Ansari L. Enhanced wound-healing efficacy of electrospun mesoporous hydroxyapatite nanoparticle-loaded chitosan nanofiber developed using pluronic F127. International Journal of Biological Macromolecules. 2023;240, 124427.
-
Holiel AA, Mahmoud EM, Abdel-Fattah WM, Kawana KY. Histological evaluation of the regenerative potential of a novel treated dentin matrix hydrogel in direct pulp capping. Clinical oral investigations. 2021;25, 2101-2112.
-
Hu L, Zhao B, Gao Z, Xu J, Fan Z, Zhang C, Wang J, Wang S. Regeneration characteristics of different dental derived stem cell sheets. Journal of oral rehabilitation.2020; 47, 66-72.
-
Hu N, Li W, Jiang W, Wen J, Gu S. Creating a Microenvironment to Give Wings to Dental Pulp Regeneration—Bioactive Scaffolds. Pharmaceutics.2023;15(1), 158.
-
Hwang Y, Lee JS, An H, Oh H, Sung D, Tae G, Choi WI. Hydroxyapatite-embedded levan composite hydrogel as an injectable dermal filler for considerable enhancement of biological efficacy. Journal of Industrial and Engineering Chemistry. 2021;104, 491-499.
-
Karobari MI, Basheer SN, Sayed FR, Shaikh S, Agwan MAS, Marya A, Messina P, Scardina GA. An in vitro stereomicroscopic evaluation of bioactivity between Neo MTA Plus, Pro Root MTA, BIODENTINE & glass ionomer cement using dye penetration method. Materials. 2021;14(12), 3159.
-
Kim J, Song YS, Min KS, Kim SH, Koh JT, Lee BN, Chang HS, Hwang IN, Oh WM, Hwang YC. Evaluation of reparative dentin formation of ProRoot MTA, Biodentine and BioAggregate using micro-CT and immunohistochemistry. Restorative dentistry & endodontics.2016;41(1), 29.
-
Kirtel O, Menéndez C, Versluys M, Van den Ende W, Hernández L, Toksoy Öner E. Levansucrase from Halomonas smyrnensis AAD6 T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized. Applied microbiology and biotechnology.2018;102, 9207-9220.
Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay. Cold spring harbor protocols. 2018;(6), pdb. prot095505.
-
Li J, Rao Z, Zhao Y, Xu Y, Chen L, Shen Z, Bai Y. Lin Z, Huang Q. A decellularized matrix hydrogel derived from human dental pulp promotes dental pulp stem cell proliferation, migration, and induced multidirectional differentiation in vitro. Journal of Endodontics. 2018;46(10), 1438-1447. e1435.
-
Lin CC, Metters AT. Hydrogels in controlled release formulations: network design and mathematical modeling. Advanced drug delivery reviews. 2006;58(12-13), 1379-1408.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods.1983; 65(1-2), 55-63.
-
Moussa DG, Aparicio C. Present and future of tissue engineering scaffolds for dentin‐pulp complex regeneration. Journal of tissue engineering and regenerative medicine.2019;13(1), 58-75.
-
Njeh A, Uzunoğlu E, Ardila-Osorio H, Simon S, Berdal A, Kellermann O, Goldberg M. Reactionary and reparative dentin formation after pulp capping: Hydrogel vs. Dycal. Evidence-Based Endodontics.2016; 1, 1-9.
-
Öner ET, Hernández L, Combie J. Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnology advances.2016; 34(5), 827-844.
Roveri N, Foresti E, Lelli M, Lesci IG. Recent advancements in preventing teeth health hazard: the daily use of hydroxyapatite instead of fluoride. Recent Patents on Biomedical Engineering (Discontinued).2009;2(3), 197-215.
-
Sarilmiser HK, Oner ET. Investigation of anti-cancer activity of linear and aldehyde-activated levan from Halomonas smyrnensis AAD6T. Biochemical engineering journal.2014; 92, 28-34.
-
Selvi SS, Hasköylü ME, Genç S, Toksoy Öner E. Synthesis and characterization of levan hydrogels and their use for resveratrol release. Journal of Bioactive and Compatible Polymers.2021;36(6), 464-480.
-
Sonmez Kaplan S, Sazak Ovecoglu H, Genc D, Akkoc T. TNF-α, IL-1B and IL-6 affect the differentiation ability of dental pulp stem cells. BMC Oral Health. 2023;23(1), 555.
-
Ueda H, Hong L, Yamamoto M, Shigeno K, Inoue M, Toba T, Yoshitani M, Nakamura T, Tabata Y, Shimizu Y. Use of collagen sponge incorporating transforming growth factor-β1 to promote bone repair in skull defects in rabbits. Biomaterials.2002; 23(4), 1003-1010.
-
Yuan Z, Nie H, Wang S, Lee CH, Li A, Fu SY, Zhou H, Chen L, Mao JJ. Biomaterial selection for tooth regeneration. Tissue Engineering Part B: Reviews.2011; 17(5), 373-388.
-
Zhao H, He W, Wang Y, Zhang X, Li Z, Yan S, Zhou W, Wang G.Biomineralization of large hydroxyapatite particles using ovalbumin as biosurfactant. Materials Letters.2008; 62(20), 3603-3605.
Evaluation of Viability Tests of Levan-Based Hydrogels on Dental Pulp-Derived Mesenchymal Stem Cells
Year 2025,
Volume: 9 Issue: 3, 173 - 178, 31.12.2025
Ezgi Altuntaş
,
Sabriye Senem Kılıç
,
Selay Tornacı
,
Pınar Yılmaz Atalı
,
Safa Tuncer
,
Tunç Akkoç
,
Ebru Öner
,
Cafer Türkmen
Abstract
Objectives: This in vitro study aimed to evaluate the effects of levan-based hydrogels recognized for their anti-inflammatory properties on the viability of dental pulp-derived mesenchymal stem cells (DPSCs), and to compare them with two commonly used pulp capping materials: mineral trioxide aggregate (MTA) and Biodentine.
Materials and Methods: Enzymatic levan (EL) hydrogel was synthesized using the levansucrase enzyme. A second formulation, EL/ nanohydroxyapatite (nHAP), was prepared by incorporating nHAP in a 1:1 ratio. MTA, Biodentine, and both hydrogel formulations were incubated in Dulbecco's Modified Eagle Medium (DMEM) for 3 days to generate extracts. DPSCs were seeded in 96-well plates at a density of 100,000 cells per plate. The extracts were applied to the cells, and viability was assessed at 24, 48, and 120 hours using the MTT assay. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (p<0.05).
Results: Levan-based hydrogels showed enhanced cell viability at 48 hours (mean: 123.4), suggesting short-term biocompatibility. However, by 120 hours, cell viability significantly declined (mean: 22.0). In contrast, MTA exhibited a gradual and sustained increase in viability over the same period, reaching a mean of 219.8 at 120 hours. Biodentine presented intermediate effects between the hydrogels and MTA.
Conclusions: Levan-based hydrogels demonstrated promising short-term biocompatibility with DPSCs. However, MTA may provide a more stable environment conducive to long-term cell viability. These findings support further investigation into the optimization of levan-based formulations for pulp capping applications.
References
-
Akkulah CY, Erginer M, Cumbul A, Kirtel O, Bayram F, Oner ET. Enhanced effects of levan hydrogels and bovine grafts on guided bone regeneration: In-vitro and in-vivo analysis. International Journal of Biological Macromolecules. 2025;292, 139129.
-
Avsar G, Agirbasli D, Agirbasli MA, Gunduz O, Oner ET. Levan based fibrous scaffolds electrospun via co-axial and single-needle techniques for tissue engineering applications. Carbohydrate polymers. 2018;193, 316-325.
-
Bai X, Gao M, Syed S, Zhuang J, Xu X, Zhang XQ. Bioactive hydrogels for bone regeneration. Bioactive materials. 2018;3(4), 401-417.
-
Combie J. Öner ET. From healing wounds to resorbable electronics, levan can fill bioadhesive roles in scores of markets. Bioinspiration & biomimetics. 2018;14(1), 011001.
-
Derakhshi M, Naseri M, Vafaeipour Z, Malaekeh-Nikouei B, Jafarian AH, Ansari L. Enhanced wound-healing efficacy of electrospun mesoporous hydroxyapatite nanoparticle-loaded chitosan nanofiber developed using pluronic F127. International Journal of Biological Macromolecules. 2023;240, 124427.
-
Holiel AA, Mahmoud EM, Abdel-Fattah WM, Kawana KY. Histological evaluation of the regenerative potential of a novel treated dentin matrix hydrogel in direct pulp capping. Clinical oral investigations. 2021;25, 2101-2112.
-
Hu L, Zhao B, Gao Z, Xu J, Fan Z, Zhang C, Wang J, Wang S. Regeneration characteristics of different dental derived stem cell sheets. Journal of oral rehabilitation.2020; 47, 66-72.
-
Hu N, Li W, Jiang W, Wen J, Gu S. Creating a Microenvironment to Give Wings to Dental Pulp Regeneration—Bioactive Scaffolds. Pharmaceutics.2023;15(1), 158.
-
Hwang Y, Lee JS, An H, Oh H, Sung D, Tae G, Choi WI. Hydroxyapatite-embedded levan composite hydrogel as an injectable dermal filler for considerable enhancement of biological efficacy. Journal of Industrial and Engineering Chemistry. 2021;104, 491-499.
-
Karobari MI, Basheer SN, Sayed FR, Shaikh S, Agwan MAS, Marya A, Messina P, Scardina GA. An in vitro stereomicroscopic evaluation of bioactivity between Neo MTA Plus, Pro Root MTA, BIODENTINE & glass ionomer cement using dye penetration method. Materials. 2021;14(12), 3159.
-
Kim J, Song YS, Min KS, Kim SH, Koh JT, Lee BN, Chang HS, Hwang IN, Oh WM, Hwang YC. Evaluation of reparative dentin formation of ProRoot MTA, Biodentine and BioAggregate using micro-CT and immunohistochemistry. Restorative dentistry & endodontics.2016;41(1), 29.
-
Kirtel O, Menéndez C, Versluys M, Van den Ende W, Hernández L, Toksoy Öner E. Levansucrase from Halomonas smyrnensis AAD6 T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized. Applied microbiology and biotechnology.2018;102, 9207-9220.
Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay. Cold spring harbor protocols. 2018;(6), pdb. prot095505.
-
Li J, Rao Z, Zhao Y, Xu Y, Chen L, Shen Z, Bai Y. Lin Z, Huang Q. A decellularized matrix hydrogel derived from human dental pulp promotes dental pulp stem cell proliferation, migration, and induced multidirectional differentiation in vitro. Journal of Endodontics. 2018;46(10), 1438-1447. e1435.
-
Lin CC, Metters AT. Hydrogels in controlled release formulations: network design and mathematical modeling. Advanced drug delivery reviews. 2006;58(12-13), 1379-1408.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods.1983; 65(1-2), 55-63.
-
Moussa DG, Aparicio C. Present and future of tissue engineering scaffolds for dentin‐pulp complex regeneration. Journal of tissue engineering and regenerative medicine.2019;13(1), 58-75.
-
Njeh A, Uzunoğlu E, Ardila-Osorio H, Simon S, Berdal A, Kellermann O, Goldberg M. Reactionary and reparative dentin formation after pulp capping: Hydrogel vs. Dycal. Evidence-Based Endodontics.2016; 1, 1-9.
-
Öner ET, Hernández L, Combie J. Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnology advances.2016; 34(5), 827-844.
Roveri N, Foresti E, Lelli M, Lesci IG. Recent advancements in preventing teeth health hazard: the daily use of hydroxyapatite instead of fluoride. Recent Patents on Biomedical Engineering (Discontinued).2009;2(3), 197-215.
-
Sarilmiser HK, Oner ET. Investigation of anti-cancer activity of linear and aldehyde-activated levan from Halomonas smyrnensis AAD6T. Biochemical engineering journal.2014; 92, 28-34.
-
Selvi SS, Hasköylü ME, Genç S, Toksoy Öner E. Synthesis and characterization of levan hydrogels and their use for resveratrol release. Journal of Bioactive and Compatible Polymers.2021;36(6), 464-480.
-
Sonmez Kaplan S, Sazak Ovecoglu H, Genc D, Akkoc T. TNF-α, IL-1B and IL-6 affect the differentiation ability of dental pulp stem cells. BMC Oral Health. 2023;23(1), 555.
-
Ueda H, Hong L, Yamamoto M, Shigeno K, Inoue M, Toba T, Yoshitani M, Nakamura T, Tabata Y, Shimizu Y. Use of collagen sponge incorporating transforming growth factor-β1 to promote bone repair in skull defects in rabbits. Biomaterials.2002; 23(4), 1003-1010.
-
Yuan Z, Nie H, Wang S, Lee CH, Li A, Fu SY, Zhou H, Chen L, Mao JJ. Biomaterial selection for tooth regeneration. Tissue Engineering Part B: Reviews.2011; 17(5), 373-388.
-
Zhao H, He W, Wang Y, Zhang X, Li Z, Yan S, Zhou W, Wang G.Biomineralization of large hydroxyapatite particles using ovalbumin as biosurfactant. Materials Letters.2008; 62(20), 3603-3605.