-
Cell Proliferation Sep 2023Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that...
Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that hyperactivated nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) inflammasomes with pyroptotic cell death was present in pulpitis tissues, especially in the odontoblast layer, and mitochondrial oxidative stress (OS) was involved in driving this NLRP3 inflammasome-induced pathology. Using bone marrow mesenchymal stem cells (BMSCs) as mitochondrial donor cells, we demonstrated that BMSCs could donate their mitochondria to odontoblasts via tunnelling nanotubes (TNTs) and, thus, reduce mitochondrial OS and the consequent NLRP3 inflammasome-induced pyroptosis in odontoblasts. These protective effects of BMSCs were mostly blocked by inhibitors of the mitochondrial function or TNT formation. In terms of the mechanism of action, TNF-α secreted from pyroptotic odontoblasts activates NF-κB signalling in BMSCs via the paracrine pathway, thereby promoting the TNT formation in BMSCs and enhancing mitochondrial transfer efficiency. Inhibitions of NF-κB signalling and TNF-α secretion in BMSCs suppressed their mitochondrial donation capacity and TNT formation. Collectively, these findings demonstrated that TNT-mediated mitochondrial transfer is a potential protective mechanism of BMSCs under stress conditions, suggesting a new therapeutic strategy of mitochondrial transfer for dental pulp repair.
Topics: Humans; Pyroptosis; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; NF-kappa B; Tumor Necrosis Factor-alpha; Pulpitis; Dental Pulp; Mitochondria
PubMed: 37086012
DOI: 10.1111/cpr.13442 -
Current Protocols Nov 2022A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately...
A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem cells (iPSC) from both skin and blood, there are still limitations to the collection, production and use of iPSC derived neurons. We have had significant success in collecting and growing human dental pulp stem cells (DPSC) from exfoliated teeth sent directly to our laboratory by the parents of children with a variety of rare neurogenetic syndromes. This protocol outlines our current methods for the growth and expansion of DPSC from exfoliated (baby) teeth. These DPSC can be differentiated into a variety of cell types including osteoblasts, chondrocytes, and mixed neuron and glial cultures. Here we provide our protocol for the differentiation of early passage DPSC cultures into neurons for molecular and cellular studies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Collection and transportation of exfoliated teeth Basic Protocol 2: Dental pulp extraction Basic Protocol 3: Passage, freezing, and thawing of DPSC cultures Basic Protocol 4: Differentiation of DPSC into mixed neuronal cultures.
Topics: Child; Humans; Dental Pulp; Cell Differentiation; Induced Pluripotent Stem Cells; Tooth, Deciduous; Neurons
PubMed: 36420818
DOI: 10.1002/cpz1.600 -
Cells Aug 2021The dental pulp can be affected by thermal, physical, chemical, and bacterial phenomena that stimulate the inflammatory response. The pulp tissue produces an...
The dental pulp can be affected by thermal, physical, chemical, and bacterial phenomena that stimulate the inflammatory response. The pulp tissue produces an immunological, cellular, and vascular reaction in an attempt to defend itself and resolve the affected tissue. The expression of different microRNAs during pulp inflammation has been previously documented. MicroRNAs (miRNAs) are endogenous small molecules involved in the transcription of genes that regulate the immune system and the inflammatory response. They are present in cellular and physiological functions, as well as in the pathogenesis of human diseases, becoming potential biomarkers for diagnosis, prognosis, monitoring, and safety. Previous studies have evidenced the different roles played by miRNAs in proinflammatory, anti-inflammatory, and immunological phenomena in the dental pulp, highlighting specific key functions of pulp pathology. This systematized review aims to provide an understanding of the role of the different microRNAs detected in the pulp and their effects on the expression of the different target genes that are involved during pulp inflammation.
Topics: Cell Differentiation; Dental Pulp; Down-Regulation; Gene Expression Regulation; Humans; Inflammation; MicroRNAs; RNA, Messenger; Signal Transduction; Up-Regulation
PubMed: 34440911
DOI: 10.3390/cells10082142 -
Journal of Dental Research Aug 2022Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop...
Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop more effective treatments. Calcitonin gene-related peptide (CGRP) and dental pulp cells are promising candidates for mediating tooth pain and generating reparative dental tissues, respectively, but their behavior in the context of pulpitis remains elusive. The mouse incisor requires Sonic hedgehog (Shh) secreted from sensory nerves to continuously regenerate. However, it is unknown whether sensory nerves also regulate the comparatively nonregenerative mouse molar through CGRP and Shh. This is an important knowledge gap to fill since mouse incisors differ biologically from human teeth, while mouse and human molars are similar. In this work, we identified that molar pulp cells express CGRP receptor and Gli1, a Hedgehog (Hh) signaling protein found to label a dental stem cell population in the mouse incisor. We also observed in a mouse molar injury model that Hh signaling was activated and Shh expression was upregulated in vivo. We then determined in vitro that Shh and CGRP regulate differentiation of primary mouse molar and incisor pulp cells and a human dental pulp stem cell line. Furthermore, conditioned media from stimulated sensory neurons induced Hh signaling activation and inflammatory gene expression in primary molar pulp cells, which was abolished by inhibition of either Shh or CGRP. Our results suggest that CGRP and Shh signaling may promote an inflammatory response after injury in the molar and that activated sensory nerves secrete CGRP and Shh to regulate molar pulp cell expansion and differentiation into odontoblast-like cells for dentin repair. Thus, CGRP/Shh signaling should be considered for new strategies that seek to manage pain or dentin regeneration in the molar.
Topics: Animals; Calcitonin Gene-Related Peptide; Dental Pulp; Hedgehog Proteins; Humans; Incisor; Mice; Neurons, Afferent; Pain
PubMed: 35403480
DOI: 10.1177/00220345221086858 -
International Dental Journal Feb 2023
Topics: Humans; Dental Materials; Materials Testing; Dental Pulp
PubMed: 36653076
DOI: 10.1016/j.identj.2022.11.013 -
Journal of Feline Medicine and Surgery Feb 2023The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency,...
OBJECTIVES
The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability.
METHODS
Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P, P and P, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P, and cells were immunophenotyped in P by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P, P and P.
RESULTS
The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P to P. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45, CD90 and CD44. Structural and numerical cytogenetic aberrations were observed in P-P.
CONCLUSIONS AND RELEVANCE
In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.
Topics: Cats; Animals; Dental Pulp; Cell Differentiation; Stem Cells; Flow Cytometry; Cells, Cultured; Cell Proliferation
PubMed: 36745130
DOI: 10.1177/1098612X221150625 -
International Journal of Molecular... Mar 2021Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types for regenerative therapies given their ability to grow in the absence of serum and... (Review)
Review
Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types for regenerative therapies given their ability to grow in the absence of serum and their realistic possibility to be used in autologous grafts. In this review, we describe the particular advantages of hDPSCs for neuroregenerative cell therapies. We thoroughly discuss the knowledge about their embryonic origin and characteristics of their postnatal niche, as well as the current status of cell culture protocols to maximize their multilineage differentiation potential, highlighting some common issues when assessing neuronal differentiation fates of hDPSCs. We also review the recent progress on neuroprotective and immunomodulatory capacity of hDPSCs and their secreted extracellular vesicles, as well as their combination with scaffold materials to improve their functional integration on the injured central nervous system (CNS) and peripheral nervous system (PNS). Finally, we offer some perspectives on the current and possible future applications of hDPSCs in neuroregenerative cell therapies.
Topics: Cell Differentiation; Cell- and Tissue-Based Therapy; Dental Pulp; Extracellular Vesicles; Humans; Nerve Regeneration; Neuroglia; Stem Cell Transplantation; Stem Cells; Tissue Engineering; Tissue Scaffolds
PubMed: 33805573
DOI: 10.3390/ijms22073546 -
International Journal of Molecular... Feb 2022Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to...
Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering.
Topics: Cell Differentiation; Cell Proliferation; Cells, Cultured; Dental Papilla; Dental Pulp; Mesenchymal Stem Cells; Osteogenesis; Stem Cells
PubMed: 35269758
DOI: 10.3390/ijms23052615 -
International Journal of Molecular... Jul 2023Protected by the surrounding mineralized barriers of enamel, dentin, and cementum, dental pulp is a functionally versatile tissue that fulfills multiple roles [...].
Protected by the surrounding mineralized barriers of enamel, dentin, and cementum, dental pulp is a functionally versatile tissue that fulfills multiple roles [...].
Topics: Dentin; Dental Pulp; Regeneration; Tissue Engineering
PubMed: 37511210
DOI: 10.3390/ijms241411453 -
Stem Cell Research & Therapy Jul 2023Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state...
BACKGROUND
Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state could contribute to improvements in the dentin-pulp complex and dentinogenesis.
METHODS
TSC1 conditional knockout (DMP1-Cre+; TSC1, hereafter CKO) mice were generated to increase the activity of mechanistic target of rapamycin complex 1 (mTORC1). H&E staining, immunofluorescence and micro-CT analysis were performed with these CKO mice and littermate controls. In vitro, exosomes were collected from the supernatants of MDPC23 cells with different levels of mTORC1 activity and then characterized by transmission electron microscopy and nanoparticle tracking analysis. DPSCs were cocultured with MDPC23 cells and MDPC23 cell-derived exosomes. Alizarin Red S staining, ALP staining, qRT‒PCR, western blotting analysis and micro-RNA sequencing were performed.
RESULTS
Our study showed that mTORC1 activation in odontoblasts resulted in thicker dentin and higher dentin volume/tooth volume of molars, and it increased the expression levels of the exosome markers CD63 and Alix. In vitro, when DPSCs were cocultured with MDPC23 cells, odontoblastic differentiation was inhibited. However, the inhibition of odontoblastic differentiation was reversed when DPSCs were cocultured with MDPC23 cells with mTORC1 overactivation. To further study the effects of mTORC1 on exosome release from odontoblasts, MDPC23 cells were treated with rapamycin or shRNA-TSC1 to inactivate or activate mTORC1, respectively. The results revealed that exosome release from odontoblasts was negatively correlated with mTORC1 activity. Moreover, exosomes derived from MDPC23 cells with active or inactive mTORC1 inhibited the odontoblastic differentiation of DPSCs at the same concentration. miRNA sequencing analysis of exosomes that were derived from shTSC1-transfected MDPC23 cells, rapamycin-treated MDPC23 cells or nontreated MDPC23 cells revealed that the majority of the miRNAs were similar among these groups. In addition, exosomes derived from odontoblasts inhibited the odontoblastic differentiation of DPSCs, and the inhibitory effect was positively correlated with exosome concentration.
CONCLUSION
mTORC1 regulates exosome release from odontoblasts to inhibit the odontoblastic differentiation of DPSCs, but it does not alter exosomal contents. These findings might provide a new understanding of dental pulp complex regeneration.
Topics: Mice; Animals; Odontoblasts; Extracellular Matrix Proteins; Dental Pulp; Exosomes; Cell Differentiation; Stem Cells; Cells, Cultured
PubMed: 37422687
DOI: 10.1186/s13287-023-03401-9