human dental pulp
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Human dental pulp stem/stromal cells (hDPSCs) derived from the permanent secondary dentition are recognised to possess certain advantageous traits, which support their potential use as a viable source of mesenchymal stem/stromal cells (MSCs) for regenerative medicine-based applications. However, the well-established heterogeneous nature of hDPSC subpopulations, coupled with their limited numbers within dental pulp tissues, has impeded our understanding of hDPSC biology and the translation of sufficient quantities of these cells from laboratory research, through successful therapy development and clinical applications. This article reviews our current understanding of hDPSC biology and the evidence underpinning the molecular basis of their heterogeneity, which may be exploited to distinguish individual subpopulations with specific or superior characteristics for regenerative medicine applications. Pertinent unanswered questions which still remain, regarding the developmental origins, hierarchical organisation, and stem cell niche locations of hDPSC subpopulations and their roles in hDPSC heterogeneity and functions, will further be explored. Ultimately, a greater understanding of how key features, such as specific cell surface, senescence and other relevant genes, and protein and metabolic markers, delineate between hDPSC subpopulations with contrasting stemness, proliferative, multipotency, immunomodulatory, anti-inflammatory, and other relevant properties is required. Such knowledge advancements will undoubtedly lead to the development of novel screening, isolation, and purification strategies, permitting the routine and effective identification, enrichment, and expansion of more desirable hDPSC subpopulations for regenerative medicine-based applications. Furthermore, such innovative measures could lead to improved cell expansion, manufacture, and banking procedures, thereby supporting the translational development of hDPSC-based therapies in the future.

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.

Abstract Objective Platelet-rich plasma (PRP) activation is an important factor in triggering the initial release of blood-derived growth factors from platelets. Vascular endothelial growth factor-A (VEGF-A) can be expressed by human dental pulp stem cells (hDPSCs) and plays an important role in dental pulp angiogenesis. The aim of this study is to analyze the effects of calcium gluconate on PRP activation in hDPSC VEGF-A expression. Materials and Methods Two types of PRP and their corresponding activators were analyzed in this study: PRP (activated using calcium chloride/CaCl2) and PRP-T (activated using CaCl2 with the addition of 10% calcium gluconate). hDPSCs were obtained by using an out-growth method (DPSCs-OG), and harvest between the fifth and sixth passages, then cultured in three different media groups: control, PRP, and PRP-T, which were planted in 96 wells (5 × 103 each well). The VEGF-A expression of hDPSCs was analyzed by using an ELISA test and observed at 24, 48, and 72 hours. Statistical Analysis This study was performed by using one-way ANOVA (p < 0.05) test. Results There were significant differences between all groups (p < 0.05) at 48 and 72 hours of observations, and no significant differences in the PRP and PRP-T groups at 48 and 72 hours of observations (p > 0.05). Conclusion PRP and PRP-T were equally effective in inducing VEGF-A expression of hDPSCs.

It is primarily important to define the standard features and factors that affect dental pulp stem cells (DPSCs) for their broader use in tissue engineering. This study aimed to verify whether DPSCs isolated from various teeth extracted from the same donor exhibit intra-individual variability and what the consequences are for their differentiation potential. The heterogeneity determination was based on studying the proliferative capacity, viability, expression of phenotypic markers, and relative length of telomere chromosomes. The study included 14 teeth (6 molars and 8 premolars) from six different individuals ages 12 to 16. We did not observe any significant intra-individual variability in DPSC size, proliferation rate, viability, or relative telomere length change within lineages isolated from different teeth but the same donor. The minor non-significant variances in phenotype were probably mainly because DPSC cell lines comprised heterogeneous groups of undifferentiated cells independent of the donor. The other variances were seen in DPSC lineages isolated from the same donor, but the teeth were in different stages of root development. We also did not observe any changes in the ability of cells to differentiate into mature cell lines—chondrocytes, osteocytes, and adipocytes. This study is the first to analyze the heterogeneity of DPSC dependent on a donor.

Abstract Background: Human dental pulp stem cells (hDPSCs) are critical for pulp generation. hDPSCs proliferate faster under hypoxia, but the regulatory mechanism of long noncoding RNAs (lncRNAs) in this process is not fully understood.Methods: Novel lncRNAs were obtained by reanalysis of transcriptome datasets coming from RNA-Seq under hypoxia compared with normoxia, and differential expression analysis of target genes were performed. Bioinformatics analyses including Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Set Enrichment Analysis (GSEA) analysis were used to understand the function of key novel lncRNA. hDPSCs were isolated from dental pulp tissue. EdU test and scratch healing test were used to detect the proliferation and migration of hDPSCs. qRT-PCR was used to detect the RNA level expression changes of selected genes. RNA fluorescence in situ hybridization (FISH), small interfering RNA (siRNA), qRT-PCR and western blot analysis were used to explore the function of key novel lncRNA. Results: We identified 496 novel lncRNAs in hDPSCs under hypoxia, including 45 expressed differentially novel lncRNAs. Of them, we focused on a key novel lncRNA, which we named HRL-SC (hypoxia related lncRNA in stem cells). Functional annotation revealed that HRL-SC was associated with hypoxic conditions and PI3K/AKT signalling pathway. HRL-SC was mainly located in the cytoplasm of hDPSCs and had stably high expression under hypoxia. Knockdown of HRL-SC inhibited proliferation and migration of hDPSCs and expression levels of PI3K/AKT related marker proteins. Furthermore, AKT activator SC79 partially offset the inhibitory effect caused by the knockdown, indicating that HRL-SC promoted hDPSCs through PI3K/AKT signalling pathway.Conclusion: Hypoxia related lncRNA HRL-SC promotes proliferation and migration of hDPSCs through PI3K/AKT signalling pathway and it may provide a better understanding for regenerative application of hDPSCs.

In this study, the preparation and characterization of three hydroxyapatite-based bioactive scaffolds, including hydroxyapatite microspheres (HAps), amoxicillin–hydroxyapatite composite (Amx–HAp), and collagen–hydroxyapatite composite (Col–HAp) were performed. In addition, their behavior in human dental pulp mesenchymal stem cell (hDPSC) culture was investigated. HAps were synthesized through the following methods: microwave hydrothermal, hydrothermal reactor, and precipitation, respectively. hDPSCs were obtained from samples of third molars and characterized by immunophenotypic analysis. Cells were cultured on scaffolds with osteogenic differentiation medium and maintained for 21 days. Cytotoxicity analysis and migration assay of hDPSCs were evaluated. After 21 days of induction, no differences in genes expression were observed. hDPSCs highly expressed the collagen IA and the osteonectin at the mRNA. The cytotoxicity assay using hDPSCs demonstrated that the Col–HAp group presented non-viable cells statistically lower than the control group (p = 0.03). In the migration assay, after 24 h HAps revealed the same migration behavior for hDPSCs observed compared to the positive control. Col–HAp also provided a statistically significant higher migration of hDPSCs than HAps (p = 0.02). Migration results after 48 h for HAps was intermediate from those achieved by the control groups. There was no statistical difference between the positive control and Col–HAp. Specifically, this study demonstrated that hydroxyapatite-based bioactive scaffolds, especially Col-Hap, enhanced the dynamic parameters of cell viability and cell migration capacities for hDPSCs, resulting in suitable adhesion, proliferation, and differentiation of this osteogenic lineage. These data presented are of high clinical importance and hold promise for application in therapeutic areas, because Col–HAp can be used in ridge preservation, minor bone augmentation, and periodontal regeneration. The development of novel hydroxyapatite-based bioactive scaffolds with clinical safety for bone formation from hDPSCs is an important yet challenging task both in biomaterials and cell biology.