The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains. Consequently, the literature was screened to identify manuscripts reporting the use of human dental pulp cells to study mineralization. This dataset was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review. Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent. While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Aim: The aim of this study was to investigate the effect of type 2 diabetes (T2D) on clinically normal dental pulp tissue by using special stains and immunohistochemistry (IHC) to determine the morphology of the coronal pulp and distribution of immune markers in non‐T2D and T2D groups.Methodology: Ethics approval for this in vitro pilot study was obtained from the University of Otago Human Ethics Committee (16/069). Twenty extracted permanent molar teeth diagnosed as having clinically normal pulp status were collected. Ten teeth were from participants with well‐controlled T2D and ten from participants without diabetes (non‐T2D). Each tooth was sectioned transversely at the cemento‐enamel junction before the crowns were decalcified and embedded in paraffin. Sections were stained with haematoxylin and eosin, Massons trichrome, and van Gieson stains for histological and morphological evaluation. IHC using anti‐CD4, anti‐CD68 and anti‐CD83 and anti‐IL1β, anti‐IL6, anti‐IL17, anti‐TNF‐α, anti‐TLR2, anti‐TLR4 and anti‐FOXP3 identified proteins of interest. Qualitative and semi‐quantitative analyses evaluated the morphology of the dental pulp and protein expression. Data analyses were performed with GraphPad Prism, using Student's t‐test and multiple regression using SPSS at p < .05.Results: Special stains demonstrated morphological differences in the T2D dental pulp compared with non‐T2D. Qualitative analysis indicated that the pulp in the T2D samples was consistently less cellular, less vascular, showed evidence of thickened blood vessel walls, increased pulp calcification and collagen deposition. Semi‐quantitative analysis of IHC samples showed the T2D pulp had significantly increased expression of macrophage and dendritic cell markers CD68 (p < .001) and CD83 (p = .04), and there was significantly greater expression of inflammatory cytokines IL1β (p = .01), IL6 (p < .0001), IL17 (p < .0001) and TNF‐α (p = .01). T2D samples showed a significant increase in markers of innate inflammation, TLR2 (p < .001) and TLR4 (p < .001) and decreased expression of regulatory T‐cell marker, FOXP3 (p = .01). Multiple regression showed that age‐corrected differences were statistically significant.Conclusion: Preliminary findings suggest that T2D may exert a similar response in the pulp to complications in other body sites. Hyperglycaemia is associated with changes in the morphology of the clinically normal dental pulp with altered immune cell and cytokine expression.

Objectives: This investigation aimed to isolate and culture human dental pulp cells from carious teeth (cHDPCs) and compare their growth characteristics, colony-forming efficiency, mineralization potential and gene expression of Toll-like receptors (TLR)-2, TLR-4, TLR-9, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-17A, 1L-17R, IL-23A, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK1), dentin matrix protein (DMP)-1, dentin sialophospho protein (DSPP), sex determining region Y-box 2 (SOX2) and marker of proliferation Ki-67 (MKi67) with cells isolated from healthy or non-carious teeth (ncHDPCs). Methods: Pulp tissues were obtained from both healthy and carious teeth (n = 5, each) to generate primary cell lines using the explant culture technique. Cell cultures studies were undertaken by generating growth curves, a colony forming unit and a mineralization assay analysis. The expression of vimentin was assessed using immunocytochemistry (ICC), and the gene expression of above-mentioned genes was determined using quantitative real-time reverse-transcription polymerase chain reaction. Results: ncHDPCs and cHDPCs were successfully isolated and cultured from healthy and inflamed human dental pulp tissue. At passage 4, both HDPC types demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed between ncHDPCs and cHDPCs in their growth characteristics or ability to differentiate into a mineralizing phenotype. ncHDPCs showed a statistically significant higher colony forming efficiency than cHDPCs. The gene expression levels of TLR-2, TLR-4, TLR-9, TNF-α, IL-6, IL-8, IL-17R, IL-23A, NF-κB, MAPK1, DMP1, DSPP and SOX2 were significantly higher in cHDPCs compared with ncHDPC cultures. Conclusion: cHDPCs retain their differentiation potential and inflammatory phenotype in vitro. The inflamed tooth pulp contains viable stem/progenitor cell populations which have the potential for expansion, proliferation and differentiation into a mineralizing lineage, similar to cells obtained from healthy pulp tissue. These findings have positive implications for regenerative endodontic procedures.