Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells

Background: Different methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O 2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells. Material and Methods: One hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed. Results: Dental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ ml of thermolysine for 40 minutes and sonicating the culture for one minute. Conclusions: Dental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agent

Bioprinting of three-dimensional dentin-pulp complex with local differentiation of human dental pulp stem cells

Numerous approaches have been introduced to regenerate artificial dental tissues. However, conventional approaches are limited when producing a construct with three-dimensional patient-specific shapes and compositions of heterogeneous dental tissue. In this research, bioprinting technology was applied to produce a three-dimensional dentin-pulp complex with patient-specific shapes by inducing localized differentiation of human dental pulp stem cells within a single structure. A fibrin-based bio-ink was designed for bioprinting with the human dental pulp stem cells. The effects of fibrinogen concentration within the bio-ink were investigated in terms of printability, human dental pulp stem cell compatibility, and differentiation. The results show that micro-patterns with human dental pulp stem cells could be achieved with more than 88% viability. Its odontogenic differentiation was also regulated according to the fibrinogen concentration. Based on these results, a dentin-pulp complex having patient-specific shape was produced by co-printing the human dental pulp stem cell-laden bio-inks with polycaprolactone, which is a bio-thermoplastic used for producing the overall shape. After culturing with differentiation medium for 15 days, localized differentiation of human dental pulp stem cells in the outer region of the three-dimensional cellular construct was successfully achieved with localized mineralization. This result demonstrates the possibility to produce patient-specific composite tissues for tooth tissue engineering using three-dimensional bioprinting technology

Application of thermosensitive-hydrogel combined with dental pulp stem cells on the injured fallopian tube mucosa in an animal model

Objectives: Fallopian tube (FT) injury is an important factor that can lead to tubal infertility. Stem-cell-based therapy shows great potential for the treatment of injured fallopian tube. However, little research has shown that mesenchymal stem cells (MSCs) can be used to treat fallopian tube damage by in situ injection. In this study, we in situ transplanted PF127 hydrogel encapsulating dental pulp stem cells (DPSCs) into the injured sites to promote the repair and regeneration of fallopian tube injury.Materials and methods: The properties of dental pulp stem cells were evaluated by flow cytometry, immunofluorescence analysis, and multi-differentiation detection. The immunomodulatory and angiogenic characteristics of dental pulp stem cells were analyzed on the basis of the detection of inflammatory factor expression and the formation of capillary-like structures, respectively. The biocompatibility of PF127 hydrogel was evaluated by using Live/Dead and CCK-8 assays. The effects of PF127 hydrogel containing dental pulp stem cells on the repair and regeneration of fallopian tube injury were evaluated by histological analysis [e.g., hematoxylin and eosin (H&E) and Masson’s trichrome staining, TUNEL staining, immunofluorescence staining, and immunohistochemistry], Enzyme-linked immunosorbent assay (ELISA), and RT-PCR detections.Results: Dental pulp stem cells had MSC-like characteristics and great immunomodulatory and angiogenic properties. PF127 hydrogel had a thermosensitive feature and great cytocompatibility with dental pulp stem cells. In addition, our results indicated that PF127 hydrogel containing dental pulp stem cells could promote the repair and regeneration of fallopian tube damage by inhibiting cell apoptosis, stimulating the secretion of angiogenic factors, promoting cell proliferation, modulating the secretion of inflammatory factors, and restoring the secretion of epithelial cells.Conclusion: In this study, our results reported that in situ injection of PF127 hydrogel encapsulating dental pulp stem cells into the injured sites could provide an attractive strategy for the future treatment of fallopian tube injury in clinical settings

TISSUE ENGINEERING IN REGENERATIVE ENDODONTICS -A REVIEW

Tissue engineering is mainly used to replace the impaired or damaged tissues with new tissues. The key cells involved for tissue engineering are stem cells, the morphogens or growth factors. They will rapidly multiply and differentiates and forms tissues. This new technique is now most commonly used in endodontics. The aim of this study was to review about the dental pulp stem cells, which are most common growth factors, and the scaffolds used to control their differentiation. To study the clinical technique for the management of immature non-vital teeth based on this novel concept.KEYWORDS: Dental pulp stem cells; Morphogens; Scaffolds; Regenerative endodontics; Pulp revascularization

TISSUE ENGINEERING IN REGENERATIVE ENDODONTICS -A REVIEW

Tissue engineering is mainly used to replace the impaired or damaged tissues with new tissues. The key cells involved for tissue engineering are stem cells, the morphogens or growth factors. They will rapidly multiply and differentiates and forms tissues. This new technique is now most commonly used in endodontics. The aim of this study was to review about the dental pulp stem cells, which are most common growth factors, and the scaffolds used to control their differentiation. To study the clinical technique for the management of immature non-vital teeth based on this novel concept.KEYWORDS: Dental pulp stem cells; Morphogens; Scaffolds; Regenerative endodontics; Pulp revascularization

Perivascular Niche and Self-renewal of Dental Pulp Stem Cells

Interactions with the microenvironment modulate the fate of stem cells in perivascular niches in tissues (e.g. bone) and organs (e.g. liver). However, the functional relevance of the molecular crosstalk between endothelial cells and stem cells within the perivascular niche in dental pulps is unclear. Here, we tested the hypothesis that endothelial cell-initiated signaling is necessary to maintain self-renewal of dental pulp stem cells. First, we show preferential localization of cells that express high levels of self-renewal stem cell markers (i.e. ALDH1 and Bmi-1) in physiological human dental pulps. Then, through an in vitro functional assay for self-renewal (i.e. secondary orosphere assay), we show that endothelial cell-derived factors promote self-renewal of dental pulp stem cells. Mechanistic studies demonstrated that endothelial cell-derived Interleukin (IL)-6 induces expression of Bmi-1 in dental pulp stem cells through the STAT3 signaling pathway. STAT3-silenced dental pulp stem cells (stably transduced with shRNA-STAT3) seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated fewer perivascular niches comprised of endothelial cells and stem cells exhibiting features of self-renewal. In vitro capillary sprouting assays revealed that inhibition of IL-6 or STAT3 signaling decreases the vasculogenic potential of dental pulp stem cells, suggesting its importance in the establishment of the perivascular niche. Collectively, these data demonstrate that endothelial cell-derived factors mediate self-renewal of dental pulp stem cells through STAT3 signaling and induction of Bmi-1. Overall, these data suggest that a crosstalk between endothelial cells and stem cells within the perivascular niche is required for the maintenance of stem cell pools in dental pulp.PHDOral Health SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155181/1/minoh_1.pd

Evaluating the efficacy of human dental pulp stem cells and scaffold combination for bone regeneration in animal models:A systematic review and meta-analysis

Abstract Introduction Human adult dental pulp stem cells (hDPSC) and stem cells from human exfoliated deciduous teeth (SHED) hold promise in bone regeneration for their easy accessibility, high proliferation rate, self-renewal and osteogenic differentiation capacity. Various organic and inorganic scaffold materials were pre-seeded with human dental pulp stem cells in animals, with promising outcomes in new bone formation. Nevertheless, the clinical trial for bone regeneration using dental pulp stem cells is still in its infancy. Thus, the aim of this systematic review and meta-analysis is to synthesise the evidence of the efficacy of human dental pulp stem cells and the scaffold combination for bone regeneration in animal bone defect models. Methodology This study was registered in PROSPERO (CRD2021274976), and PRISMA guideline was followed to include the relevant full-text papers using exclusion and inclusion criteria. Data were extracted for the systematic review. Quality assessment and the risk of bias were also carried out using the CAMARADES tool. Quantitative bone regeneration data of the experimental (scaffold + hDPSC/SHED) and the control (scaffold-only) groups were also extracted for meta-analysis. Results Forty-nine papers were included for systematic review and only 27 of them were qualified for meta-analysis. 90% of the included papers were assessed as medium to low risk. In the meta-analysis, qualified studies were grouped by the unit of bone regeneration measurement. Overall, bone regeneration was significantly higher (p < 0.0001) in experimental group (scaffold + hDPSC/SHED) compared to the control group (scaffold-only) (SMD: 1.863, 95% CI 1.121–2.605). However, the effect is almost entirely driven by the % new bone formation group (SMD: 3.929, 95% CI 2.612–5.246) while % BV/TV (SMD: 2.693, 95% CI − 0.001–5.388) shows a marginal effect. Dogs and hydroxyapatite-containing scaffolds have the highest capacity in % new bone formation in response to human DPSC/SHED. The funnel plot exhibits no apparent asymmetry representing a lack of remarkable publication bias. Sensitivity analysis also indicated that the results generated in this meta-analysis are robust and reliable. Conclusion This is the first synthesised evidence showing that human DPSCs/SHED and scaffold combination enhanced bone regeneration highly significantly compared to the cell-free scaffold irrespective of scaffold type and animal species used. So, dental pulp stem cells could be a promising tool for treating various bone diseases, and more clinical trials need to be conducted to evaluate the effectiveness of dental pulp stem cell-based therapies

EFFECTS OF DIFFERENT MICROENVIRONMENTAL CONDITIONS ON THE GROWTH AND DIFFERENTIATION OF DENTAL PULP STEM CELLS

Human teeth are very complex structures that are susceptible to many different pathologies due to poor dental health. Currently, there are many restorative methods to reestablish some of the function that teeth have, but the materials used in these methods all have drawbacks and cannot fully mimic the native teeth. Tissue engineering research groups have begun to explore regenerating bone or dental tissue using mesenchymal stem cells derived from the bone marrow. However, our group focuses on regenerating dental tissues using multipotent stem cells from dental pulp. Dental pulp stem cells (DPSCs) have shown similarities to bone marrow stem cells in in that they can differentiate into many cell types. Also, stem cells in general have shown that differentiation can be induced with microenvironmental factors such as growth factors and substrate properties. If enough is known about the cues that cells receive that induces differentiation, tissues could be engineered using the constructs and growth conditions necessary. To determine the effect of substrate stiffness on human DPSCs, cells were placed on polyacrylamide gels of varying stiffness and in varying growth factor conditions. The cells were then observed with light and confocal microscopy, and the amount of alkaline phosphatase (ALP) activity was measured. These tests gave an indication of growth and differentiation. It was seen that the growth patterns were different on the gels than they were on a glass control, but there was little difference between the two gels. Also, the growth factors did not appear to have a significant contribution to differentiation. Much work has been done to determine the effects of mechanical compression muscleoskeletal tissues, such as cartilage and bone. Dental tissues are also subject to loading throughout the day. Therefore, it was hypothesized that if dental pulp stem cells are compressed with pressure similar to that seen physiologically, it will induce differentiation to a bone or tooth-like lineage. To determine the effects of static compression on dental pulp stem cells, a custom compressive device was fabricated. The device was tested for usability and it was deemed acceptable for use. ALP assays were performed similar to the previous studies. Preliminary results showed that that after only 1 day of culture time, the compression did not have much of an effect on dental pulp stem cells, while it did have an effect on osteoblasts. More work is to be done to determine the effects of compressive forces on dental pulp stem cells

Histomorphometric evaluation of bone regeneration induced by biodegradable scaffolds as carriers for dental pulp stem cells in a rat model of calvarial "critical size" defect

Objective: The aim of this study was to test specific stem cells that could enhance bone formation in combination with specific scaffolds. Methods: Dental Pulp Stem Cells (DPSCs) were seeded with Granular Deproteinized Bovine Bone (GDPB) or Beta-Tricalcium Phosphate (ß-TCP) in a rat model of calvarial "critical size" defect. DPSCs were isolated from permanent human teeth, obtained and characterized using specific stem cells markers (Nanog and Oct-4) by real time-PCR and immunofluorescence. Cells were differentiated for 10-15 days towards the osteoblastic phenotype with 100μM L-ascorbic acid, added every day in culture medium and 20 vol. percentage of FBS in α-MEM medium. Osteogenic commitment was evaluated with real time-PCR by measuring the expression of specific markers (osteonectin and runx2). When a sufficient cell number was obtained, DPSCs were trypsinized, washed in culture medium and seeded onto the GDPB and ß-TCP scaffold sat a density of 0.5-1×106 cells/scaffold. Two bilateral critical-size circular defects (5 mm diameter; 1 mm thickness) were created from the parietal bone of the 8 athymic T-cell deficient nude rats. One cranial defect for each rat was filled with the scaffold alone and the other defect with the scaffold seeded with stem cells. After 12 weeks post-surgery animals were euthanized and histomorphometric analysis was performed. Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's Protected Least Significant Difference (PLSD) post-hoc test. A p-value &lt;0.05 was considered statistically significant. Results: GDPB group presented higher percentage of lamellar bone than that of GDPB/DPSC, ß-TCP alone had lower levels as compared to ß-TCP/DPSC. The addition of stem cells significantly increased woven bone formation in both scaffold-based implants, although still higher in GDPB based implants. Conclusion: Our findings indicate that GDPB and ß-TCP used as scaffold to induce bone regeneration may benefit from adding DPSC to tissue-engineered constructs