Relevance of Cellular Redox Homeostasis for Vital Functions of Human Dental Pulp Cells

Odontogenic MSCs are vulnerable to LPS-triggered bacterial infections, and they respond by secreting inflammatory mediators, such as IL-6, and with mineralization. Since both processes might be prone to a disturbance of the redox homeostasis, the oxidative stress influence on vital functions of human dental pulp cells (HPCs) was investigated. With these aims, a model of LPS-stimulated primary HPCs was established, and anti- and pro-oxidant substances were administered up to 21 days to measure inflammation and mineralization parameters. LPS-stimulated HPCs retained mineralization potential, which was decreased with the antioxidants NAC and fisetin and the pro-oxidant BSO. The expression of surface markers related to odontogenic commitment was influenced accordingly but counteracted by the enhanced expression of BMP2 and ALP at the transcriptional level. LPS triggers an early IL-6 production in non-odontogenic conditions, while it can be measured only after 15 days in the presence of the differentiation medium. The present study shows that HPCs functions causally depend on a tightly regulated cellular redox balance. Our data demonstrate a redox control of pulp MSC odontogenic commitment along with a potential association between an IL-6 late secretion and mineralization. These findings lay the groundwork for investigations on the molecular role of IL-6 in dental hard tissue metabolism.

Evaluation of Injectable Hyaluronic Acid-Based Hydrogels for Endodontic Tissue Regeneration

Dental pulp tissue engineering (TE) endeavors to regenerate dentin/pulp complex by combining a suitable supporting matrix, stem cells, and biochemical stimuli. Such procedures foresee a matrix that can be easily introduced into the root canal system (RCS) and tightly adhere to dentin walls to assure the dentin surface’s proper colonization with progenitor cells capable of restoring the dentin/pulp complex. Herein was investigated an injectable self-setting hyaluronic acid-based (HA) hydrogel system, formed by aldehyde-modified (a-HA) with hydrazide-modified (ADH), enriched with platelet lysate (PL), for endodontic regeneration. The hydrogels’ working (wT) and setting (sT) times, the adhesion to the dentine walls, the hydrogel’s microstructure, and the delivery of human dental pulp cells (DPCs) were studied in vitro. Hydrogels incorporating PL showed a suitable wT and sT and a porous microstructure. The tensile tests showed that the breaking point occurs after 4.3106 ± 1.8677 mm deformation, while in the indentation test after 1.4056 ± 0.3065 mm deformation. Both breaking points occur in the hydrogel extension. The HA/PL hydrogels exhibited supportive properties and promoted cell migration toward dentin surfaces in vitro. Overall, these results support using PL-laden HA injectable hydrogels (HA/PL) as a biomaterial for DPCs encapsulation, thereby displaying great clinical potential towards endodontic regenerative therapies.

Effect of Magnesium on Dentinogenesis of Human Dental Pulp Cells

Introducing therapeutic ions into pulp capping materials has been considered a new approach for enhancing regeneration of dental tissues. However, no studies have been reported on its dentinogenic effects on human dental pulp cells (HDPCs). This study was designed to investigate the effects of magnesium (Mg2+) on cell attachment efficiency, proliferation, differentiation, and mineralization of HDPCs. HDPCs were cultured with 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM concentrations of supplemental Mg2+ and 0 mM (control). Cell attachment was measured at 4, 8, 12, 16, and 20 hours. Cell proliferation rate was evaluated at 3, 7, 10, 14, and 21 days. Crystal violet staining was used to determine cell attachment and proliferation rate. Alkaline phosphatase (ALP) activity was assessed using the fluorometric assay at 7, 10, and 14 days. Mineralization of cultures was measured by Alizarin red staining. Statistical analysis was done using multiway analysis of variance (multiway ANOVA) with Wilks’ lambda test. Higher cell attachment was shown with 0.5 mM and 1 mM at 16 hours compared to control ( P < 0.0001 ). Cells with 0.5 mM and 1 mM supplemental Mg2+ showed significantly higher proliferation rates than control at 7, 10, 14, and 21 days ( P < 0.0001 ). However, cell proliferation rates decreased significantly with 4 mM and 8 mM supplemental Mg2+ at 14 and 21 days ( P < 0.0001 ). Significantly higher levels of ALP activity and mineralization were observed in 0.5 mM, 1 mM, and 2 mM supplemental Mg2+ at 10 and 14 days ( P < 0.0001 ). However, 8 mM supplemental Mg2+ showed lower ALP activity compared to control at 14 days ( P < 0.0001 ), while 4 mM and 8 mM supplemental Mg2+showed less mineralization compared to control ( P < 0.0001 ). The study indicated that the optimal (0.5–2 mM) supplemental Mg2+ concentrations significantly upregulated HDPCs by enhancing cell attachment, proliferation rate, ALP activity, and mineralization. Magnesium-containing biomaterials could be considered for a future novel dental pulp-capping additive in regenerative endodontics.

Evaluation of Injectable Hyaluronic Acid-Based Hydrogels for Endodontic Tissue Regeneration

Dental pulp tissue engineering (TE) quests to regenerate dentin/pulp complex by combining a suitable supporting matrix, stem cells, and biochemical stimuli. Such procedures foresee a matrix that can be easily introduced into the root canal system (RCS) and tightly adhere to dentin walls to assure the dentin surface's proper colonization with progenitor cells capable of restoring the dentin/pulp complex. Herein was investigated an injectable self-setting hyaluronic acid-based (HA) hydrogel system, formed by aldehyde-modified (a-HA) with hydrazide-modified (ADH), enriched with platelet lysate (PL), for endodontic regeneration. The hydrogels' working (wT) and setting (sT) times, the adhesion to the dentine walls, the hydrogel's microstructure, and the delivery of human Dental Pulp Cells (DPCs) were studied in vitro. Hydrogels incorporating PL showed a suitable wT and sT and a porous microstructure. The tensile tests showed that the breaking point occurs after 4.13 mm deformation. While in the indentation test after 1.3 mm deformation. Both breaking points occur in the hydrogel extension. The HA/PL hydrogels exhibited supportive properties and promoted cell migration toward dentin surfaces in vitro. Overall, these results support using PL-laden HA injectable hydrogels (HA/PL) as a biomaterial for DPCs encapsulation, thereby displaying great clinical potential towards endodontic regenerative therapies.

Platelet-Rich Plasma Induces Autophagy and Promotes Regeneration in Human Dental Pulp Cells

Regenerative endodontic procedures using autologous platelet-rich plasma (PRP) can improve the biologic outcome of treatment. However, its mechanism of action on improving pulp regeneration is not fully elucidated. Autophagy was recently shown to be related to tissue repair and osteogenesis. Therefore, the objective of this study was to investigate the effect of PRP in dental pulp regeneration and to elucidate the role of autophagy involved in this process. Human dental pulp cells (hDPCs) were isolated from healthy dental pulp and co-cultured with an increasing concentration of PRP. Cellular migration and proliferation were determined by scratch assay, transwell assay, and cell-counting kit 8 assay. Osteogenic differentiation was clarified by using alkaline phosphatase staining, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) to measure the gene expression levels of alkaline phosphatase, collagen-1, osteocalcin, dentin matrix protein 1, and dentin sialophosphoprotein. Autophagic bodies were observed by transmission electron microscopy and the expression of autophagy marker light chain 3B (LC3B) was determined by immunofluorescence staining. The mRNA and protein expression level of LC3B and Beclin-1 were quantified by qRT-PCR and western blotting. The effect of PRP on cellular migration, proliferation, and osteogenic differentiation was further investigated in the milieu of autophagy activator, rapamycin, and inhibitor, 3-methyladenine. Results showed that PRP promoted cell migration, proliferation, and osteogenic differentiation. Autophagic bodies were strongly activated and the expression level of LC3B and Beclin-1 was significantly promoted by PRP. Autophagy inhibition suppressed PRP-induced hDPCs migration, proliferation, and osteogenic differentiation, whereas autophagy activator substantially augmented PRP-stimulated migration, proliferation, and differentiation. Taken together, these findings suggested that PRP could effectively promote regenerative potentials associated with autophagy.

Effects of different aperture-sized type I collagen/silk fibroin scaffolds on the proliferation and differentiation of human dental pulp cells

This study aimed at evaluate the effects of different aperture-sized type I collagen/silk fibroin (CSF) scaffolds on the proliferation and differentiation of human dental pulp cells (HDPCs). The CSF scaffolds were designed with 3D mapping software Solidworks. Three different aperture-sized scaffolds (CSF1–CSF3) were prepared by low-temperature deposition 3D printing technology. The morphology was observed by scanning electron microscope (SEM) and optical coherence tomography. The porosity, hydrophilicity and mechanical capacity of the scaffold were detected, respectively. HDPCs (third passage, 1 × 105 cells) were seeded into each scaffold and investigated by SEM, CCK-8, alkaline phosphatase (ALP) activity and HE staining. The CSF scaffolds had porous structures with macropores and micropores. The macropore size of CSF1 to CSF3 was 421 ± 27 μm, 579 ± 36 μm and 707 ± 43 μm, respectively. The porosity was 69.8 ± 2.2%, 80.1 ± 2.8% and 86.5 ± 3.3%, respectively. All these scaffolds enhanced the adhesion and proliferation of HDPCs. The ALP activity in the CSF1 group was higher than that in the CSF3 groups (P &lt; 0.01). HE staining showed HDPCs grew in multilayer within the scaffolds. CSF scaffolds significantly improved the adhesion and ALP activity of HDPCs. CSF scaffolds were promising candidates in dentine-pulp complex regeneration.