Exploring the staining potential of GSK-3 inhibitors in bovine teeth: a one-year laboratory investigation

GSK-3 inhibitors, such as Tideglusib (TG) and CHIR-99021 (CHIR), show promise in stimulating reparative dentin formation. The aim of this study was to assess the discoloration potential of TG and CHIR in an established in vitro model.

Enamel-dentin specimens made from bovine incisors were randomly allocated to five groups (n=15 each): group bovine blood (BB), group dimethyl sulfoxide (DMSO), group TG, group CHIR, and group mineral trioxide aggregate (MTA). Each specimen had a central cavity in which the respective material was applied and sealed with resin-based luting material. Color determination was conducted using a dental spectrophotometer at t0 (before filling), t1 (immediately after filling), t2 (after one week), t3 (after one month), t4 (after three months), t5 (after six months), and t6 (after one year). Statistical analysis involved descriptive statistics, Kruskal-Wallis tests, and analysis of variance (alpha=0.05).

Group BB and group CHIR exhibited the most significant decrease in lightness after one year, whereas groups DMSO, TG, and MTA showed minimal changes. Group BB and CHIR exhibited the highest DeltaE values.

Unlike CHIR, TG did not result in discoloration exceeding the threshold of visual perception, defined by a DentalE value of 5.5, during the one-year observation period. This laboratory study therefore suggests that TG could be utilized for indirect or direct pulp capping without major discoloration concerns. However, additional research is required to corroborate these findings.

Accepted for publication: August 7, 2023

Published online: August 8, 2023

The final edited and typeset version will appear in the future.


Dental injuries and deep carious lesions can lead to pulp exposure. When signs and symptoms are absent, the primary objective of treating such lesions is to preserve the vitality and health of the pulp tissue (Ricucci et al. 2014; Bjørndal et al. 2019). Various operative treatment approaches are available for maintaining pulp vitality, including indirect and direct pulp capping, partial pulpotomy, and complete pulpotomy. Preserving pulp vitality offers numerous benefits. The dentin-pulp complex can continue fulfilling its developmental, defensive, and proprioceptive functions. In addition, teeth with a vital pulp have a more favorable long-term prognosis regarding tooth survival compared with those that have undergone root canal treatment (Caplan et al. 2005; Leong & Yap 2021).

The gold standard materials for indirect and direct pulp capping comprise calcium hydroxide pastes and hydraulic silicate cements, including but not limited to mineral trioxide aggregate (MTA), Biodentine, Endocem MTA, EndoSequence, and others (Duncan et al. 2019; Dammaschke et al. 2019; Parirokh et al. 2018). Hydraulic silicate cements exhibit high mechanical resistance and can set in both dry and moist environments (Camilleri & Pitt Ford 2006; Parirokh & Torabinejad 2010; Torabinejad & Parirokh 2010; Dammaschke et al. 2019). On the other hand, calcium hydroxide pastes are less mechanically stable and susceptible to resorption and porosities over time at the site of pulp capping (Dammaschke et al. 2019). Both hydraulic silicate cements and calcium hydroxide pastes release calcium hydroxide ions during the setting process, which contribute to their high antibacterial properties but may also cause local tissue damage owing to chemical irritation (Spångberg 1969; Meadow et al. 1984; Briseño & Willershausen 1992).

It has been observed that localized cell death resulting from this mechanism leads to the release of inflammatory mediators, which subsequently activate tissue regeneration pathways and promote the formation of reparative dentin (Babb et al.  2017). This process involves the mobilization and proliferation of mesenchymal stem cells from the pulp tissue, which can differentiate into new odontoblast-like cells and initiate the secretion of reparative dentin (Babb et al. 2017).

Based on a molecular understanding of the cellular signaling pathways involved in regenerative/reparative dentin formation, researchers have investigated pharmacological treatments that directly activate these pathways using specific molecules, inducing regulated reparative processes and promoting cellular differentiation (Nakashima & Reddi 2003; Thesleff & Tummers 2008; Galler et al. 2014). Collagen sponges loaded with these molecules have been utilized to administer the treatment by placing them on the exposed pulp. Following application, controlled degradation of these collagen sponges allows for their integration within the structure of reparative dentin (Neves et al. 2017; Zaugg et al. 2020).

The novel treatment approach under investigation involves the use of glycogen synthase kinase 3 (GSK-3) inhibitors as pharmacological agents. GSK-3, a protein kinase, modulates the Wnt signaling cellular pathway (Vishwakarma et al. 2015). Laboratorystudies have demonstrated that GSK-3 inhibitors stimulate the proliferation and viability of human dental pulp stem cells(Hanna et al. 2023; Kornsuthisopon et al.  2023) and elicit the formation of reparative dentin in animal models following pulp exposure (Neves et al. 2017; Zaugg et al. 2020). The Wnt signaling cellular pathway assumes a crucial role in reparative dentin development in cases of pulp exposure. Remarkably, the activity of the Wnt signaling pathway can also be activated in dentin damage without pulp exposure, as observed in indirect pulp capping, resulting in the formation of reactionary dentin and thickening of the dentin wall beneath the "near exposure" (Neves & Sharpe 2018). These findings suggest that novel medicaments utilizing GSK-3 inhibitors hold promise for both direct and indirect pulp capping in the future.

Tideglusib (TG), a non-competitive GSK-3 inhibitor, has been the subject of clinical trials for Alzheimer therapy (Eldar-Finkelman & Martinez 2011; Wang et al. 2021). Notably, no adverse reaction profile was described for TG (Eldar-Finkelman & Martinez 2011). Another potent ATP-competitive GSK-3 inhibitor, CHIR-99021 (CHIR), an aminopyrimidine, has demonstrated its ability to activate the Wnt signaling pathway, leading to enhanced self-renewal and pluripotency in mouse stem cells (Eldar-Finkelman & Martinez 2011; Wu et al. 2013). Both CHIR and TG have exhibited promising outcomes in preclinical research regarding reparative dentin formation, prompting considerations for their potential clinical application in dentistry (Neves et al.  2017; Neves & Sharpe 2018; Zaugg et al.  2020; Alaohali et al.  2022).

One drawback associated with early market-stage MTA products was their propensity for significant tooth discoloration (Naik & Hegde 2005; Lenherr et al. 2012; Krastl et al. 2013; Dettwiler et al. 2016; Abuelniel et al. 2020). Recent investigations attribute this discoloration to the presence of the radio-opaque marker bismuth oxide, prompting the modification of formulations using alternative, non-discoloring radio-opaque markers (Kang et al. 2015; Keskin et al. 2015; Xuereb et al. 2016; Marciano et al. 2019). These findings underscore the importance of laboratory research in evaluating dental materials to identify potential adverse effects, including their propensity for discoloration.

In recent years, there has been a surge in studies exploring the molecular mechanisms and potential therapeutic applications of GSK-3 inhibitors. However, as of today, there is a lack of published evidence regarding the discoloration potential of these inhibitors. Thus, the objective of this study was to evaluate the discoloration potential of GSK-3 inhibitors in a well-established in vitro setup.

Material and methods

Sample size calculation

Based on data reported in a previous study, an a priori sample size calculation was performed (Lenherr et al.  2012). A change of luminosity mean values (Lmean) of 92 to 90 with a standard deviation of 2.2 was assumed as the effect size between the control group and the test groups. By setting the type I (two-sided) and type II error rates at 5% and 20% respectively, establishing a significance level of 5%, and aiming for a test power of 80%, a minimum of 10 samples per group was determined as necessary. To ensure a prudent safety margin, accounting for the possibility of subtle variations in discoloration outcomes, a total of 15 specimens per group were included.

Specimen preparation

A total of 75 specimens were produced using a methodology previously described in detail by Lenherr et al. (2012). The source material consisted of bovine incisors obtained from calves slaughtered in a commercial slaughterhouse and stored in tap water at room temperature. The crown and root of each tooth were separated at the cemento-enamel junction by using a diamond-coated cutting disk, which was continuously cooled with water. From the middle third of each crown, rectangular enamel-dentin slabs measuring 10 mm in length, 10 mm in width, and 3 mm in thickness were excised. Subsequently, an oral surface cavity with dimensions of 2.5 mm in diameter and 2.0 mm in depth was created in each enamel-dentin slab using a water-cooled cylindrical diamond bur. To ensure uniformity, the length, width, and thickness dimensions of all specimens were verified using a digital caliper.

Disinfection procedure

The specimens were treated by immersing them in 1% sodium hypochlorite (NaOCl; Toppharm Apotheke Hersberger, Basel, Switzerland) for a period of 30 minutes. Subsequently, they were thoroughly rinsed with tap water, dried using compressed air, and submerged in 20% ethylenediaminetetraacetic acid (EDTA, Toppharm Apotheke Hersberger, Basel, Switzerland) for 2 minutes to eliminate the smear layer. After rinsing with copious amounts of tap water and drying with air, the specimens were immersed in 1% NaOCl for an additional 3 minutes. Finally, the specimens were thoroughly washed with tap water and placed in 0.9% saline (Grosse Apotheke Dr. G. Bichsel, Interlaken, Switzerland) for storage in an area with indirect sunlight.

Experimental groups

The specimens were randomly allocated to five groups using computer-generated sequences created with free online software (www.randomizer.org). Each group consisted of 15 specimens (Tab. I), and the same amount of liquid (1µL) and collagen sponges (Kolspon; Eucare, Chennai, India) were used. The sponges were cut according to the dimensions 2 mm x 2 mm x 2 mm and placed in the cylindrical cavity prior to injecting the liquid. Group BB was designated as positive control group using bovine blood (Bell Schweiz, Oensingen, Switzerland). In group DMSO, dimethyl sulfoxide (DMSO, Sigma-Aldrich/ Merck, Darmstadt, Germany), a polar aprotic solvent and organosulfur compound, was dispensed onto the sponge previously placed in the cylindrical cavity of the specimens. In groups TG and CHIR, 50nM TG (Sigma-Aldrich/ Merck, Darmstadt, Germany) and 5µM CHIR (Sigma-Aldrich/ Merck, Darmstadt, Germany), respectively, were dissolved in DMSO and subsequently administered into each cavity. In group MTA, the non-discoloring formula of mineral trioxide aggregate (MTA) powder and liquid (PD-MTA, Produits Dentaires SA, Vevey, Switzerland) were thoroughly mixed at a ratio of 3:1 until a homogeneous mixture was achieved. The resulting mixture had a creamy consistency and was promptly applied into the cylindrical cavities without any sponge.

After the application of the material in each group, the cylindrical cavities were sealed with self-adhesive resin-based luting material (RelyX Unicem2 Aplicap, 3M, St. Paul, MN, USA). The luting material was light cured for 20 s at an irradiance of 907 mW/cm2 (SmartLite Focus, Dentsply Sirona, Charlotte, NC, USA). The tip of the curing light, whose emission spectrum ranged from 420 nm to 540 nm, was positioned as close as possible to the resin-based material without touching it during light curing. After light curing, the specimens were placed in individual test tubes (Standard Micro Test Tube 3810, Eppendorf, Hamburg, Germany) containing 0.9% saline (Grosse Apotheke Dr. G. Bichsel, Interlaken, Switzerland). The test tubes were then stored at room temperature, ensuring they were kept away from direct sunlight.

Spectrophotometric color determination

Color determination was performed using a dental spectrophotometer (Vita EasyShade Compact, Vita, Bad Säckingen, Germany) (Lehmann et al. 2011). The measurements were performed exclusively under the illumination provided by the experimental setup's lamp (Lamp Trio 1 [max. 40W, 230V/50Hz] with a light-emitting diode bulb [OSRAM, 40W, Classic B, 230V, E14, SES], both purchased from Migros, Zurich, Switzerland) within a dark room. The spectrophotometric measurements were taken at multiple time points, including baseline (t0), immediately after filling (t1), and subsequently after one week (t2), one month (t3), three months (t4), six months (t5), and one year (t6). Before each group measurement, the spectrophotometer was calibrated following the manufacturer's instructions. The color parameters recorded and measured according to the Lab* color space were as follows: L* (lightness), a* (red-green coordinates), and b* (blue-yellow coordinates).

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ABUELNIEL GM, DUGGAL MS, KABEL N: A comparison of mta and biodentine as medicaments for pulpotomy in traumatized anterior immature permanent teeth: A randomized clinical trial. Dent Traumatol 36:400-410 (2020)

ALAOHALI A, SALZLECHNER C, ZAUGG LK, SUZANO F, MARTINEZ A, GENTLEMAN E, SHARPE PT:  Gsk3 inhibitor-induced dentinogenesis using a hydrogel. J Dent Res 101:46-53 (2022)

ATILA D, CHEN CY, LIN CP, LEE YL, HASIRCI V, TEZCANER A, LIN FH: In vitro evaluation of injectable tideglusib-loaded hyaluronic acid hydrogels incorporated with rg1-loaded chitosan microspheres for vital pulp regeneration. Carbohydr Polym 278:118976 (2022)

BABB R, CHANDRASEKARAN D, CARVALHO MORENO NEVES V, SHARPE PT: Axin2-expressing cells differentiate into reparative odontoblasts via autocrine wnt/beta-catenin signaling in response to tooth damage. Sci Rep 7:3102 (2017)

BJORNDAL L, SIMON S, TOMSON PL, DUNCAN HF: Management of deep caries and the exposed pulp. Int Endod J 52:949-973 (2019)

BRISEÑO BM, WILLERSHAUSEN B: Root canal sealer cytotoxicity with human gingival fibroblasts. Iii. Calcium hydroxide-based sealers. J Endod 18:110-113 (1992)

CAMILLERI J, PITT FORD TR: Mineral trioxide aggregate: A review of the constituents and biological properties of the material. Int Endod J 39:747-754 (2006)

CAPLAN DJ, CAI J, YIN G, WHITE BA: Root canal filled versus non-root canal filled teeth: A retrospective comparison of survival times. J Public Health Dent 65:90-96 (2005)

DAMMASCHKE T, GALLER K, KRASTL G: Aktuelle Empfehlungen zur Vitalerhaltung der Pulpa. Wissenschaftliche Mitteilung der deutschen Gesellschaft für Endodontologie und zahnärztliche Traumatologie. Zahnarztl Mitt 109:40-49  (2019)

DAY PF, DUGGAL MS, HIGH AS, ROBERTSON A, GREGG TA, ASHLEY PF, WELBURY RR, COLE BO, WESTLAND S: Discoloration of teeth after avulsion and replantation: Results from a multicenter randomized controlled trial. J Endod 37:1052-1057 (2011)

DEL SER T, STEINWACHS KC, GERTZ HJ, ANDRES MV, GOMEZ-CARRILLO B, MEDINA M, VERICAT JA, REDONDO P, FLEET D, LEON T: Treatment of Alzheimer's disease with the GSK-3 inhibitor tideglusib: A pilot study. J Alzheimers Dis 33:205-215 (2013)

DETTWILER CA, WALTER M, ZAUGG LK, LENHERR P, WEIGER R, KRASTL G: In vitro assessment of the tooth staining potential of endodontic materials in a bovine tooth model. Dental Traumatol 32:480-487 (2016)

DUNCAN HF, GALLER KM, TOMSON PL, SIMON S, EL-KARIM I, KUNDZINA R, KRASTL G, DAMMASCHKE T, FRANSSON H, MARKVART M: European society of endodontology position statement: Management of deep caries and the exposed pulp. Int Endod J 52:923-934 (2019)

EGGMANN F, RIHS J, LENHERR P, WEIGER R, KRASTL G, ZAUGG LK: Spectrophotometric insights: Calcium hydroxide influences tooth discolorations induced by short-term application of antibiotic/corticosteroid pastes. Clin Oral Investig 25:1141-1149 (2021)

ELDAR-FINKELMAN H, MARTINEZ A: Gsk-3 inhibitors: Preclinical and clinical focus on cns. Front Mol Neurosci 4:32 (2011)

GALLER KM, EIDT A, SCHMALZ G: Cell-free approaches for dental pulp tissue engineering. J Endod 40:41-45 (2014)

HANNA S, ALY R, ELDEEN GN, ADANERO VELASCO A, PÉREZ ALFAYATE R: Small molecule gsk-3 inhibitors safely promote the proliferation and viability of human dental pulp stem cells-in vitro. Biomedicines 11(2) (2023)

KANG SH, SHIN YS, LEE HS, KIM SO, SHIN Y, JUNG IY, SONG JS: Color changes of teeth after treatment with various mineral trioxide aggregate-based materials: An ex vivo study. J Endod 41:737-741 (2015)

KESKIN C, DEMIRYUREK EO, OZYUREK T: Color stabilities of calcium silicate-based materials in contact with different irrigation solutions. J Endod 41:409-411 (2015)

KORNSUTHISOPON C, TOMPKINS KA, OSATHANON T: Tideglusib enhances odontogenic differentiation in human dental pulp stem cells in vitro. Int Endod J 56:369-384 (2023)

KRASTL G, ALLGAYER N, LENHERR P, FILIPPI A, TANEJA P, WEIGER R: Tooth discoloration induced by endodontic materials: A literature review. Dent Traumatol 29:2-7 (2013)

LEHMANN KM, DEVIGUS A, IGIEL C, WENTASCHEK S, AZAR MS, SCHELLER H: Repeatability of color-measuring devices. Eur J Esthet Dent 6:428-435 (2011)

LENHERR P, ALLGAYER N, WEIGER R, FILIPPI A, ATTIN T, KRASTL G: Tooth discoloration induced by endodontic materials: A laboratory study. Int Endod J 45:942-949 (2012)

LEONG DJX, YAP AU: Vital pulp therapy in carious pulp-exposed permanent teeth: An umbrella review. Clin Oral Investig 25:6743-6756 (2021)

MARCIANO MA, CAMILLERI J, LUCATELI RL, COSTA RM, MATSUMOTO MA, DUARTE MAH: Physical, chemical, and biological properties of white mta with additions of alf(3). Clin Oral Investig 23:33-41 (2019)

MEADOW D, LINDNER G, NEEDLEMAN H: Oral trauma in children. Pediatr Dent 6:248-251 (1984)

NAIK S, HEGDE AH: Mineral trioxide aggregate as a pulpotomy agent in primary molars: An in vivo study. J Indian Soc Pedod Prev Dent 23:13-16 (2005)

NAKASHIMA M, REDDI AH: The application of bone morphogenetic proteins to dental tissue engineering. Nat Biotechnol 21:1025-1032 (2003)

NEVES VC, BABB R, CHANDRASEKARAN D, SHARPE PT: Promotion of natural tooth repair by small molecule gsk3 antagonists. Sci Rep 7:39654 (2017)

NEVES VCM, SHARPE PT: Regulation of reactionary dentine formation. J Dent Res 97:416-422 (2018)

PARIROKH M, TORABINEJAD M: Mineral trioxide aggregate: A comprehensive literature review—part i: Chemical, physical, and antibacterial properties. J Endod 36:16-27 (2010)

PARIROKH M, TORABINEJAD M, DUMMER PMH: Mineral trioxide aggregate and other bioactive endodontic cements: an updated overview - part I: vital pulp therapy. Int Endod J 51:177-205 (2018)

RICUCCI D, LOGHIN S, SIQUEIRA JF, JR: Correlation between clinical and histologic pulp diagnoses. J Endod 40:1932-1939 (2014)

SPÅNGBERG L: Biological effects of root canal filling materials. 7. Reaction of bony tissue to implanted root canal filling material in guineapigs. Odontol Tidskr 77:133-159 (1969)

THESLEFF I, TUMMERS M: Tooth organogenesis and regeneration. Stembook, Cambridge (2008)
TORABINEJAD M, PARIROKH M: Mineral trioxide aggregate: A comprehensive literature review—part ii: Leakage and biocompatibility investigations. J Endod 36:190-202 (2010)

VISHWAKARMA A, SHARPE P, SHI S, RAMALINGAM M: Stem cell biology and tissue engineering in dental sciences. Elsevier, Waltham (2015)

WANG L, BHARTI, KUMAR R, PAVLOV PF, WINBLAD B: Small molecule therapeutics for tauopathy in alzheimer's disease: Walking on the path of most resistance. Eur J Med Chem 209:112915 (2021)

WESTLAND S, LUO W, LI Y, PAN Q, JOINER A: Investigation of the perceptual thresholds of tooth whiteness. J Dent 67:11-14 (2017)

WU Y, AI Z, YAO K, CAO L, DU J, SHI X, GUO Z, ZHANG Y: Chir99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression. Exp Cell Res 319:2684-2699 (2013)

XUEREB M, SORRENTINO F, DAMIDOT D, CAMILLERI J: Development of novel tricalcium silicate-based endodontic cements with sintered radiopacifier phase. Clin Oral Investig 20:967-982 (2016)

ZAUGG LK, BANU A, WALTHER AR, CHANDRASEKARAN D, BABB RC, SALZLECHNER C, HEDEGAARD MAB, GENTLEMAN E, SHARPE PT: Translation approach for dentine regeneration using GSK-3 antagonists. J Dent Res 99:544-551 (2020)




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