This study aimed to compare the biological properties of newly synthesized cements based on calcium phosphate with a commercially used cement, mineral trioxide aggregate (MTA). Strontium (Sr)-, Copper (Cu)-, and Zinc (Zn)-doped hydroxyapatite (miHAp) powder was obtained through hydrothermal synthesis and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectrometry (EDX). Calcium phosphate cement (CPC) was produced by mixing miHAp powder with a 20 wt.% citric acid solution, followed by the assessment of its compressive strength, setting time, and in vitro bioactivity. Acetylsalicylic acid (ASA) was added to the CPC, resulting in CPCA. Biological tests were conducted on CPC, CPCA, and MTA. The biocompatibility of the cement extracts was evaluated in vitro using human dental pulp stem cells (hDPSCs) and in vivo using a zebrafish model. Antibiofilm and antimicrobial effect (quantified by CFUs/mL) were assessed against Streptococcus mutans and Lactobacillus rhamnosus. None of the tested materials showed toxicity, while CPCA even increased hDPSCs proliferation. CPCA showed a better safety profile than MTA and CPC, and no toxic or immunomodulatory effects on the zebrafish model. CPCA exhibited similar antibiofilm effects against S. mutans and L. rhamnosus to MTA.

Regenerative endodontics (REP) is a new clinical modality aiming to regenerate damaged soft and hard dental tissues, allowing for root completion in young adults\' teeth. Effective disinfection is crucial for REP success, but commonly used antimicrobials often harm the niche dental pulp stem cells (DPSCs). To our knowledge, this is the first study to explore the biocompatibility and antimicrobial potential of pectin as a potential natural intracanal medicament for REPs. Low methoxyl commercial citrus pectin (LM) (pectin CU701, Herbstreith&Fox.de) was used in all experiments. The pectin\'s antibacterial activity against single species biofilms (E. faecalis and F. nucleatum) was assessed using growth curves. The pectin\'s antimicrobial effect against mature dual-species biofilm was also evaluated using confocal laser scanning microscopy (CLSM) after 30 min and 7 days of treatment. The DPSC biocompatibility with 2% and 4% w/v of the pectin coatings was evaluated using live/dead staining, LDH, and WST-1 assays. Pectin showed a concentration-dependent inhibitory effect against single-species biofilms (E. faecalis and F. nucleatum) but failed to disrupt dual-species biofilm. Pectin at 2% w/v concentration proved to be biocompatible with the HDPSCs. However, 4% w/v pectin reduced both the viability and proliferation of the DPSCs. Low concentration (2% w/v) pectin was biocompatible with the DPSCs and showed an antimicrobial effect against single-species biofilms. This suggests the potential for using pectin as an injectable hydrogel for clinical applications in regenerative endodontics.

Vascularization is a key step to achieve pulp tissue regeneration and in vitro pre-vascularized dental pulp tissue could be applied as a graft substitute for dental pulp tissue repair. In this study, human dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (hUVECs) were co-cultured in 3D Matrigel and 150 mV/mm electric fields (EFs) were used to promote the construction of pre-vascularized dental pulp tissue. After optimizing co-cultured ratio of two cell types, immunofluorescence staining, and live/dead detection were used to investigate the effect of EFs on cell survival, differentiation and vessel formation in 3D engineered dental pulp tissue. RNA sequencing was used to investigate the potential molecular mechanisms by which EF regulates vessel formation in 3D engineered dental pulp tissue. Here we identified that EF-induced pre-vascularized engineered dental pulp tissue not only had odontoblasts, but also had a rich vascular network, and smooth muscle-like cells appeared around the blood vessels. The GO enrichment analysis showed that these genes were significantly enriched in regulation of angiogenesis, cell migration and motility. The most significant term of the KEGG pathway analysis were NOTCH signaling pathway and Calcium signaling pathway etc. The PPI network revealed that NOTCH1 and IL-6 were central hub genes. Our study indicated that EFs significantly promoted the maturation and stable of blood vessel in 3D engineered pulp tissue and provided an experimental basis for the application of EF in dental pulp angiogenesis and regeneration.

Reprograming of the dental pulp somatic cells to endothelial cells is an attractive strategy for generation of new blood vessels. For tissue regeneration, vascularization of engineered constructs is crucial to improve repair mechanisms. In this study, we show that dentin matrix protein 1 (DMP1) and HUVEC-ECM scaffold enhances the differentiation potential of dental pulp stem cells (DPSCs) to an endothelial phenotype. Our results show that the differentiated DPSCs expressed endothelial markers CD31 and VE-Cadherin (CD144) at 7 and 14 days. Expression of CD31 and VE-Cadherin (CD144) were also confirmed by immunofluorescence. Furthermore, flow cytometry analysis revealed a steady increase in CD31 and VE-Cadherin (CD144) positive cells with DMP1 treatment when compared with control. In addition, integrins specific for endothelial cells were highly expressed during the differentiation process. The endothelial cell signature of differentiated DPSCs were additionally characterized for key endothelial cell markers using gene expression by RT-PCR, Western blotting, immunostaining, and RNA-seq analysis. Furthermore, the angiogenic phenotype was confirmed by tubule and capillary sprout formation. Overall, stimulation of DPSCs by DMP1 and use of HUVEC-ECM scaffold promoted their differentiation into phenotypically, transcriptionally, and functionally differentiated bonafide endothelial cells. This study is novel, physiologically relevant and different from conventional strategies.

Dentin-pulp regeneration through stem/progenitor cell transplantation represents a promising frontier in regenerative endodontics. This systematic review meticulously evaluates animal studies to investigate the efficacy of stem cell therapy in repairing/regenerating the dentine-pulp complex in mature/immature animal teeth. Employing a comprehensive electronic search of PubMed and Scopus databases up to October 2023, relevant English studies were identified/assessed. Evaluation parameters encompassed radiographic and histological assessments of dentin-pulp complex formation. Outcome measures included pulp-like and dentin-like tissues regeneration, apical healing, dentin thickening, apical closure, and dentinal bridge formation. The risk-of-bias assessment adhered to the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) guidelines. Out of 3250 identified articles, 23 animal experiments were included, categorized into regenerative procedures in mature teeth (n=11), regenerative procedures in immature teeth (n=4), and vital pulp therapy (n=8). Despite the promising potential, the bias in the included studies was high. Notably, Various scaffolds, and growth factors were employed, highlighting the heterogeneity across the studies. Dental pulp stem cells (DPSCs) and bone marrow stem cells, especially specific subfractions, demonstrated notable regenerative potential: hypoxic conditions and extracellular vesicles from preconditioned DPSCs enhanced regeneration, with considerations of cell fate. Donor age impacted regeneration, and challenges persisted in pulpotomy and direct pulp capping. Scaffold and growth factor choices influenced outcomes, underscoring the need for standardized strategies. Despite the promise, clinical viability faces hurdles, necessitating further investigation into adverse effects, optimized scaffolds, and regulatory considerations. This systematic review illuminates the potential of stem cell transplantation for dentin-pulp complex regeneration. The overall evidence quality, influenced by study heterogeneity and biases, underscores the need for cautious interpretation of findings. Future studies should refine methodologies and establish reliable histological parameters for meaningful advancements in dentin-pulp regeneration.

This systematic review evaluates clinical studies investigating regenerative endodontic procedures for mature/immature teeth utilizing stem cell transplantation. An electronic search of Scopus, PubMed, ISI Web Science, and Google Scholar was conducted up to January 2023. Outcome measures encompassed radiographic (periapical lesion, root length, apical foramen width, volume of the regenerated pulp) and clinical (post-operative pain, sensibility test) parameters. Among 3250 identified articles, five clinical studies were selected, comprising two randomized controlled trials (RCTs) for mature/immature teeth, and three case reports/series for mature teeth. Despite the promising potential, the included studies exhibited a notable risk of bias. The diversity in stem cells (e.g., dental pulp stem cells [DPSCs], umbilical cord mesenchymal stem cells [UC-MSCs]), scaffolds (Atecollagen, collagen membrane, platelet-poor plasma [PPP], leukocyte platelet-rich in fibrin [L-PRF]), and growth factors (granulocyte colony-stimulating factor [G-CSF]) emphasized the heterogeneity across interventions. In RCTs, DPSCs application increased root length and reduced apical foramen width in immature teeth, while UC-MSCs transplantation reduced apical lesions in mature teeth. Transplantation of DPSCs aggregates or UC-MSCs/PPP also elicited positive pulp responses and increased blood flow. In case reports/series, DPSCs application in teeth with irreversible pulpitis resulted in mineralization and increased the regenerated pulp\' volume. Furthermore, transplantation of DPSCs with G-CSF/atelocollagen or L-PRF/collagen membrane led to positive pulp responses. While underscoring the potential of stem cell transplantation for regenerative endodontics in mature/immature teeth, the overall evidence quality and the limited number of available studies emphasize the need for cautious interpretation of results. Future well-designed clinical studies are essential to validate these findings further.

UNASSIGNED: The aim of the study was to evaluate the ability of cultivated odontoblast to form dentin-like tissue using fibroblast growth factor (FGF) and insulin-like growth factor (IGF).
UNASSIGNED: Dental pulp stem cells (DPSCs) were extracted from 10 human teeth. They were isolated and cultivated in vitro with the use of stem cell markers. The human DPSCs were characterized for trilineage differentiation. They were then differentiated into odontoblasts. The ability of cultivated odontoblasts to form dentin-like tissue was evaluated using FGF and IGF.
UNASSIGNED: IGF showed superior ability to form dentin-like tissue as compared to FGF. The addition of FGF showed no significant difference in the formation of dentin-like tissue. A combination of FGF and IGF in odontoblast showed an enhanced ability to form dentin-like tissue.
UNASSIGNED: The use of growth factors IGF and FGF with dental stem cells showed a greater potential to form dentin-like tissue. This can profoundly alter the paradigms of conservative vital pulp therapy, which may eventually make it possible to treat dental diseases by regeneration of lost dentine.

BACKGROUND: Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches.
OBJECTIVE: To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR.
METHODS: We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups.
RESULTS: All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms\' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking.
CONCLUSIONS: For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

The aim of this study is to introduce a dental capping agent for the treatment of pulp inflammation (pulpitis). Nanohydroxyapatite with Elaeagnus angustifolia L. extract (nHAEA) loaded with metronidazole (nHAEA@MTZ) was synthesized and evaluated using a lipopolysaccharide (LPS) in vitro model of pulpitis. nHAEA was synthesized through sol-gel method and analyzed using Scanning Electron Microscopy, Transmission Electron Microscopy, and Brunauer Emmett Teller. Inflammation in human dental pulp stem cells (HDPSCs) induced by LPS. A scratch test assessed cell migration, RT PCR measured cytokines levels, and Alizarin red staining quantified odontogenesis. The nHAEA nanorods were 17-23 nm wide and 93-146 nm length, with an average pore diameter of 27/312 nm, and a surface area of 210.89 m2/g. MTZ loading content with controlled release, suggesting suitability for therapeutic applications. nHAEA@MTZ did not affect the odontogenic abilities of HDPSCs more than nHAEA. However, it was observed that nHAEA@MTZ demonstrated a more pronounced anti-inflammatory effect. HDPSCs treated with nanoparticles exhibited improved migration compared to other groups. These findings demonstrated that nHAEA@MTZ could be an effective material for pulp capping and may be more effective than nHAEA in reducing inflammation and activating HDPSCs to enhance pulp repair after pulp damage.

Introduction: Facial nerve injury significantly impacts both the physical and psychological] wellbeing of patients. Despite advancements, there are still limitations associated with autografts transplantation. Consequently, there is an urgent need for effective artificial grafts to address these limitations and repair injuries. Recent years have witnessed the recognition of the beneficial effects of chitosan (CS) and graphene in the realm of nerve repair. Dental pulp stem cells (DPSCs) hold great promise due to their high proliferative and multi-directional differentiation capabilities. Methods: In this study, Graphene/CS (G/CST) composite tubes were synthesized and their physical, chemical and biological properties were evaluated, then DPSCs were employed as seed cells and G/CST as a scaffold to investigate their combined effect on promoting facial nerve injury repair. Results and Disscussion: The experimental results indicate that G/CST possesses favorable physical and chemical properties, along with good cyto-compatibility. making it suitable for repairing facial nerve transection injuries. Furthermore, the synergistic application of G/CST and DPSCs significantly enhanced the repair process for a 10 mm facial nerve defect in rabbits, highlighting the efficacy of graphene as a reinforcement material and DPSCs as a functional material in facial nerve injury repair. This approach offers an effective treatment strategy and introduces a novel concept for clinically managing facial nerve injuries.