A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BACKGROUND: 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. OBJECTIVES: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes. METHOD: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it 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. RESULTS: 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. DISCUSSION: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. CONCLUSION: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Potentiality and Inflammatory Marker Expression Are Maintained in Dental Pulp Cell Cultures from Carious Teeth.

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)-1ß, 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.

The relative frequency of paediatric oral and maxillofacial pathology in New Zealand: A 10-year review of a national specialist centre.

BACKGROUND: There has been no previous report of the prevalence of paediatric oral and maxillofacial pathology in a New Zealand oral pathology diagnostic service. AIM: The aim of this study was to review cases of paediatric oral pathology to determine relative frequencies of oral lesions in this age group. DESIGN: Paediatric oral pathology cases (≤15 years of age) received between 2007 and 2016 were retrieved from the electronic database of the Oral Pathology Centre, University of Otago. Data collected included diagnoses (categorised into 12 groups), age at diagnosis, and gender. The prevalence of each diagnosis was calculated in terms of percentage of all diagnoses made. Male-to-female ratio and mean age at diagnosis were also determined. RESULTS: A total of 1139 paediatric cases were identified representing 5.2% of all cases. The most common diagnostic group was salivary gland pathology (25.4%), followed by dental (24.8%) pathology. The most prevalent lesion was mucocoele (23%), followed by dental follicle (14.1%). Malignancies were rare with only two cases identified. CONCLUSION: The findings provide an insight into the prevalence of paediatric oral pathology for clinicians. Mucocoele was the most common diagnosis made, suggesting a high prevalence of soft tissue injury as a main presenting concern warranting diagnosis and management through biopsy.

The nexus between periodontics and oral pathology.

A wide variety of lesions may arise from the oral mucosa, fibrous connective tissue, bone and cementum of the periodontium. The commonest pathology occurs as a result of bacterial infection and is very well known to dentists and periodontists, but rarer conditions present as gingival pathology. The pathogenesis of these conditions ranges from genetic to traumatic to immunological to neoplastic, and includes benign, malignant and metastatic lesions. This paper outlines some of these conditions and describes how the periodontist and oral pathologist can work together using a framework, and how with careful consideration of the clinical features and the use of appropriate special tests, including obtaining an adequate tissue specimen, a timely and accurate diagnosis can be obtained.

Concordance of clinician, Chat-GPT4, and ORAD diagnoses against histopathology in Odontogenic Keratocysts and tumours: a 15-Year New Zealand retrospective study.

BACKGROUND: This research aimed to investigate the concordance between clinical impressions and histopathologic diagnoses made by clinicians and artificial intelligence tools for odontogenic keratocyst (OKC) and Odontogenic tumours (OT) in a New Zealand population from 2008 to 2023. METHODS: Histopathological records from the Oral Pathology Centre, University of Otago (2008-2023) were examined to identify OKCs and OT. Specimen referral details, histopathologic reports, and clinician differential diagnoses, as well as those provided by ORAD and Chat-GPT4, were documented. Data were analyzed using SPSS, and concordance between provisional and histopathologic diagnoses was ascertained. RESULTS: Of the 34,225 biopsies, 302 and 321 samples were identified as OTs and OKCs. Concordance rates were 43.2% for clinicians, 45.6% for ORAD, and 41.4% for Chat-GPT4. Corresponding Kappa value against histological diagnosis were 0.23, 0.13 and 0.14. Surgeons achieved a higher concordance rate (47.7%) compared to non-surgeons (29.82%). Odds ratio of having concordant diagnosis using Chat-GPT4 and ORAD were between 1.4 and 2.8 (p < 0.05). ROC-AUC and PR-AUC were similar between the groups (Clinician 0.62/0.42, ORAD 0.58/0.28, Char-GPT4 0.63/0.37) for ameloblastoma and for OKC (Clinician 0.64/0.78, ORAD 0.66/0.77, Char-GPT4 0.60/0.71). CONCLUSION: Clinicians with surgical training achieved higher concordance rate when it comes to OT and OKC. Chat-GPT4 and Bayesian approach (ORAD) have shown potential in enhancing diagnostic capabilities.

Epidemiology of odontogenic tumours and selected cysts diagnosed at a single New Zealand oral pathology centre- A 15-year retrospective study.

PURPOSE: This research aimed to investigate the relative frequency of odontogenic tumours (OT) and selected odontogenic cysts in a single oral pathology center in New Zealand from 2008 to 2023. METHODS: Histopathological records from the Oral Pathology Centre, University of Otago (2008-2023) were examined to identify OT. Odontogenic keratocyst (OKC) and calcifying odontogenic cyst (COC), previously classified as OT were also included. Patient demographics, clinical details and histopathologic diagnoses were recorded. Data were analyzed using SPSS. RESULTS: Of the 34,225 biopsies over the 15-year period, 1.8% were identified as OTs, COC and OKCs and accounted for 47%, 1.5% and 51.5% respectively. The most prevalent OT types were odontoma (43.7%), ameloblastoma (27%) and cemento-ossifying fibroma (7.5%). Malignant OT, ameloblastic carcinoma, constituted 1.4% of OT. The average age at diagnosis for OKC, COC and OT patients were 48.2 ± 20.9, 33.7 ± 23.3 and 28.9 ± 19.3 years. Overall, male and mandibular site predilections were observed. Recurrence of OKC and ameloblastoma occurred in 15.2% and 13.7% of patients. The time for recurrence for OKC and Ameloblastoma were 61.7 ± 56.5 months and 122 ± 152 months respectively. CONCLUSION: The demographic features and range of OT, COC and OKC in New Zealand align with those of other western countries. The study also confirms need for long term follow up for patient with OKC and ameloblastoma.

In vivo healing of low temperature deproteinized bovine bone xenograft in a rabbit cranial model.

The physicochemical properties of grafting materials affect the quality of the osteointegration, resorption rate, and the new bone (NB) formation. This study assessed the physicochemical properties and integration of a low temperature deproteinized bovine bone xenograft (BBX), referred to as optimized MoaBone® (OMB). This novel BBX was physiochemically characterized both pre and post chemical bleaching and sterilization by gamma irradiation. OMB was compared to two commercial BBX; Bio-Oss® (BO) and MoaBone® (MB) using a rabbit cranial model. Residual graft and NB were quantified using histology and micro-computed tomography. Results showed that chemical treatment and gamma irradiation had limited effect on the surface texture. A significant decrease in the collagen content was detected post chemical treatment and in the carbonate content post gamma irradiation. There was no evidence of inflammatory infiltrate, necrosis, or connective tissue encapsulation, and a significant increase of NB in all grafted sites as compared to untreated defects could be observed. However, there was no statistically significant difference between the grafted sites. We conclude that chemical treatment and terminal sterilization strongly impact the final graft's properties. OMB graft showed equivalence with BO for in vivo bone formation and potentially results in lower levels of graft retention.

A Cell Culture Method for the Isolation and Study of Primary Human Dental Pulp Cells.

Primary human dental pulp cell (HDPC) cultures contain dental pulp stem cell (DPSC) populations. DPSCs are multipotent mesenchymal cells residing inside the dental pulp where they can contribute to the regenerative potential of this and other tissues throughout the body. These cells are promising tools for cell-based therapies, including regenerative endodontic procedures. HDPCs can be readily isolated and expanded from extracted teeth either by the dental tissue explant method or enzymatic digestion method. This chapter describes the explant method, whereby cells outgrow from dissected pulp tissue, to generate HDPC cultures. We also provide protocols for HDPC passaging, cryopreservation, and basic immunocytochemical characterization.

Vascularity and Angiogenic Signaling in the Dentine-Pulp Complex of Immature and Mature Permanent Teeth.

ABSTRACT. OBJECTIVE: This study aimed to examine the protein and gene expression of vascular endothelial growth factor (VEGF) and angiopoietins-1 and 2 in tissue from healthy and inflamed dental pulps. METHODS: Permanent teeth with pulps diagnosed as healthy or reversible pulpitis were used for immunohistochemistry (IHC) and gene expression experiments. For IHC, a whole pulp tissue was excavated from the pulp chamber, and it was formalin-fixed and processed for routine IHC with angiogenic markers anti-VEGF, anti-Ang1, and anti-Ang2. Staining was visualized with diaminobenzidine (DAB), and examined using light microscopy. The distribution of markers in healthy and inflamed pulps was qualitatively and quantitatively analyzed. Real-time quantitative polymerase chain reaction (RT qPCR) was used to ascertain the gene expression levels of ANGPT1, ANGPT2, and TEK in the presence of inflammation. Statistical analysis was performed using the Mann-Whitney test with the statistical significance level set at 0.05. RESULTS: There was increased protein and mRNA expression of VEGF and Ang-1 markers in inflamed pulp samples as compared with that in the healthy pulp tissue. IHC demonstrated intense expression of the VEGF protein on endothelial cells (EC) and some non-ECs, and there was significantly more staining on ECs associated with inflamed tissue (P<0.001). Ang-1 and Ang-2 were significantly expressed on ECs and non-ECs (P<0.05). RT qPCR did not show significant differences in gene expression between healthy and inflamed samples although similar trends were observed to IHC. CONCLUSION: The presence of Ang-1, Ang-2, VEGF, and TEK gene in healthy and mildly inflamed pulp tissue associated with reversible pulpitis indicates that these angiogenic factors may participate in physiological and pathological angiogenesis and healing. The inflammatory process may regulate Ang-1/Ang2/Tie2 signaling; and together with VEGF, these growth factors have an important role in modulating pulp angiogenesis.

Unfolded protein response-related gene regulation in inflamed periodontal tissues with and without Russell bodies.

OBJECTIVE: To examine the expression of unfolded protein response (UPR) genes, a set of genes that are activated to assist in protein trafficking and cellular homeostasis when endoplasmic reticulum (ER) stress occurs, in inflamed and uninflamed periodontal tissues, with or without Russell bodies (RB). RB are a histologically apparent extension of the ER that represents an accumulation of abnormal proteins that cannot be secreted or degraded and may serve as a marker of ER stress. DESIGN: Periodontal tissue specimens were collected and categorised histologically based on the presence of inflammation and the quantity of RB. The differential regulation of 84 UPR-related genes was examined by qRT(2)-PCR. RESULTS: UPR genes related to the inositol-requiring ER-to-nucleus signal kinase (IRE)-1 pathway, molecular chaperones and ER quality control were up-regulated in RB(+) tissues compared with RB(-) tissues, irrespective of inflammation. Inflamed periodontal tissues showed a marked down-regulation of heat shock protein (HSP)-70 family members. CONCLUSION: The presence of RB in inflamed periodontal tissues correlated with the expression of a unique set of ER stress-related genes and therefore may serve as a marker of UPR response in periodontal inflammation. Inflamed periodontal tissues showed a marked down-regulation of UPR genes, in particular HSP70. This may be contributory to disease progression in periodontal disease.