Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

Human dental pulp stem cells differentiation into odontoblast and osteoblast-like cells on scaffolds contain bioactive materials.

Epigenetic change has been found to play an important role in cell differentiation and regulation and the dental pulp stem cell in tissue engineering is gaining attention due to the ability of cells to differentiate into odontoblast and other cells. This study evaluated the influence of poly L- lactic acid with hydroxyapatite-coated with polyaniline scaffold (PLLA/HA/PANI) on dental pulp stem cell (DPSC) proliferation and differentiation. After scaffold preparation and DPSCs seeding, the cells proliferation and differentiation were evaluated by immunocytochemistry assay and cell viability was measured by cytotoxicity / MTT assay. The results showed (PLLA/HA/PANI) scaffold facilitates DPSC proliferation and differentiation with gene expression. This finding underscores the promise of this biomaterial combination as a scaffold for dental tissue regeneration and application.

An innovative cell-based transplantation therapy for an immature permanent tooth in an adult: a case report.

BACKGROUND: Immature teeth with necrotic pulps present multiple challenges to clinicians. In such cases, regenerative endodontic procedures (REPs) may be a favorable strategy. Cells, biomaterial scaffolds, and signaling molecules are three key elements of REPs. Autologous human dental pulp cells (hDPCs) play an important role in pulp regeneration. In addition, autologous platelet concentrates (APCs) have recently been demonstrated as effective biomaterial scaffolds in regenerative dentistry, whereas the latest generation of APCs-concentrated growth factor (CGF), especially liquid phase CGF (LPCGF)-has rarely been reported in REPs. CASE PRESENTATION: A 31-year-old woman presented to our clinic with the chief complaint of occlusion discomfort in the left mandibular posterior region for the past 5 years. Tooth #35 showed no pulp vitality and had a periodontal lesion, and radiographic examination revealed that the tooth exhibited extensive periapical radiolucency with an immature apex and thin dentin walls. REP was implemented via transplantation of autologous hDPCs with the aid of LPCGF. The periodontal lesion was managed with simultaneous periodontal surgery. After the treatment, the tooth was free of any clinical symptoms and showed positive results in thermal and electric pulp tests at 6- and 12-month follow-ups. At 12-month follow-up, radiographic evidence and three-dimensional models, which were reconstructed using Mimics software based on cone-beam computed tomography, synergistically confirmed bone augmentation and continued root development, indicating complete disappearance of the periapical radiolucency, slight lengthening of the root, evident thickening of the canal walls, and closure of the apex. CONCLUSION: hDPCs combined with LPCGF represents an innovative and effective strategy for cell-based regenerative endodontics.

The Effect of Glycyrrhizin on the Viability and Proliferation of Dental Pulp Stem Cells Compared to Intracanal Medicaments.

AIM: To study the effect of glycyrrhizin (GA) on the viability and proliferation of dental pulp stem cells (DPSCs) compared with intracanal medicaments. MATERIALS AND METHODS: Third molars of an adult donor were used to obtain the DPSCs. Flow cytometry was utilized to conduct phenotypic analysis for DPSCs. The methyl-thiazol tetrazolium (MTT) test was used to detect the cell viability. Cell proliferation assay was conducted at distinct time intervals: 3, 5, and 7 days. RESULTS: The flow cytometry analysis verified the positive expression of mesenchymal cell surface antigen molecules (CD73, CD90, and CD105) and the absence of hematological markers (CD14, CD34, and CD45) in the DPSCs. The cells that treated with concentrations more than 0.5 mg/mL of Ca(OH2) and triple antibiotic paste (TAP) gave significant decrease in viability in comparison to the untreated cells (p < 0.05). Also, the cells treated with concentrations 50 and 25 µM of GA showed no significant difference compared with the untreated cells (p > 0.05), while concentrations 12.5 and 6.25 µM expressed a significant increase in viability compared with the untreated cells (p < 0.05). At 7 days, cells treated with the three different concentrations of GA (12.5, 25, and 50 µM) demonstrated a significant increase in cell density compared with Ca(OH)2 and TAP-treated cells (p < 0.05). CONCLUSION: Based upon the potential of GA on DPSCs proliferation compared with Ca(OH)2 and TAP, It is conceivable to acknowledge that GA could be used as an intracanal medicaments for revascularization process of necrotic immature teeth. CLINICAL SIGNIFICANCE: This study emphasizes the significance of assessing alternative root canal medicaments and their impact on the proliferation and viability of DPSCs. The results regarding GA, specifically its impact on the viability and growth of DPSCs, provide essential understanding for its potential application as an intracanal medicine. This study adds to the continuous endeavors in identifying safer and more efficient intracanal therapies, which are essential for improving patient outcomes in endodontic operations. How to cite this article: Alrashidi MA, Badawi MF, Elbeltagy MG, et al. The Effect of Glycyrrhizin on the Viability and Proliferation of Dental Pulp Stem Cells Compared to Intracanal Medicaments. J Contemp Dent Pract 2024;25(3):267-275.

Electrical Stimulations Generated by P(VDF-TrFE) Films Enhance Adhesion Forces and Odontogenic Differentiation of Dental Pulp Stem Cells (DPSCs).

Biophysical and biochemical cues of biomaterials can regulate cell behaviors. Dental pulp stem cells (DPSCs) in pulp tissues can differentiate to odontoblast-like cells and secrete reparative dentin to form a barrier to protect the underlying pulp tissues and enable complete pulp healing. Promotion of the odontogenic differentiation of DPSCs is essential for dentin regeneration. The effects of the surface potentials of biomaterials on the adhesion and odontogenic differentiation of DPSCs remain unclear. Here, poly(vinylidene fluoride-trifluoro ethylene) (P(VDF-TrFE)) films with different surface potentials were prepared by the spin-coating technique and the contact poling method. The cytoskeletal organization of DPSCs grown on P(VDF-TrFE) films was studied by immunofluorescence staining. Using atomic force microscopy (AFM), the lateral detachment forces of DPSCs from P(VDF-TrFE) films were quantified. The effects of electrical stimulation generated from P(VDF-TrFE) films on odontogenic differentiation of DPSCs were evaluated in vitro and in vivo. The unpolarized, positively polarized, and negatively polarized films had surface potentials of -52.9, +902.4, and -502.2 mV, respectively. DPSCs on both negatively and positively polarized P(VDF-TrFE) films had larger cell areas and length-to-width ratios than those on the unpolarized films (P < 0.05). During the detachment of DPSCs from P(VDF-TrFE) films, the average magnitudes of the maximum detachment forces were 29.4, 72.1, and 53.9 nN for unpolarized, positively polarized, and negatively polarized groups, respectively (P < 0.05). The polarized films enhanced the mineralization activities and increased the expression levels of the odontogenic-related proteins of DPSCs compared to the unpolarized films (P < 0.05). The extracellular signal-regulated kinase (ERK) signaling pathway was involved in the odontogenic differentiation of DPSCs as induced by surface charge. In vivo, the polarized P(VDF-TrFE) films enhanced adhesion of DPSCs and promoted the odontogenic differentiation of DPSCs by electrical stimulation, demonstrating a potential application of electroactive biomaterials for reparative dentin formation in direct pulp capping.

An injectable dental pulp-derived decellularized matrix hydrogel promotes dentin repair through modulation of macrophage response.

Maintaining the viability of damaged pulp is critical in clinical dentistry. Pulp capping, by placing dental material over the exposed pulp, is a main approach to promote pulp-dentin healing and mineralized tissue formation. The dental materials are desired to impact on intricate physiological mechanisms in the healing process, including early regulation of inflammation, immunity, and cellular events. In this study, we developed an injectable dental pulp-derived decellularized matrix (DPM) hydrogel to modulate macrophage responses and promote dentin repair. The DPM derived from porcine dental pulp has high collagen retention and low DNA content. The DPM was solubilized by pepsin digestion (named p-DPM) and subsequently injected through a 25G needle to form hydrogel facilely at 37 °C. In vitro results demonstrated that the p-DPM induced the M2-polarization of macrophages and the migration, proliferation, and dentin differentiation of human dental pulp stem cells from deciduous teeth (SHEDs). In a mouse subcutaneous injection test, the p-DPM hydrogel was found to facilitate cell recruitment and M2 polarization during the early phase of implantation. Additionally, the acute pulp restoration in rat models proved that injectable p-DPM hydrogel as a pulp-capping agent had excellent efficacy in dentin regeneration. This study demonstrates that the DPM promotes dentin repair by modulating macrophage responses, and has a potential for pulp-capping applications in dental practice.

Understanding the multi-functionality and tissue-specificity of decellularized dental pulp matrix hydrogels for endodontic regeneration.

Dental pulp is the only soft tissue in the tooth which plays a crucial role in maintaining intrinsic multi-functional behaviors of the dentin-pulp complex. Nevertheless, the restoration of fully functional pulps after pulpitis or pulp necrosis, termed endodontic regeneration, remained a major challenge for decades. Therefore, a bioactive and in-situ injectable biomaterial is highly desired for tissue-engineered pulp regeneration. Herein, a decellularized matrix hydrogel derived from porcine dental pulps (pDDPM-G) was prepared and characterized through systematic comparison against the porcine decellularized nerve matrix hydrogel (pDNM-G). The pDDPM-G not only exhibited superior capabilities in facilitating multi-directional differentiation of dental pulp stem cells (DPSCs) during 3D culture, but also promoted regeneration of pulp-like tissues after DPSCs encapsulation and transplantation. Further comparative proteomic and transcriptome analyses revealed the differential compositions and potential mechanisms that endow the pDDPM-G with highly tissue-specific properties. Finally, it was realized that the abundant tenascin C (TNC) in pDDPM served as key factor responsible for the activation of Notch signaling cascades and promoted DPSCs odontoblastic differentiation. Overall, it is believed that pDDPM-G is a sort of multi-functional and tissue-specific hydrogel-based material that holds great promise in endodontic regeneration and clinical translation. STATEMENT OF SIGNIFICANCE: Functional hydrogel-based biomaterials are highly desirable for endodontic regeneration treatments. Decellularized extracellular matrix (dECM) preserves most extracellular matrix components of its native tissue, exhibiting unique advantages in promoting tissue regeneration and functional restoration. In this study, we prepared a porcine dental pulp-derived dECM hydrogel (pDDPM-G), which exhibited superior performance in promoting odontogenesis, angiogenesis, and neurogenesis of the regenerating pulp-like tissue, further showed its tissue-specificity compared to the peripheral nerve-derived dECM hydrogel. In-depth proteomic and transcriptomic analyses revealed that the activation of tenascin C-Notch axis played an important role in facilitating odontogenic regeneration. This biomaterial-based study validated the great potential of the dental pulp-specific pDDPM-G for clinical applications, and provides a springboard for research strategies in ECM-related regenerative medicine.

Effects of Ibuprofen and Diclofenac Pre-Treatment on Viability and Apoptosis Processes in Human Dental Pulp Stem Cells.

Background and Objectives: Stem cell-based regeneration strategies have shown therapeutic efficacy in various fields of regenerative medicine. These include bone healing after bone augmentation, often complicated by pain, which is managed by using nonsteroidal anti-inflammatory drugs (NSAIDs). However, information is limited about how NSAIDs affect the therapeutic potential of stem cells. Materials and Methods: We investigated the effects of ibuprofen and diclofenac on the characteristics, morphology, and immunophenotype of human mesenchymal stromal cells isolated from the dental pulp (DPSCs) and cultured in vitro, as well as their effects on the expression of angiogenic growth factors (VEGFA and HGF) and selected genes in apoptosis signalling pathways (BAX, BAK, CASP3, CASP9, and BCL2). Results: Ibuprofen and diclofenac significantly reduced the viability of DPSCs, while the expression of mesenchymal stem cell surface markers was unaffected. Both ibuprofen and diclofenac treatment significantly upregulated the expression of HGF, while the expression of VEGFA remained unchanged. Ibuprofen significantly altered the expression of several apoptosis-related genes, including the upregulation of CASP9 and BCL2, with decreased CASP3 expression. BAK, CASP3, CASP9, and BCL2 expressions were significantly increased in the diclofenac-treated DPSCs, while no difference was demonstrated in BAX expression. Conclusions: Our results suggest that concomitant use of the NSAIDs ibuprofen or diclofenac with stem cell therapy may negatively impact cell viability and alter the expression of apoptosis-related genes, affecting the efficacy of stem cell therapy.

Dental pulp stem cells ameliorate D-galactose-induced cardiac ageing in rats.

Background: Ageing is a key risk factor for cardiovascular disease and is linked to several alterations in cardiac structure and function, including left ventricular hypertrophy and increased cardiomyocyte volume, as well as a decline in the number of cardiomyocytes and ventricular dysfunction, emphasizing the pathological impacts of cardiomyocyte ageing. Dental pulp stem cells (DPSCs) are promising as a cellular therapeutic source due to their minimally invasive surgical approach and remarkable proliferative ability. Aim: This study is the first to investigate the outcomes of the systemic transplantation of DPSCs in a D-galactose (D-gal)-induced rat model of cardiac ageing. Methods. Thirty 9-week-old Sprague-Dawley male rats were randomly assigned into three groups: control, ageing (D-gal), and transplanted groups (D-gal + DPSCs). D-gal (300 mg/kg/day) was administered intraperitoneally daily for 8 weeks. The rats in the transplantation group were intravenously injected with DPSCs at a dose of 1 × 106 once every 2 weeks. Results: The transplanted cells migrated to the heart, differentiated into cardiomyocytes, improved cardiac function, upregulated Sirt1 expression, exerted antioxidative effects, modulated connexin-43 expression, attenuated cardiac histopathological alterations, and had anti-senescent and anti-apoptotic effects. Conclusion: Our results reveal the beneficial effects of DPSC transplantation in a cardiac ageing rat model, suggesting their potential as a viable cell therapy for ageing hearts.

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

The profile of cytokines against bacterial infection in dental pulp.

BACKGROUND: Dental pulp in a confined environment, with little connection to the outside and only a small distribution of immune cells, provides a good research model for investigating how cells respond to bacterial infections through cytokines. METHODS: The data of single-cell transcriptome sequencing of healthy and inflamed pulp tissue were downloaded from the GEO dataset. The expression character of 79 cytokines was analyzed based on the expression matrix. RESULTS: The cytokine secretion profiles of the two populations of pulp cells in healthy dental pulp were associated with vascularization and nervous system development, as well as immune cell regulation. For the three populations of pulp stem cells with stem cell activity in the dental pulp, the secretion of cytokines related to nervous system development, regulation of endothelial cell proliferation and migration, and regulation of immune cell function comprised the characteristics that we observed. The cytokines secreted by T cells and macrophages were more of an immune reserve against pathogenic microorganisms. In the inflammatory state, the spectrum of cytokines secreted by various types of cells in the dental pulp tended to be identical, such that it mainly resisted pathogenic microorganisms. CONCLUSIONS: The cytokine secretion profiles of various cell types in healthy and inflamed dental pulp at the single-cell level are summarized.

Comparison of the vitality test with sensitivity tests in mature and immature teeth: clinical trial.

BACKGROUND: One of the most important steps in deciding on the treatment of a tooth is to determine the vitality and health status of the pulp. Since immature teeth innervation is not completed, the response to sensitivity tests may not yield definite results. Pulse oximetry (PO) which is considered as a vitality test, measures the arterial oxygen saturation (SpO2). This study aims to compare PO, electric pulp test (EPT) and cold test in mature and immature permanent teeth. METHODS: 20 immature and 20 mature permanent incisors of 6-12-year-old ASA1 children who did not use any analgesics, were included in the study. Pulp vitality of the teeth was determined by EPT, cold test and PO. An infant probe of PO device (CMS60D, Contec Medical Systems Co. Ltd, China) was used to determine the SpO2 of the teeth. The SpO2 level is controlled on the patient's finger by a children's probe and an infant probe of PO. Shapiro-Wilk, Spearman rank correlation test and Kruskal-Wallis test/Dunn post-hoc analysis were used for statistical comparisons. RESULTS: There was no significant correlation between finger SpO2 and the mature/immature teeth SpO2 (r=-0.026, r = 0.253). Arterial oxygen saturation values in the immature teeth were significantly higher than in the mature teeth (p = 0.002). There was a high correlation between the vitality response of the EPT, cold test and PO. CONCLUSIONS: Pulse oximetry can be used as an effective vitality test compared to sensitivity tests in both immature and mature permanent incisors.

Effect of submucosal cryotherapy compared with steroids and NSAIDs injections on Substance P and Interleukin 6 pulpal release in experimentally induced pulpal inflammation in rabbits.

OBJECTIVE: To compare the effect of submucosal cryotherapy using cold saline to dexamethasone sodium phosphate and diclofenac sodium injections on substance P and interleukin 6 release in experimentally induced pulpal inflammation in rabbits' molar teeth. METHODOLOGY: Fifteen rabbits were randomly classified into 3 groups according to the submucosal injection given: cold saline, dexamethasone sodium phosphate, and diclofenac sodium. A split-mouth design was adopted, the right mandibular molars were experimental, and the left molars served as the control without injections. Intentional pulp exposures were created and left for 6 hours to induce pulpitis. Pulpal tissue was extracted and examined for SP and IL-6 levels using ELISA. Within each group, the level of cytokines released was measured for both control and experimental groups for intragroup comparison to determine the effect of injection. The percentage reduction of each mediator was calculated compared with the control side for intergroup comparison then the correlation between SP and IL-6 levels was analyzed using Spearman's rank order correlation coefficient. Statistical analysis was performed, and the significance level was set at p<0.05. RESULTS: Submucosal cryotherapy, dexamethasone sodium phosphate, and diclofenac sodium significantly reduced SP and IL-6 pulpal release. Submucosal cryotherapy significantly reduced SP more than and IL-6 more than dexamethasone sodium phosphate and diclofenac sodium. Pulpal reduction of SP and IL-6 showed a strong positive significant correlation. CONCLUSIONS: Submucosal cryotherapy reduces the pulpal release of SP and IL-6 and could be tested as an alternative to premedication to potentiate the effect of anesthesia and control postoperative endodontic pain.

Macrophage M2 polarization promotes pulpal inflammation resolution during orthodontic tooth movement.

Mechanical force induces hypoxia in the pulpal area by compressing the apical blood vessels of the pulp, triggering pulpal inflammation during orthodontic tooth movement. However, this inflammation tends to be restorable. Macrophages are recognized as pivotal immunoreactive cells in the dental pulp. Whether they are involved in the resolution of pulpal inflammation in orthodontic teeth remains unclear. In this study, we investigated macrophage polarization and its effects during orthodontic tooth movement. It was demonstrated that macrophages within the dental pulp polarized to M2 type and actively participated in the process of pulpal inflammation resolution. Inflammatory reactions were generated and vascularization occurred in the pulp during orthodontic tooth movement. Macrophages in orthodontic pulp show a tendency to polarize towards M2 type as a result of pulpal hypoxia. Furthermore, by blocking M2 polarization, we found that macrophage M2 polarization inhibits dental pulp-secreting inflammatory factors and enhances VEGF production. In conclusion, our findings suggest that macrophages promote pulpal inflammation resolution by enhancing M2 polarization and maintaining dental health during orthodontic tooth movement.

Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells.

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

Deciduous pulp tissue implantation into the root canal of mandibular incisor resulted in pulp revascularization: a case report with a 5-year follow-up.

To explore a new method to implant deciduous tooth pulp into the canal of young permanent teeth with necrotic pulps and apical periodontitis for the regenerative endodontic treatment of tooth no: 41 in a 7-year-old male. Briefly, 1.5% Sodium Hypochlorite (NaOCl) irrigation and calcium hydroxide-iodoform paste were used as root canal disinfectant at the first visit. After 2 weeks, the intracanal medication was removed, and the root canal was slowly rinsed with 17% Ethylene Diamine Tetraacetic Acid (EDTA), followed by flushing with 20 mL saline and then drying with paper points. Tooth no: 72 was extracted, and its pulp was extracted and subsequently implanted into the disinfected root canal along with induced apical bleeding. Calcium hydroxide iodoform paste was gently placed over the bleeding clot, and after forming a mineral trioxide aggregate (MTA) coronal barrier, the accessed cavities were restored using Z350 resin composite. The root developments were evaluated via radiographic imaging at 6 months, 1 year and 5 years after treatment. Imaging and clinical analysis showed closure of the apical foramen, thickening of the root canal wall, and satisfactory root length growth. Autologous transplantation might be useful to regenerate dental pulp in necrotic young permanent teeth.

Human dental pulp stem cells mitigate the neuropathology and cognitive decline via AKT-GSK3ß-Nrf2 pathways in Alzheimer's disease.

Oxidative stress is increasingly recognized as a major contributor to the pathophysiology of Alzheimer's disease (AD), particularly in the early stages of the disease. The multiplicity advantages of stem cell transplantation make it fascinating therapeutic strategy for many neurodegenerative diseases. We herein demonstrated that human dental pulp stem cells (hDPSCs) mediated oxidative stress improvement and neuroreparative effects in in vitro AD models, playing critical roles in regulating the polarization of hyperreactive microglia cells and the recovery of damaged neurons. Importantly, these therapeutic effects were reflected in 10-month-old 3xTg-AD mice after a single transplantation of hDPSCs, with the treated mice showing significant improvement in cognitive function and neuropathological features. Mechanistically, antioxidant and neuroprotective effects, as well as cognitive enhancements elicited by hDPSCs, were at least partially mediated by Nrf2 nuclear accumulation and downstream antioxidant enzymes expression through the activation of the AKT-GSK3ß-Nrf2 signaling pathway. In conclusion, our findings corroborated the neuroprotective capacity of hDPSCs to reshape the neuropathological microenvironment in both in vitro and in vivo AD models, which may be a tremendous potential therapeutic candidate for Alzheimer's disease.

Mesenchymal Stem Cells from the Deciduous Tooth Pulp Lose their Ability to Suppress the Differentiation of Dendritic Cells during Long-Term Culturing.

A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products.

Functional extracellular vesicles from SHEDs combined with gelatin methacryloyl promote the odontogenic differentiation of DPSCs for pulp regeneration.

BACKGROUND: Pulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration. RESULTS: We successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). CONCLUSIONS: The effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.

Comparative proteomic analysis of dental pulp from supernumerary and normal permanent teeth.

OBJECTIVES: To obtain and compare the protein profiles of supernumerary and normal permanent dental pulp tissues. MATERIALS AND METHODS: Dental pulp tissues were obtained from supernumerary and normal permanent teeth. Proteins were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS). Protein identification and quantification from MS data was performed with MaxQuant. Statistical analysis was conducted using Metaboanalyst to identify differentially expressed proteins (DEPs) (P-value < 0.05, fold-change > 2). Gene Ontology enrichment analyses were performed with gProfiler. RESULTS: A total of 3,534 proteins were found in normal dental pulp tissue and 1,093 in supernumerary dental pulp tissue, with 174 DEPs between the two groups. This analysis revealed similar functional characteristics in terms of cellular component organization, cell differentiation, developmental process, and response to stimulus, alongside exclusive functions unique to normal permanent dental pulp tissues such as healing, vascular development and cell death. Upon examination of DEPs, these proteins were associated with the processes of wound healing and apoptosis. CONCLUSIONS: This study provides a comprehensive understanding of the protein profile of dental pulp tissue, including the first such profiling of supernumerary permanent dental pulp. There are functional differences between the proteomic profiles of supernumerary and normal permanent dental pulp tissue, despite certain biological similarities between the two groups. Differences in protein expression were identified, and the identified DEPs were linked to the healing and apoptosis processes. CLINICAL RELEVANCE: This discovery enhances our knowledge of supernumerary and normal permanent pulp tissue, and serves as a valuable reference for future studies on supernumerary teeth.