A comparison of lithium-substituted phosphate and borate bioactive glasses for mineralised tissue repair

ObjectivesWnt/β-catenin signalling plays important roles in regeneration, particularly in hard tissues such as bone and teeth, and can be regulated by small molecule antagonists of glycogen synthase kinase 3 (GSK3); however, small molecules can be difficult to deliver clinically. Lithium (Li) is also a GSK3 antagonist and can be incorporated into bioactive glasses (BG), which can be used clinically in dental and bone repair applications and tuned to quickly release their constituent ions.MethodsHere, we created phosphate (P)- and borate (B)-based BG that also contained Li (LiPBG and LiBBG) and examined their ion release kinetics and the toxicity of their dissolution ions on mouse 17IA4 dental pulp cells.ResultsWe found that although LiPBG and LiBBG can both quickly release Li at concentrations known to regulate Wnt/β-catenin signalling, the P and B ions they concomitantly release are highly toxic to cells. Only when relatively low concentrations of LiPBG and LiBBG were placed in cell culture medium were their dissolution products non-toxic. However, at these concentrations, LiPBG and LiBBG’s ability to regulate Wnt/β-catenin signalling was limited.SignificanceThese data suggest that identifying a BG composition that can both quickly deliver high concentrations of Li and is non-toxic remains a challenge.     

The role of canonical Wnt signaling in dentin bridge formation

ObjectiveWnt signaling has been reported to be involved in dentin bridge formation. However, the detailed mechanism has not yet been clarified. We elucidated the localization of canonical Wnt signaling molecules during dentin bridge formation.MethodsPulp of the maxillary first molar in mice was exposed and directly capped with MTA cement. Maxillae were collected on the 1st, 4th, 7th, 14th, and 28th days after treatment. After μCT analysis, immunohistochemistry for Wnt3a, Wnt10a, β-catenin, F4/80, and osterix was performed in paraffin-embedded sections.ResultsOn the 4th and 7th days after pulp capping, odontoblasts and dental pulp cells expressed Wnt3a, Wnt10a, and β-catenin. On the 14th day, reactionary dentin was formed around the pulp exposure area. Odontoblasts and dental pulp cells express Wnt3a, Wnt10a, and β-catenin. Additionally, F4/80- and Wnt10a-positive macrophages were observed at the center of the dental pulp. When the dentin bridge was formed on the 28th day, reparative odontoblasts expressed Wnt3a, β-catenin and osterix.ConclusionWnt ligands derived from odontoblasts and dental pulp cells are important for the activation of odontoblasts and the differentiation of reparative odontoblasts during dentin bridge formation. Macrophage-derived Wnts are also involved in reparative odontoblast differentiation.     

Wnt/β-catenin信号通路影响牙本质形成及再生的研究进展

Wnt/β-catenin信号通路是Wnt通路中的经典途径,在牙的发生中发挥着重要作用。研究表明,Wnt/β-catenin信号通路在牙本质的形成过程中扮演着重要角色,本文就近年来相关研究对Wnt/β-catenin信号通路在成牙本质细胞分化和牙本质发生及再生中的作用及影响作一阐述。     

Effects of lithium on extraction socket healing in rats assessed with micro-computed tomography

Abstract

Objective. Lithium is an activator of β-catenin signaling and β-catenin plays an important role in regulating bone formation and remodeling. The purpose of this study was to investigate the effects of lithium on bone repair in tooth extraction sockets in rats. Material and methods. Twenty male Wistar rats were subjected to maxillary left second molar extraction. The animals received a daily injection of lithium chloride (LiCl) or the same dose of sodium chloride (NaCl) starting 7 days before tooth extraction until sacrifice 14 days after extraction. Rats were randomly divided into: (1) a pre-treated group that received LiCl injection from 7 days before to 3 days after tooth extraction; (2) a post-treated group that received LiCl injection starting 4 days after tooth extraction; (3) a continuously treated group that received LiCl injection for the entire 21 days; and (4) a control group that received NaCl injection only. The volume of new bone and the bone density in the extraction socket were quantified by micro-computed tomography. Results. The percentage of new bone formation in the extraction socket was as follows: 63.2 ± 13.4% (pre-treated group), 53.9 ± 9.8% (post-treated), 23.8 ± 8.0% (continuously treated) and 37.5 ± 4.2% (control). The difference in percentage was statistically significant between each pair of groups. Pre- and post-treated groups also showed a significant increase in the density of new bone. Conclusions. Lithium enhances bone repair in extraction sockets when delivered before or after tooth extraction. Tooth extraction during lithium treatment may impair bone healing.     

Alteration of odontoblast osteonectin expression following dental cavity preparation.

Cavity preparation can increase the active synthesis and secretion of non-collagenous proteins by odontoblasts, thus resulting in the deposition of tertiary dentine. In this study, the effect of cavity preparation on osteonectin expression was examined in odontoblasts of the rat tooth pulp. A class V cavity was prepared in rat first molars to stimulate odontoblastic secretory activity, and the animals were killed at various intervals. In the normal pulp, osteonectin immunoreactivity was detected in odontoblasts but not other cells. At 1 day after cavity preparation, immunoreactivity had diminished beneath the cavity. At 3 days, strong immunoreactivity could be detected in odontoblasts beneath the cavity. Numerous round cells underlying the odontoblastic layer also demonstrated immunoreactivity. Thereafter, the intensity of osteonectin immunoreactivity in odontoblasts beneath tertiary dentine decreased gradually, and at 30 and 60 days, it was weaker than in normal pulp. These findings suggest that osteonectin is actively synthesized by odontoblasts underlying a cavity in the initial stage of tertiary dentine formation.     

Expression of Wnt3a,Wnt10b, β-catenin and DKK1 in periodontium during orthodontic tooth movement in rats

Objective: To investigate the expression of Wnt3a, Wnt10b, β-catenin and DKK1 in the periodontal ligament (PDL) during orthodontic tooth movement (OTM) in rats. Materials and methods: Nickel-titanium closed-coil springs were used to deliver an initial 50 g mesial force to the left maxillary first molars in 30 rats. The force was kept constant for 1, 3, 5, 7, 10 and 14 days until the animals were sacrificed. The right maxillary molars without force application served as control. Paraffin-embedded sections of the upper jaws were prepared for histological and immunohistochemical analyses to detect Wnt3a, Wnt10b, β-catenin and DKK1 expression in PDL. Results: Wnt3a, Wnt10b, β-catenin and DKK1 were expressed on both the ipsilateral and contralateral sides of PDL in each group. After the application of orthodontic force, the expression of β-catenin and DKK1 was initially increased and then decreased on both sides, with maximal levels of expression at day 7 and day 10, respectively. On the compression side, Wnt3a and Wnt10b levels started to increase at day 5, while on the tension side, these two molecules began to increase at day 1. Furthermore, the expression levels of Wnt3a, Wnt10b, and β-catenin were much stronger on the tension side than on the compression side at any of the observation points, while DKK1 level was much higher on the compression side. Conclusion: Wnt3a, Wnt10b, β-catenin and DKK1 expression may be related to the periodontal tissue remodeling following the application of an orthodontic force in rats. These observations suggest that the Wnt/β-catenin signaling pathway may play a crucial role in periodontal tissue remodeling during OTM.     

Dental stem cell signaling pathway activation in response to hydraulic calcium silicate-based endodontic cements: A systematic review of in vitro studies

ObjectiveTo present a qualitative synthesis of in vitro studies which analyzed human dental stem cell (DSC) molecular signaling pathway activation in response to hydraulic calcium silicate-based cements (HCSCs).MethodsA systematic electronic search was performed in Medline, Scopus, Embase, Web of Science and SciELO databases on January 20 and last updated on March 20, 2020. In vitro studies assessing the implication of signaling pathways in activity related marker (gene/protein) expression and mineralization induced by HCSCs in contact with human DSCs were included.ResultsThe search identified 277 preliminary results. After discarding duplicates, and screening of titles, abstracts, and full texts, 13 articles were considered eligible. All of the materials assessed by the included studies showed positive results in cytocompatibility and/or bioactivity assays. ProRoot MTA and Biodentine were the modal HCSCs studied, hDPSCs were the modal cell variant used, and the most studied signaling pathway was MAPK. In vitro assays measuring the expression of activity-related markers and mineralized nodule formation evidenced the involvement of MAPK (and its subfamilies ERK, JNK and P38), NF-κB, Wnt/β-catenin, BMP/Smad and CAMKII pathways in the biological response of DSCs to HCSCs.SignificanceHCSCs considered in the present review elicited a favorable biological response from a variety of DSCs in vitro, thus supporting their use in biologically-based endodontic procedures. MAPK, NF-κβ, Wnt/β-catenin, BMP/Smad and CAMKII signaling pathways have been proposed as potential mediators in the biological interaction between DSCs and HCSCs. Understanding the signaling processes involved in tissue repair could lead to the development of new biomaterial compositions targeted at enhancing these mechanisms through biologically-based procedures.     

Pulpal blood flow recorded from exposed dentine with a laser Doppler flow meter using red or infrared light

ObjectiveTo determine the percentage of the blood flow signal that is derived from dental pulp when recording from exposed dentine in a human premolar.DesignRecordings were made from 7 healthy teeth in 5 subjects (aged 22–33 yr.) with a laser Doppler flow meter (Periflux 4001) using either a red (635 nm) or an infrared (780 nm) laser. After exposing dentine above the buccal pulpal horn (cavity diam. 1.6 mm, depth 3 mm) and isolating the crown with opaque rubber dam, blood flow was recorded alternately with infrared or red light from the exposed dentine under four conditions: before and after injecting local anaesthetic (3% Mepivacaine without vasoconstrictor) (LA) over the apex of the root of the tooth; after exposing the pulp by cutting a buccal, class V cavity in the tooth; and after sectioning the coronal pulp transversely through the exposure.ResultsThere was no significant change in mean blood flow recorded with either light source when the tooth was anaesthetized or when the pulp was exposed. After the pulp had been sectioned, the blood flow recorded with infrared light fell by 67.8% and with red light, by 68.4%. The difference between these effects was not significant.ConclusionsWhen recording blood flow from exposed coronal dentine with either infrared or red light in a tooth isolated with opaque rubber dam, about 68% to the signal was contributed by the pulp. The signal:noise ratio was better with infrared than red light, and when recording from dentine than enamel.     

Dentine-pulp tissue engineering in miniature swine teeth by set calcium silicate containing bioactive molecules

Objective: The present study aims to investigate whether reparative dentinogenesis could be guided at central pulpal sites or at a distance from the amputated pulp of miniature pig teeth, by using set calcium silicate-based carriers containing human recombinant bioactive molecules.DesignPulp exposures were performed in 72 permanent teeth of 4 healthy miniature swine. The teeth were capped with pre-manufactured implants of set calcium silicate-based material containing BMP-7, TGFβ1 or WnT-1, for 3 weeks. Conical-shaped intrapulpal implants were exposed in the central pulp core, while disc-shaped extrapulpal implants were placed at a distance from the amputated pulp. Implants without bioactive molecules were used as controls. Thickness and forms of new matrix mineralized deposition were assessed histologically at post-operative periods of 3 weeks by light microscopy.ResultsIntrapulpal applications: Calcified structures composed of osteodentine were found in contact with the BMP-7 implants. An inhomogeneous calcified tissue matrix was found around the WnT-1 carriers. A two-zone calcified structure composed of osteodentine and a thicker tubular matrix zone was seen at the TGFβ1 carrier-pulp interface. Extrapulpal applications: The space between WnT-1 implants and pulp periphery had been invaded by soft tissue with traces of calcified foci. Thick calcified structures composed of osteodentine were found surrounding pulp exposure sites in response to application of BMP-7. Spindle-shaped cells associated with atubular calcified matrix or elongated polarized cells associated with tubular dentine-like matrix were found along the cut dentinal walls of the TGFβ1 group.ConclusionThe present experiments indicated that set calcium silicate could be used as carrier for biologically active molecules. TGFβ1 was shown to be an effective bioactive molecule in guiding tertiary dentine formation.     

Stimulation of the rat dentine-pulp complex by bone morphogenetic protein-7 in vitro

Human recombinant bone morphogenetic protein-7 (BMP-7), when applied to freshly cut dentine in monkey teeth, stimulated tertiary dentine formation, but it is unclear whether this response involved upregulation of the synthetic and secretory activity of existing odontoblasts or the induction of differentiation of new odontoblast-like cells. Using a recently developed organ-culture system for whole tooth slices, the aim here was to examine the effects of BMP-7 on the stimulation and modulation of existing odontoblasts in the absence of tissue injury. Agarose beads were soaked in a 500 ng/ml or 100 ng/ml solution of BMP-7 in culture medium and placed on the odontoblast area of the dentine pulp complex of rat tooth slices. The slices were embedded in a semisolid agar-based medium and cultured at the liquid gas interface for 7 days. Results showed that beads soaked in 500 ng/ml BMP-7 stimulated a localized increase in extracellular matrix secretion by odontoblasts at the site of application, with greater stimulatory effects than from the lower concentration. These effects may be important in the reparative processes after tissue injury within the dentine-pulp complex and may be useful in the therapeutic induction of tertiary dentinogenesis.     

Wnt/β-catenin通路与NF-κB通路的交叉调控对间充质干细胞分化的影响

Wnt/β-catenin信号通路与NF-κB信号通路都是高度保守的通路，调节多种生物学过程，二者之间的交叉影响已在多个生物和医学研究领域引起重视。该文将就Wnt/β-catenin信号通路与NF-κB信号通路的相互作用及对间充质干细胞多向分化的影响进行综述。     

The use of mineral trioxide aggregate to achieve root end closure: three case reports

Abstract – The use of mineral trioxide aggregate (MTA) to achieve root end closure has many advantages over the traditional calcium hydroxide (Ca(OH)₂) technique including the reduced number of visits and the reduced mechanical damage to dentine. Limited studies have reported the outcome of using MTA as an apexification material and a one‐stage obturation technique in non‐vital immature teeth. This article illustrates three successful clinical cases where MTA was used as an apexification material. In case study one: Type 1 Dens Invaginatus tooth with incomplete root formation, case study two: an immature tooth that suffered pulp necrosis following an enamel and dentine fracture trauma and case study three: a non‐vital tooth following an apical root fracture.     

Dickkopf-1 may regulate bone coupling by attenuating wnt/β-catenin signaling in chronic apical periodontitis

ObjectiveAlveolar bone loss is a common outcome of chronic apical periodontitis. In this study, we investigated the involvement of the Dickkopf-1-Wnt/β-catenin signaling pathway in the attenuation of osteogenic differentiation induced by Escherichia coli lipopolysaccharide, and we evaluated the use of Dickkopf-1 inhibitor and Dickkopf-1 recombinant protein to reverse bone loss in different phases of osteogenic differentiation.MethodsMC3T3-E1 cells grown in osteogenic medium were treated with Escherichia coli lipopolysaccharide for 24 h during osteogenic induction on days 0, 1, 7, 14 and 21. Dickkopf-1 siRNA was added on days 0 and 1, and Dickkopf-1 recombinant was added on days 7, 14, and 21. Quantitative real-time PCR, Western blotting and alkaline phosphatase activity assays were performed to measure osteogenic marker expression and Wnt/β-catenin signaling. A rat apical periodontitis model was used to further evaluate the function of Dickkopf-1 in relation to bone loss.ResultsMC3T3-E1 cells treated with Escherichia coli lipopolysaccharide showed decreased mRNA expression of osteogenic markers. Wnt/β-catenin signaling was also inhibited, and Dickkopf-1 showed corresponding variations as quantified by Western blotting. Using Dickkopf-1 inhibitor or Dickkopf-1 recombinant protein at different phases of osteogenic differentiation in vitro partially reversed the decrease in osteogenic marker expression. The rat apical periodontitis model indicated that the Dickkopf-1 inhibitor could restore bone loss in the periapical area in vivo.ConclusionsDickkopf-1 may play a key regulatory role in determining the outcome for bone in inflammatory environments, and modulating the Wnt/β-catenin signaling pathway via Dickkopf-1 inhibitor or recombinant protein may provide a potential therapeutic option to prevent bone destruction in endodontic disease.     

Disclosing the physiology of pulp tissue for vital pulp therapy

The discovery that dentine is a reservoir of bioactive molecules that can be recruited on demand has attracted efforts to develop new protocols and materials for vital pulp therapy (VPT). The noncollagenous proteins (NCPs) present in the dentine extracellular matrix (ECM) include growth factors (TGF‐β1, BMP‐7, FGF‐2, IGF‐1 and IGF‐2, NGF and GDNF), extracellular matrix molecules (DSP, DPP, BSP, DMP‐1 and DSPP) and both anti‐inflammatory and pro‐inflammatory chemokines and cytokines (TNF‐α, IL‐1, IL‐6 and IL‐10). Molecules such as DSP and DPP are mainly expressed by odontoblasts, and they are cleaved products from dentine sialophosphoprotein (DSPP). Some molecules, such as TGF‐β1, specifically interact with decorin/biglycan in dentine. Although TGF‐β1 increases the expression and secretion of NGF in human pulp cells, NGF induces mineralization and increases the expression of DSPP and DMP‐1. Furthermore, GDNF may act as a cell survival factor and mitogen during tooth injury and repair. Pulp capping materials, such as MTA and calcium hydroxide, can solubilize bioactive dentine molecules (TGF‐β1, NGF and GDNF) that stimulate tertiary dentinogenesis. The binding of these signalling molecules leads to activation of several signalling transduction pathways involved in dentinogenesis, odontoblast differentiation and inflammatory responses, such as the p38 MAPK, NF‐kβ and Wnt/β‐catenin signalling pathways. Understanding the cascade of cellular and molecular events underlying the repair and regeneration processes provides a reasonable new approach to VPT through a targeted interaction between tooth tissue and bioactive molecules.     

Effect of novel chitosan-fluoroaluminosilicate glass ionomer cement with added transforming growth factor beta-1 on pulp cells

Introduction

Vital pulp therapy might benefit from the sustained release of transforming growth factor beta-1 (TGF-β1) from dental restorative materials. Chitosan has previously been shown to enable sustained release of bovine serum albumin (BSA) from glass ionomer cement (GIC). Because BSA can prolong release of growth factor, chitosan-fluoroaluminosilicate GIC with albumin (BIO-GIC) should sustain the effect of growth factor. This study investigated the effect of BIO-GIC with added TGF-β1 on pulp cells.

Methods

BIO-GIC was prepared from GIC (conventional type) incorporated with 15% of chitosan and 10% of BSA. TGF-β1 (100 ng) was added in BIO-GIC+TGF-β1 and GIC+TGF-β1 groups during each disk specimen (10 mm diameter, 1 mm high) preparation. Two control groups were BIO-GIC and GIC. The effect of each specimen on pulp cells was investigated by using the Transwell plate technique. Cell proliferation was determined by MTT assay at 2 time periods (each period lasting 3 days). Pulp cell differentiation was examined by alkaline phosphatase activity and also by cell mineralization, which was measured by calculating the area of mineralization with von Kossa staining.

Results

Percentage of viable cells of GIC+TGF-β1 group was the highest after the first period. This might suggest an initial rapid release of TGF-β1 from GIC. After the second period, BIO-GIC, BIO-GIC+TGF-β1, and GIC+TGF-β1 had more than 90% cell survival. It was significantly greater than GIC (82% ± 2%). There was no significant difference in alkaline phosphatase activity. BIO-GIC+TGF-β1 had the highest mineralization area during 21 days.

Conclusions

BIO-GIC could retain the effect of TGF-β1.     

Endogenous hydrogen sulfide is involved in osteogenic differentiation in human periodontal ligament cells

ObjectiveEndogenous hydrogen sulfide (H₂S) has recently emerged as an important intracellular gaseous signaling molecule within cellular systems. Endogenous H₂S is synthesized from l-cysteine via cystathionine β-synthase and cystathionine γ-lyase and it regulates multiple signaling pathways in mammalian cells. Indeed, aberrant H₂S levels have been linked to defects in bone formation in experimental mice. The aim of this study was to examine the potential production mechanism and function of endogenous H₂S within primary human periodontal ligament cells (PDLCs).DesignPrimary human PDLCs were obtained from donor molars with volunteer permission. Immunofluorescent labeling determined expression of the H₂S synthetase enzymes. These enzymes were inhibited with D,L-propargylglycine or hydroxylamine to examine the effects of H₂S signaling upon the osteogenic differentiation of PDLCs. Gene and protein expression levels of osteogenic markers in conjunction with ALP staining and activity and alizarin red S staining of calcium deposition were used to assay the progression of osteogenesis under different treatment conditions. Cultures were exposed to Wnt3a treatment to assess downstream signaling mechanisms.ResultsIn this study, we show that H₂S is produced by human PDLCs via the cystathionine β-synthase/cystathionine γ-lyase pathway to promote their osteogenic differentiation. These levels must be carefully maintained as excessive or deficient H₂S levels temper the observed osteogenic effect by inhibiting Wnt/β-catenin signaling.ConclusionsThese results demonstrate that optimal concentrations of endogenous H₂S must be maintained within PDLCs to promote osteogenic differentiation by activating the Wnt/β-catenin signaling cascade.     

Post-natal effect of overexpressed DKK1 on mandibular molar formation

Dickkopf-related protein 1 (DKK1) is a potent inhibitor of Wnt/β-catenin signaling. Dkk1-null mutant embryos display severe defects in head induction. Conversely, targeted expression of Dkk1 in dental epithelial cells leads to the formation of dysfunctional enamel knots and subsequent tooth defects during embryonic development. However, its role in post-natal dentinogenesis is largely unknown. To address this issue, we studied the role of DKK1 in post-natal dentin development using 2.3-kb Col1a1-Dkk1 transgenic mice, with the following key findings: (1) The Dkk1 transgene was highly expressed in pulp and odontoblast cells during post-natal developmental stages; (2) the 1(st) molar displayed short roots, an enlarged pulp/root canal region, and a decrease in the dentin formation rate; (3) a small malformed second molar and an absent third molar; (4) an increase of immature odontoblasts, few mature odontoblasts, and sharply reduced dentinal tubules; and (5) a dramatic change in Osx and nestin expression. We propose that DKK1 controls post-natal mandibular molar dentin formation either directly or indirectly via the inhibition of Wnt signaling at the following aspects: (i) post-natal dentin formation, (ii) formation and/or maintenance of the dentin tubular system, (iii) mineralization of the dentin, and (iv) regulation of molecules such as Osx and nestin.