Human dental pulp stem cells (hDPSCs) are a promising cell source for tooth regeneration therapies. However, conventional culture scaffold materials are often animal-derived, leading to immunogenicity concerns and limited availability. In this study, we explored phosphorylated cellulose nanofibers (P-CNFs), which have a fine fiber morphology and phosphate groups, as a novel scaffold material for cell culture. Immortalized hDPSCs were cultured on P-CNF scaffolds with different phosphate contents (0-1.42 mmol g-1) prepared by varying the molar ratio of urea and diammonium hydrogen phosphate and the reaction time. Cells cultured on unmodified CNFs exhibited poor adhesion and formed spheroids, indicating low bioadaptability. In contrast, P-CNF scaffolds with moderate phosphate content (0.54-0.78 mmol g-1) significantly improved cell adhesion; further increases in phosphate content decreased cell adhesion, indicating a strong dependence on phosphate content. Intriguingly, even in the absence of differentiation inducers, hDPSCs on P-CNF scaffolds with an optimal phosphate content of 0.78 mmol g-1 showed equal or higher expression of hard tissue marker genes compared to collagen scaffolds with differentiation inducers, suggesting that P-CNFs can directly promote hard tissue differentiation. These findings highlight plant-derived, animal-free P-CNFs as a promising biomaterial for advanced dental tissue engineering.

Stem cell treatment shows promise in treating conditions such as neurodegenerative disorders and spinal injuries, but its effectiveness is hampered by cell death and apoptosis. Improving the differentiation of MSCs into neural cells could enhance their therapeutic potential. The role of miR-22-3p in human dental pulp stem cells (HDPSCs), a superior alternative to treat neurodegenerative disorders, and its molecular mechanisms during neural differentiation remain elusive. Therefore, we investigated the miR-22-3p transfections during HDPSC differentiation into neural progenitor-like cells (NPCs) and elucidated the molecular processes through transcriptomic analysis. HDPSCs were differentiated into NPCs after transfection with a miR-22-3p mimic and inhibitor; the differentiation process was assessed by cell viability and expression of Nestin protein. mRNA sequencing on days 1, 3, and 7 of the differentiation process identified several differentially expressed genes (DEGs). Cytoscape and functional enrichment analysis pinpointed central hub genes among the DEGs and uniquely expressed genes. miR-22-3p mimic hindered HDPSC differentiation by reducing proliferation and increasing apoptosis. It downregulated genes linked to extracellular matrix, synaptic and vesicle functions, lipid metabolism, JAK-STAT, and cell cycle pathways across all days while activating proteasome and digestion pathways. In contrast, miR-22-3p inhibition boosts NPC proliferation and elevates Nestin neural marker protein expression. Altogether, miR-22-3p disrupts synapse functioning and lipid metabolism pathways, resulting in apoptosis and death. Conversely, inhibiting miR-22-3p enhances neural differentiation and proliferation of HDPSCs, suggesting its potential application in generating a greater quantity of NPCs and neurons.

Calcium silicate-based bioactive glass shows enhanced ion release capabilities and promotes the formation of hydroxyapatite (HA). This study aimed to synthesize a sol-gel-derived 68S bioactive glass (BAG) incorporating lithium (Li) and evaluate its structural, biological, and osteoinductive properties using human dental pulp stem cells (hDPSCs).

Two types of 68S BAG were synthesized using the sol-gel method: one containing 5 mol.% lithium nitrate (BGLi5) and a lithium-free control (BG). Structural characterization and HA formation were assessed using field emission scanning electron microscopy (FESEM) and Fourier-transform infrared spectroscopy (FTIR) before and after immersion in simulated body fluid (SBF) on Days 1, 3, and 7. The dissolution rates of the specimens were evaluated using inductively coupled plasma atomic emission spectroscopy (ICP-AES) and pH analysis. Biological activities were investigated through cell viability (MTT assay), alkaline phosphatase (ALP) enzyme activity, and alizarin red staining to assess mineralization. Additionally, the antimicrobial efficacy of the materials was tested against Streptococcus mutans (SM).

FTIR and FESEM analyses confirmed the formation of HA crystals in BGLi5 specimens by Day 3 and in BG specimens by Day 7. The MTT assay demonstrated enhanced cell viability in both BG and BGLi5 compared to the control group. ALP activity, a marker of cell differentiation, was significantly elevated in the BGLi5-DM group by Day 14. Alizarin red staining on Day 21 revealed a marked increase in mineralization in both BG and BGLi5, with the BGLi5-DM group showing the highest mineralization levels. Furthermore, both BG and BGLi5 demonstrated significant antimicrobial activity against SM.

The sol-gel-derived 68S BAG containing 5 mol.% Li is a biocompatible material that enhances cell proliferation, differentiation, and mineralization. The combination of BGLi5 with differentiation-specific culture medium synergistically promotes osteogenic differentiation and mineralization, making it a promising candidate for dental and bone tissue engineering applications.

To develop and characterize a novel bioactive polydopamine (PDA)-containing glass-ionomer cement (Dopamer) with enhanced mechanical, antibacterial, and mineralization properties for use as a restorative dental material.

Dopamer was developed by coating fluoroaluminosilicate glass particles with polydopamine (PDA) via dopamine polymerization in alkaline solution. The PDA-coated glass particles were then mixed with a polyacrylic polymer. Mechanical properties were assessed through compressive strength, flexural strength, and Vickers microhardness testing using standardized specimens. Fuji XI and Herculite composite resin were used as the control groups. The adhesion to dentin was evaluated using shear bond strength test. Mineralization potential was investigated using Raman spectroscopy and scanning electron microscopy (SEM) to detect apatite formation on the surface and at the dentin-material interface. Cytocompatibility was evaluated using viability and proliferation assays on human dental pulp stem cells (DPSCs). Antibacterial activity against Streptococcus mutans was examined using both colony-forming unit (CFU) counts and live/dead bacterial staining assays on biofilms formed on the material surfaces. Additionally, odontogenic differentiation was examined using gene expression analysis. An in vivo mice molar pulp capping model was used to assess tertiary dentin formation and inflammatory response after placement of the material. All quantitative data were analyzed using one- or two-way ANOVA followed by Tukey's post hoc test, with significance set at p < 0.05. Kruskal-Wallis Test was utilized to evaluate pulp inflammation scores analysis.

Dopamer exhibited significantly enhanced (p < 0.001) mechanical properties, including improved compressive strength, flexural strength, and microhardness, compared to the conventional glass-ionomer cement (GIC). Shear bond strength to dentin also improved significantly (p < 0.05), demonstrating stronger adhesion. In vitro analyses confirmed in situ mineral formation and dentin mineralization capacity of Dopamer. Raman spectroscopy and SEM-EDS analyses revealed extensive mineral deposition at the interface between Dopamer and dentin, including calcium phosphate-rich layers suggestive of hydroxyapatite formation. Moreover, antibacterial testing demonstrated that Dopamer significantly (p < 0.001) inhibited Streptococcus mutans colonization compared to control (p < 0.001), reducing the risk of recurrent caries. Biocompatibility assays revealed high viability of DPSCs cultured on Dopamer, comparable to or better than the control groups. Dopamer also significantly upregulated odontogenic markers in vitro. In vivo studies showed formation of a continuous layer of tertiary dentin beneath the placed Dopamer, with minimal inflammatory response indicating excellent biocompatibility and regenerative potential.

By combining enhanced mechanical strength, mineralization capacity, and antibacterial properties, Dopamer addresses critical limitations of existing glass-ionomer dental restorative materials, offering a bioactive, durable solution for restorative dentistry. This multifunctional material represents a promising advancement in dental restoration, supporting both clinical performance and long-term oral health.

Maintaining the vitality and functionality of dental pulp is paramount for tooth integrity, longevity, and homeostasis. Aiming to treat irreversible pulpitis and necrosis, there has been a paradigm shift from conventional root canal treatment towards regenerative endodontic therapy.

This extensive and multipart review presents crucial laboratory and practical issues related to pulp-dentin complex regeneration aimed towards advancing clinical translation of regenerative endodontic therapy and enhancing human life quality.

In this multipart review paper, we first present a panorama of emerging potential tissue engineering strategies for pulp-dentin complex regeneration from cell transplantation and cell homing perspectives, emphasizing the critical regenerative components of stem cells, biomaterials, and conducive microenvironments. Then, this review provides details about current clinically practiced pulp regenerative/reparative approaches, including direct pulp capping and root revascularization, with a specific focus on the remaining hurdles and bright prospects in developing such therapies. Next, special attention was devoted to discussing the innovative biomimetic perspectives opened in establishing functional tissues by employing exosomes and cell aggregates, which will benefit the clinical translation of dental pulp engineering protocols. Finally, we summarize careful consideration that should be given to basic research and clinical applications of regenerative endodontics. In particular, this review article highlights significant challenges associated with residual infection and inflammation and identifies future insightful directions in creating antibacterial and immunomodulatory microenvironments so that clinicians and researchers can comprehensively understand crucial clinical aspects of regenerative endodontic procedures.

Fine particulate matter (PM2.5) exposure significantly exacerbates respiratory morbidity, particularly in asthmatic individuals, highlighting an urgent need for effective therapeutic interventions. In this study, we evaluated the therapeutic potential and underlying mechanisms of human dental pulp stem cells (hDPSCs), a promising mesenchymal stem cell population, in mitigating airway inflammation in mice with PM2.5-induced asthma exacerbation.

In a PM2.5-exacerbated ovalbumin (OVA)-asthma murine model, hDPSCs were intravenously administered with MCC950 (NLRP3 inhibitor) as positive control, systematically evaluating their therapeutic effects on airway inflammation and pyroptosis through pulmonary function tests, histopathological examination, cytological and molecular analyses.

The administration of hDPSCs ameliorated airway inflammation. Moreover, hDPSCs further alleviated Th2 inflammation and decreased serum IgE concentrations, along with a decrease in eosinophils in BALF. At the same time, interleukin-1β (IL-1β) and IL-18 levels in BALF and caspase-1 activity in lung tissues were reduced. In addition, immunohistochemistry showed that the expression levels of NLRP3, caspase-1, GSDMD, cleaved capsase-1 and IL-1β were reduced. The western blot results also showed that the expression level of NLRP3/caspase-1/GSDMD/cleaved capsase-1 in the classical pathway of pyroptosis decreased after hDPSCs intervention.

These findings provided the first evidence that hDPSCs transplantation attenuated allergic airway inflammation and mucus secretion in mice with PM2.5-induced asthma exacerbation. Thus, hDPSCs exert these protective effects through suppression of the NLRP3/caspase-1/GSDMD-mediated pyroptosis pathway, suggesting their potential as a novel cell-based therapy for PM2.5 inhalation-mediated asthma.

The inflammatory microenvironment in pulpitis and periapical periodontitis critically impairs dental pulp stem cells (DPSCs) functionality, yet the underlying molecular mechanisms remain poorly defined. This study aimed to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) on human DPSCs behavior and elucidate the transcriptomic changes underlying LPS-induced inflammation.

Human DPSCs were exposed to 1 µg/mL LPS for 24 hours to establish an inflammatory model, with subsequent evaluation of proliferation (CCK-8), migration (Transwell), and odontogenic differentiation (ALP staining, mineralization assays and odontoblast markers expression). Cell cycle progression was quantified through flow cytometry, while RNA sequencing analysis delineated transcriptional alterations.

LPS exposure significantly attenuated DPSCs proliferative capacity (P < 0.01), suppressed migration (P < 0.01), and impaired odontogenic differentiation, evidenced by diminished ALP activity, mineralized nodule formation and odontoblast markers expression. Cell cycle analysis revealed G0/G1 phase arrest (P < 0.01), indicating proliferative quiescence. Transcriptomic profiling identified 467 differentially expressed genes. P. gingivalis LPS exerts its effects on DPSCs by concurrent activation of both canonical (Toll-like receptors, TLRs) and non-canonical (Nucleotide-binding oligomerization domain-like receptor) signaling pathway. Pathway enrichment revealed the enrichment of the several important signaling pathways such as Phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), tumor necrosis factor (TNF) /Nuclear factor kappa B (NF-κB) and interleukin (IL) -17 signaling under LPS stimulation.

P. gingivalis LPS disrupts DPSCs regenerative functions by modulating inflammatory and developmental signaling pathways, providing mechanistic insights into impaired pulp repair during oral infections.

Hydraulic calcium silicate-based materials are widely used in the dental field of Endodontics and metallic radiopacifiers are added to these materials enabling imageology identification. Bismuth oxide was added in ProRoot MTA®, a Portland cement-based material available since the 90's, for radiopacifying purposes. This additive (around 20 % weight/weight replacement) elicits crucial local drawbacks such as dentine discolouration, cytotoxicity and gene expression upregulation of metallothioneins (MT1 and MT2A), which indicates a defensive cellular mechanism against metals. Besides, an in vivo study also indicated the presence of bismuth in blood and organs accumulation (liver, brain and kidney) after bismuth-containing materials implantations in an animal model; thus, mechanisms of bismuth accumulation are here proposed for these substrates. For this purpose, an open literature review methodology in databases (PubMed and Embase) was performed for each topic focusing on relevance of bismuth oxide in mechanisms of dentine discolouration, its lack of beneficial role within reparative dentine pathways, its leaching from the material and detection in blood, and accumulation in liver, brain and - mainly - in the kidney. The accumulation of bismuth on kidney from this material was reported as 334.42 and 279.38 ng/g, after 30 days of implantation in subcutaneous tissue and bone, respectively. Worryingly, after long-term implantation mass fractions were still considerably higher than non-exposed controls. Kidney accumulation represented a 160-fold average higher accumulation in comparison with the liver. Other chemical compounds are available as radiopacifiers (i.e., tantalum oxide, calcium tungstate and zirconium oxide) for dental materials. Recent studies pointed out tantalum oxide and zirconium oxide with lower accumulative pattern in the kidneys when compared to controls. Worryingly, these recent studies analyses were performed with several already marketed materials indicating a disregard from the manufacturers towards systemic testing prior to product launching. A stricter testing is advised. As bismuth oxide appeared to be the most systemically unsafe radiopacifier, mechanistic pathways for each site of detected accumulation are here presented.

Dental pulp cells-derived small extracellular vesicles (DPCs-sEVs) had shown immunomodulatory, anti-inflammatory, and tissue function restorative abilities. Therefore, DPCs-sEVs should be considered as a promising regenerative tool for dentin-pulp complex or whole pulp regeneration. This study aimed to evaluate the effect of DPCs-sEVs on the proliferation rate, migration capability, and expression pattern of DPCs for osteo/odontogenic gene markers in comparison with mineral trioxide aggregate (MTA).

DPCs-sEVs were isolated from rats' incisors by ultracentrifugation technique. Immunophenotypic characterization, morphology, size, and protein concentration of DPCs-sEVs were monitored and analyzed using flow cytometry (FC), transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and bicinchoninic acid assay (BCA). In addition, the TSG101, CD63, and the cytosolic protein syntenin of sEVs markers were immunodetected using Western blotting. Cell cultures of DPCs from the third passage were left untreated and considered as a control (group I), whereas other cultured cells were treated with 50 µg/mL DPCs-sEVs (group II), 0.2 mg/mL MTA extract (group III), or their combination (50 µg/mL DPCs-sEVs + 0.2 mg/mL MTA extract (group IV). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, transwell migration assay, and real-time polymerase chain reaction were used for assessing proliferation, migration, and specific gene expression patterns.

The DPCs-sEVs increased DPCs proliferation, and MTA enhanced their effects. The viability and proliferative capacity of DPCs treated with 50 µg/mL DPCs-sEVs + 0.2 mg/mL MTA-conditioned medium was significantly higher when compared with the other groups. The cell migration was more prominent in the group treated with 0.2 mg/mL MTA-conditioned medium than in the group treated with 50 µg/mL DPCs-sEVs. DPCs treated with 50 µg/mL DPCs-sEVs + 0.2 mg/mL MTA extract showed a significant increase in the migration ability of DPCs in comparison with other ones. Moreover, the combination group showed the greatest expression of dentin sialophosphoprotein (Dspp), osteocalcin (Ocn), collagen type I (Col1), and runt-related transcription factor 2 (Runx2).

MTA and sEVs together could be a powerful combination for regenerative endodontics.

Not applicable.

The regulatory processes in developmental biology research are significantly influenced by long non-coding RNAs (lncRNAs). However, the dynamics of lncRNA expression during human tooth development remain poorly understood. In this research, we examined the lncRNAs present in the dental epithelium (DE) and dental mesenchyme (DM) at the late bud, cap, and early bell stages of human fetal tooth development through bulk RNA sequencing. Developmental regulators co-expressed with neighboring lncRNAs were significantly enriched in odontogenesis. Specific lncRNAs expressed in the DE and DM, such as PANCR, MIR205HG, DLX6-AS1, and DNM3OS, were identified through a combination of bulk RNA sequencing and single-cell analysis. Further subcluster analysis revealed lncRNAs specifically expressed in important regions of the tooth germ, such as the inner enamel epithelium and coronal dental papilla (CDP). Functionally, we demonstrated that CDP-specific DLX6-AS1 enhanced odontoblastic differentiation in human tooth germ mesenchymal cells and dental pulp stem cells. These findings suggest that lncRNAs could serve as valuable cell markers for tooth development and potential therapeutic targets for tooth regeneration.

Dental stem cells are widely viewed as good options for bone regeneration because of their ease of acquisition, innate ability to renew themselves, and ability to differentiate into different types of cells. However, the process of osteogenic differentiation of dental stem cells is orchestrated by an intricate system of regulatory mechanisms. Recent studies have demonstrated the critical impacts of circular RNAs (circRNAs) on osteogenic differentiation of dental stem cells. Exploring the roles and regulatory pathways of circRNAs in dental stem cells could identify novel targets and approaches for utilizing dental stem cell therapy in clinical settings. This review provides a comprehensive overview of the functions and mechanisms of circRNAs, with a particular focus on their expression patterns and regulatory roles in osteogenic differentiation of various dental stem cell types. Furthermore, this review discusses current research challenges in this field and proposes future directions for advancing our understanding of circRNA-mediated regulation in dental stem cell biology.

Bone tissue engineering (BTE) emerged as a practical approach to tackle prosthetic industry limitations. We merge aspects from developmental biology, engineering and medicine with the aim to produce fully functional bone tissue. Mesenchymal stem cells have the capability of self-renewal and specific lineage differentiation. Herein lies their potential for BTE. Among MSCs, human dental pulp stem cells have a higher proliferation rate, shorter doubling times, lower cellular senescence, and enhanced osteogenesis than hBM-SCs under specific conditions. In addition, these cells are readily accessible and can be extracted through a subtle extraction procedure. Thus, they garner fewer moral concerns than most MSCs available and embody a promising cell source for BTE therapies able to replace hBM-MSCs. Interestingly, their study has been limited. Conversely, there is a need for their further study to harness their true value in BTE, with special emphasis in the design of bioprocesses able to produce viable, homogenous bone constructs in a clinical scale. Here, we study the osteogenic differentiation of hDPSCs encapsulated in alginate hydrogels under suspended culture in a novel perfusion bioreactor. The system is compared with traditional 3D static and fed-batch culture methodologies. The novel system performed better, producing higher alkaline phosphatase activity, and more homogeneous, dense and functional bone constructs. Additionally, cell constructs produced by the in-house-designed system were richer in mature osteoblast-like and mineralizing osteocyte-like cells. In conclusion, this study reports the development of a novel bioprocess able to produce hDPSC-based bone-like constructs, providing new insights into hDPSCs' therapeutic potential and a system able to be transferred from the laboratory bench into medical facilities.

Severe dental pulp inflammation can lead to tissue lysis and destruction, underscoring the necessity for effective treatment of pulpitis. N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification has recently emerged as a key regulator in inflammatory processes. However, whether NAT10 affects the inflammatory response in human dental pulp stem cells (hDPSCs) remains unelucidated. In this study, elevated NAT10 expression was observed in pulpitis tissues and LPS-stimulated hDPSCs. Knockdown of NAT10 led to reduced inflammatory gene expression and lower reactive oxygen species (ROS) production in LPS-stimulated hDPSCs, while the chemotactic migration of macrophages was also suppressed. Similar results were observed when hDPSCs were treated with Remodelin, an inhibitor of NAT10. Differentially expressed genes identified through RNA sequencing were significantly enriched in inflammatory signaling pathways after NAT10 depletion. Among the differential genes, pentraxins 3 (PTX3) was identified as the potential target gene due to the presence of the ac4C modification site and its known ability to regulate dental pulp inflammation. The mRNA and protein levels of PTX3 were reduced in NAT10-deficient cells, along with a decrease in its mRNA stability. Exogenous PTX3 supplementation partially reversed the inflammatory inhibition induced by NAT10 knockdown. Further evidence in vivo revealed that Remodelin treatment attenuated the severity of dental pulp inflammation in rats with pulpitis. In summary, these data indicated that NAT10 deficiency inhibited the stability of PTX3 mRNA and further inhibited hDPSC inflammation, while Remodelin might be a potential therapeutic agent for pulp capping.

Histone lysine demethylases (KDMs), as important epigenetic regulators, are involved in various biological processes such as energy metabolism, apoptosis, and autophagy. Recent research shows that KDMs activate or silence downstream target genes by removing lysine residues from histone tails, and participate in the regulation of bone marrow mesenchymal stem cells (BM-MSCs), osteoblasts (OB), osteoclasts (OC), chondrocytes and other skeletal cell development, differentiation and formation. Moreover, several members of the KDM family affect the occurrence and development of bone diseases such as osteoporosis (OP), osteoarthritis (OA), osteosarcoma (OS), by regulating target genes. Specific regulation mechanisms of KDMs suggest new strategies for bone disease treatment and prevention. Despite the unique function and importance of KDMs in the skeletal system, previous studies have never systematically summarized their specific role, molecular mechanism, and clinical treatment in bone physiology and pathology. Therefore, this review summarises the expression pattern, intracellular signal transduction, and mechanism of action of the KDM family in several bone physiological and pathological conditions, aiming to highlight the important role of KDMs in bone diseases and provide a reference for the future treatment of bone diseases.

This study aimed to explore how amelogenin can improve stem cells from human exfoliated deciduous teeth (SHED)-based bone regeneration and promote tissue healing as a treatment for critical-sized bone defects. SHED was induced into bone differentiation by using osteogenic differentiation medium. Real-time polymerase chain reaction, alkaline phosphatase (ALP) staining and quantification, and Alizarin Red S staining, as well as calcium and osteocalcin quantification were performed to assess differentiation. On day 18, a significant increase was observed in the expression of RUNX2, CBFB, BGLAP, COL1, BMP2, BMP4, NOTCH1, NOTCH2, and NES. Osteocalcin gene expression continued to increase significantly. ALP activity was significantly higher in the amelogenin-treated group than in the control group on days 7, 10, and 14. On day 14, enhanced ALP staining was observed in the amelogenin-treated group. Calcium and osteocalcin levels were significantly higher in the amelogenin-treated group than in the control group on day 21. This study suggests that combining SHED and amelogenin may be effective for bone regeneration, offering a potential new approach in regenerative medicine.

Angiogenesis represents a critical challenge in dental pulp regeneration due to the tissue's restricted nutrient supply through a 0.5-mm apical foramen. While dental pulp stem cells (DPSCs) hold regenerative potential, their limited vascularization capacity impedes clinical applications. Through Single-cell RNA sequencing (scRNA-seq) analysis of human dental pulp, we discovered a PDGF (+) mesenchymal subset exhibiting enhanced angiogenic signatures, suggesting targeted cell selection could overcome this bottleneck.

ScRNA-seq identified PDGF (+) subpopulation in human pulp samples, validated through multiplex immunohistochemical of the localization of PDGF/CD73/CD31. PDGF-BB-overexpressing DPSCs were engineered via lentiviral vectors. Functional assessments included: 1) CCK-8/Edu/cell cycle/transwell assays for proliferation and migration ability 2) HUVECs co-culture models analyzing chemotaxis and tube formation 3) Vascularized tissue formation in rat kidney capsule transplants.

The CD73 (+) PDGF (+) subpopulation demonstrated spatial correlation with CD31 (+) vasculature. PDGF-BB overexpression enhanced DPSCs' proliferative capacity and migration capacity. Co-cultured HUVECs exhibited increased tube formation with PDGF-BB group. In vivo transplants generated more vascular structures containing CD31 (+) endothelia. These findings establish PDGF-BB engineering as an effective strategy to amplify DPSCs' angiogenic potential, while emphasizing the therapeutic value of functionally-defined stem cell subpopulations in pulp regeneration.

Ellagic acid (EA) is a potent antioxidant that reduces oxidative stress and promotes differentiation. By lowering the harmful levels of reactive oxygen species (ROS), EA fosters an environment conducive to the osteoblastic differentiation (OB) of stem cells. In addition, it promotes autophagy and mitophagy, which are vital for promoting differentiation. Effective autophagic activity recycles damaged organelles and proteins, meeting the energy required during differentiation and shielding from apoptosis. However, molecular mechanisms underlying the osteogenic differentiation of mesenchymal stem cells remain inadequately explored. Therefore, the current study aims to define the regulatory role of EA during the OB of dental pulp-derived stem cells (DPSC) and to study how autophagy and mitophagy are being modulated during this differentiation process. Herein, we showed that the expression level of osteoblast-specific markers, autophagy, and mitophagy-associated markers was significantly elevated during EA-mediated OB differentiation of DPSC. Moreover, we found that the EA induced the osteoblastic-specific markers through canonical BMP2 pathway molecules, reduced ROS in both basal and activated states, and induced autophagy and mitophagy molecules along with enhanced mitochondrial functions. Cell cycle analysis revealed that the G1 phase was arrested via phosphorylation of γ-H2AX, ATM, and CHK2 proteins. Furthermore, in silico analysis revealed that EA strongly binds with osteonectin, a crucial noncollagen protein involved in bone remodeling, and confirmed by Western blot analysis. These results support that EA could be a promising natural compound for bone repair and regeneration applications.

Regenerative endodontic procedures using blood clots (BC-REP) for immature teeth typically exhibit a periodontal ligament-like healing pattern. However, a pulp-like healing pattern is observed in the presence of residual pulp. This study aimed to clarify the healing phenotype according to tooth maturity when performing BC-REP in the presence of residual pulp, focusing on migrated mesenchymal stem/stromal cells (MSCs). BC-REP rat molar models were created in the presence of residual pulp at ages corresponding to tooth developmental stages, from immature to mature (5 wk: immature, root is still growing; 8 wk: near mature, root has finished growing in length but the apex is not formed; 11 wk: mature, the apex is formed). The healing pattern and histological MSC markers (α-smooth muscle actin [α-SMA], CD73, CD90, and CD146) were investigated. The frequency of periodontal ligament-like healing was higher in mature teeth than in immature teeth. In addition, more healing macrophages were observed at the apical site 28 d after BC-REP, which is the final stage of healing. In immature teeth, double-immunopositive cells for proliferation markers (ki67 and proliferating cell nuclear antigen [PCNA]) and α-SMA were frequently observed in the vicinity of the root canal orifice 7 d after treatment, which is the early stage of healing. By contrast, in mature teeth, the number of CD73-, CD90-, and CD146-immunopositive cells increased at the apical site after 7 and 28 d. CD90- and CD146-immunopositive cells expressed cell proliferation markers (ki67 or PCNA) after 7 d. MSC migration after BC-REP likely varies based on tooth maturity, resulting in different healing phenotypes.

Stem cells play a crucial role in maintaining tissue regenerative capacity and homeostasis. However, mechanisms associated with stem cell senescence require further investigation. In this study, we conducted a proteomic analysis of human dental pulp stem cells (HDPSCs) obtained from individuals of various ages. Our findings showed that the expression of NUP62 was decreased in aged HDPSCs. We discovered that NUP62 alleviated senescence-associated phenotypes and enhanced differentiation potential both in vitro and in vivo. Conversely, the knocking down of NUP62 expression aggravated the senescence-associated phenotypes and impaired the proliferation and migration capacity of HDPSCs. Through RNA-sequence and decoding the epigenomic landscapes remodeled induced by NUP62 overexpression, we found that NUP62 helps alleviate senescence in HDPSCs by enhancing the nuclear transport of the transcription factor E2F1. This, in turn, stimulates the transcription of the epigenetic enzyme NSD2. Finally, the overexpression of NUP62 influences the H3K36me2 and H3K36me3 modifications of anti-aging genes (HMGA1, HMGA2, and SIRT6). Our results demonstrated that NUP62 regulates the fate of HDPSCs via NSD2-dependent epigenetic reprogramming.

The purpose of this study was to investigate the effects and mechanism of artemisinin (ART) on the proliferation, apoptosis, and inflammatory response of human dental pulp stem cells (HDPSCs) under lipopolysaccharide (LPS)-induced inflammation.

HDPSCs were isolated, cultured, and identified by flow cytometry and three-directional differentiation induction. A suitable concentration of LPS was selected to mimic the inflammatory condition in vitro. After culturing with ART and LPS for 48 h, cell proliferation was observed by CCK-8 assay; cell apoptosis was observed by flow cytometry, western blot, and Caspase-3 activity; and the inflammatory response was observed by qRT-PCR and ELISA. Transcriptome sequencing, immunofluorescence staining, qRT-PCR, western blot, and RITA were used to explore the underlying mechanism.

HDPSCs were successfully isolated and exhibited the potential for multilineage differentiation. 0.1 μg/mL of LPS was utilized to mimic the inflammatory condition. ART promoted HDPSCs proliferation but repressed apoptosis and the inflammatory response under LPS-induced inflammation. Further, ART exerted its effect through the p53 signaling pathway.

ART inhibited the p53 signaling pathway to promote HDPSCs proliferation, but hinder apoptosis and the inflammatory response under LPS-induced inflammation.

This study demonstrates that ART facilitates the alleviation of inflammation and preserves the viability of HDPSCs. Therefore, ART may serve as a promising therapeutic drug for the repair and regeneration of dental pulp in the treatment of deep caries and reversible pulpitis.

While mesenchymal stem/stromal cells (MSCs) exhibit the ability to self-renew, they are not immortal; they eventually reach a point of irreversible growth cessation and functional deterioration following a limited series of population doublings, referred to as replicative senescence. When evaluated according to the criteria set by the International Society for Cell Therapy (ISCT), MSCs show significant differences in their senescence patterns and other characteristics related to their phenotype and function. These differences are attributed to the source of the MSCs and the conditions in which they are grown. MSCs derived from fetal or adult sources have variations in their genome stability, as well as in the expression and epigenetic profile of the cells, which in turn affects their secretome. Understanding the key factors of MSC senescence based on cell source can help to develop effective strategies for regulating senescence and improving the therapeutic potential.

Stroke occurs when the blood flow to the brain is interrupted due to a rupture of blood vessels or blockage in the brain. It is the major cause of physical disabilities in adulthood. Despite advances in surgical and pharmacological therapy, functional recovery from stroke is limited, affecting quality of life. Stem cell therapy, which may treat neurological disorders associated with brain traumas, including stroke, is an important focus in stroke research and treatment. Stem cell therapy has primarily used a type of adult stem cells called mesenchymal stem cells (MSCs) due to their universality and ability to develop into multiple lineages to regenerate brain cells and repair brain tissues. A significant number of clinical studies provide evidence of the potential of MSCs to treat stroke. This review summarizes the therapeutic mechanism and applications of MSCs in stroke treatment. We also highlight the current challenges and future prospects of adult MSC therapy for stroke treatment.

Human dental pulp stem cells (hDPSCs) have emerged as a promising resource in regenerative medicine due to their unique ability to secrete exosomes containing a diverse array of bioactive molecules, particularly microRNAs (miRNAs). These exosomes appear to be essential for stimulating regenerative mechanisms, especially those associated with stem cell pluripotency and tissue repair. However, several challenges such as cargo specificity and delivery efficiency need to be addressed to maximise the therapeutic potential of hDPSC-derived exosomes and miRNA-based therapies. This narrative review explores hDPSCs' potential in regenerative medicine by examining their role in tissue engineering, secretome composition, exosome function, exosomal miRNA in diverse models, and miRNA profiling. Therefore, it is imperative to sustain ongoing research on miRNA to advance clinical applications in the field of regenerative medicine and dentistry. A comprehensive understanding of the specific miRNA composition within hDPSC-derived exosomes is essential to elucidate their mechanistic roles in diverse disease states and to inform the development of innovative therapeutic strategies. These findings hold significant potential for the development of innovative regenerative therapies and emphasises the importance of establishing a strong connection between translational research discoveries and clinical applications. hDPSC-derived exosomes and miRNA-based therapies play a crucial role in immune modulation, regenerative dentistry, and tissue repair.

Dentin sialoprotein (DSP), a major dentin extracellular matrix noncollagenous protein, is well recognized as an important regulator for dentinogenesis. DSP as a secreted protein can interact with membrane receptors, activate intracellular signaling, and initiate the odontoblastic differentiation of dental papilla cells. In a recent study, we have demonstrated that DSP can induce the endothelial differentiation of dental pulp stem cells (DPSCs), a type of tooth pulp-derived multipotent stem cells, dependent on membrane receptor endoglin (ENG). However, the intimate mechanisms by which DSP-ENG association facilitates the endothelial differentiation of DPSCs remain enigmatic. Here, we find that the amino acid (aa) residues 34-50 of DSP (DSPaa34-50) is responsible for its association with ENG using a series of co-immunoprecipitation assays. Immunofluorescent staining and in situ proximity ligation assay demonstrate that overexpressed ENG in human embryonic kidney 293T cells shows codistribution and proximity ligation assay signals to the supplemented DSPaa34-50 protein but not to DSP without aa34-50 (DSPΔ34-50) on cell surfaces. Moreover, the zona pellucida domain of ENG mediates its association with DSPaa34-50. Further experiments indicate that DSPaa34-50 exhibits equivalent effects to the full-length DSP on the migration and endothelial differentiation of DPSCs dependent on ENG but DSPΔ34-50 does not. Mechanistically, DSPaa34-50 activates AKT1 and triggers the expression of blood vessel development-related genes in DPSCs. Multiple experiments demonstrate that AKT1 inhibition suppresses the DSPaa34-50-induced migration and endothelial differentiation of DPSCs. Thus, AKT1 mediates the cellular and molecular functions of DSPaa34-50-ENG association. Collectively, these findings identify that DSP promotes the endothelial differentiation of DPSCs through the DSPaa34-50-ENG-AKT1 signaling axis.

LepR-expressing cells (LepR+ cells), a critical subpopulation of mesenchymal stem cells, have gained increasing attention in the last decade. LepR+ cells have been found to play a crucial role in maintaining bone and periodontal homeostasis. This review summarizes current research advances focusing on the role of LepR+ cells and their underlying regulatory molecular mechanisms in bones and periodontium, aiming to provide a better understanding of the therapeutic potential of this cell lineage.

A literature review was conducted based on publications in PubMed over the past 20 years, summarizing the research progress on LepR+ cells in bone and periodontal tissues.

Current evidence revealed that LepR+ cells possess the ability of self-renewal and multilineage differentiation and are essential for bone turnover and periodontal tissue remodeling. In addition, LepR+ cells participate in the processes of bone fracture healing and alveolar socket healing. Moreover, under pathological conditions such as osteoporosis, bone marrow fibrosis, and periodontitis, LepR+ cells exhibit enhanced adipogenic or fibrogenic differentiation abilities.

Therapeutic approaches targeting the cell fate of LepR+ cells hold the potential to provide novel insights into bone/periodontal repair and regeneration therapy.

Stem cell-based therapies have raised considerable interest to develop regenerative treatment for neurological disorders with high disability. In this review, we focus on recent preclinical and clinical evidence of stem cell therapy in the treatment of degenerative neurological diseases and discuss different cell types, delivery routes and biodistribution of stem cell therapy. In addition, recent advances of mechanistic insights of stem cell therapy, including functional replacement by exogenous cells, immunomodulation and paracrine effects of stem cell therapies are also demonstrated. Finally, we also highlight the adjunction approaches that has been implemented to augment their reparative function, survival and migration to target specific tissue, including stem cell preconditioning, genetical engineering, co-transplantation and combined therapy.

Human dental pulp stem cells (hDPSCs) exhibit amazing therapeutic abilities in a variety of diseases due to their remarkable self-renewal capacity and multi-differentiation potential. However, their therapeutic potential could be weakened by various factors such as oxidative stress in cell survival microenvironment In Vivo. Here, we explored the protective effect and mechanism of melatonin (Mel) on hDPSCs transplanted in a type 1 diabetes mellitus (T1DM) rat model. Nicotinamide adenine dinucleotide (NAD+) metabolism and mitochondrial function were remarkably impaired in T1DM rats caused by oxidative stress, while the combination of Mel and post-hDPSCs transplantation could rebalance NAD+ homeostasis through regulating NAMPT-NAD+-SIRT1 axis. Furthermore, Mel significantly reduced intracellular and mitochondrial reactive oxygen species, and alleviated cell senescence and apoptosis of hDPSCs exposed to hydrogen peroxide through ameliorating NAD+ depletion and mitochondrial dysfunction. The protective role of Mel could be extremely essential to stem cells in tissue engineering and regenerative medicine.

Regeneration of dentin and preserving pulp vitality are essential targets for vital pulp therapy. Our study aimed to evaluate a novel biomimetic pulp capping agent with increased dentin regenerative activities. To produce demineralised dentin matrix (DDM) particles, human extracted teeth were ground and treated with ethylene diamine tetra-acetic acid solution. DDM particles were added to sodium alginate and this combination was dripped into a 5% calcium chloride to obtain DDM hydrogel (DDMH). The eluants of both DDMH and mineral trioxide aggregate (MTA) were tested using an MTT assay to detect their cytotoxic effect on dental pulp stem cells (DPSC). Collagen-I (COL-I) gene expression was analysed on DPSC exposed to different dilutions of pulp capping material eluants by real-time quantitative polymerase chain reaction. Acridine orange staining was used to monitor the cell growth over the tested materials. Agar diffusion assay was utilised to test the antibacterial effect of DDMH and MTA compared to controls. MTT assay revealed that neat eluates of DDMH promoted DPSC viability. However, neat eluates of MTA were cytotoxic on DPSC after 72 h of culture. Moreover, DPSC were capable of growth and attached to the surface of DDMH, while they showed a marked reduction in their number when cultured on the MTA surface for one week, as shown by the acridine orange stain. In DPSC cultured with DDMH eluates, the COL-I gene was overexpressed compared to those cultured with MTA eluants. DDMH had significant antimicrobial activity in comparison to MTA after 24 h incubation. This in vitro study showed that DDMH could be an alternative pulp capping agent for regenerative endodontics.

The enteric nervous system (ENS) consists of a network of neurons and glia that control numerous complex functions of the gastrointestinal tract. Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of ENS along variable lengths of distal intestine due to failure of neural crest-derived cells to colonize the distal intestine during embryonic development. A patient with HSCR usually presents with severe constipation in the neonatal period and is diagnosed by rectal suction biopsy, followed by pull-through procedure to surgically remove the affected segment and reconnect the proximal ganglionated intestine to the anus. Outcomes after pull-through surgery are suboptimal and many patients suffer from ongoing issues of dysmotility and bowel dysfunction, suggesting there is room for optimizing the management of this disease. This review focuses on discussing the recent advances to better understand HSCR and leverage them for more accurate and potentially less invasive diagnosis. We also discuss the potential future management of HSCR, particularly cell-based approaches for the treatment of HSCR.

The aim was to evaluate the effect of single and double doses of low-level laser irradiation on proliferation of human dental pulp stem cells (DPSC) and expression of vascular endothelial growth factor (VEGF) and dentine sialoprotein (DSP).

In this experimental in vitro study, after confirming the stemness of DPSCs, the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) for MTT assay and VEGF-ELISA and osteogenic medium for DSP-ELISA. The wells containing DPSCs were divided into three main groups and 9 subgroups (n = 7). In groups with single low-level laser, 660-nm diode laser was irradiated at 100 mW and 3 J/cm2 energy density for 15 s. In groups with double doses of low-level laser the second identical irradiation was after 48 h. The MTT-assay and ELISA for DSP/VEGF (dentinogenic/angiogenic differentiation) were performed at 1, 7 and 14 days post irradiation. Using the SPSS software 20 (SPSS, Chicago, Ill, USA) with 95% confidence interval (P = 0.05), a two-way ANOVA test with Tukey's post hoc test was used for the effect of LLLI on VEGF and DSP. The One-Way ANOVA was used for of cell proliferation.

Higher proliferation rate in both single and double low-level laser was reported. The difference was statistically significant for double doses of low-level laser (P = 0.001, P = 0.020 and P = 0.000 for 1, 7 and 14 days, respectively). Also after one, 7 and 14 days, cells in significant increase in DSP (P > 0.05) and VEGF (P > 0.05) was observed that was significantly higher for double doses of low-level laser.

Low level laser enhanced the mitochondrial activity and proliferation of DPSCs. Increased production of DSP/VEGF indicates dentinogenic/angiogenic activity.

Low level laser increases the proliferation of DPSCs, elevates the production of VEGF (which means better angiogenesis in regenerative treatments) and increases the production of DSP (which means better dentinogenesis in vital pulp treatments).

CaMKII is a serine/threonine-specific protein kinase that plays a crucial role in normal and pathological conditions. However, limited information is available regarding the roles of CaMKII in dentinogenesis, particularly in an inflammatory context. Previously, we demonstrated the pivotal role of TrkB in inflammation-induced differentiation of hDPSCs into odontoblast-like cells. Here, we investigate the interaction between CaMKII and TrkB during hDPSCs odontogenic differentiation. hDPSCs were cultured and subjected to CaMKII knockdown using siRNA, followed by treatment with dentinogenic media. TNFα-stimulated cells were treated with CaMKII- inhibitor, -protein, or TrkB antagonist, CTX-B. Immunocytochemistry and ARS were used to visualize targeted proteins and calcium deposits. Real-time PCR detected expression levels of odontogenic and mineralization markers such as DSPP and DMP-1. Our data indicate that CaMKII inhibition enhances TrkB protein levels and promotes TNFα-induced transcriptional activation of genes associated with odontogenic differentiation. CaMKII knockdown via siRNA and pharmacological inhibition elevated DSPP and DMP-1 protein levels, whereas CaMKII overexpression suppressed their expression. Notably, treatment with TNF-α and a CaMKII inhibitor upregulated DSPP and DMP-1 expression, while co-treatment with CTX-B abolished this effect. Similarly, mRNA expression of DSPP and DMP-1 was reduced at day 10. Mineralization activity exhibited a similar pattern to the expression of these markers. Our findings unveil a novel mechanism underlying the role of CaMKII via TrkB in dentinogenesis, which is vital for the success of hDPSCs engineering strategies.

Alternative splicing not only expands the genetic encoding of genes but also determines cellular activities. This study aimed to elucidate the regulation mechanism and biological functions of lincRNA-ASAO in the process of odontogenesis-related genes alternative splicing mediated odontogenic differentiation of hDPSCs.

RACE, RNA-seq, FISH and bioinformatics techniques were used to identify novel lincRNA-ASAO. ALP staining, alizarin red staining, qRT-PCR and western blot were used to identify the role of lincRNA-ASAO in regulating the odontoblast differentiation of hDPSCs. The binding protein PTBP1 of lincRNA-ASAO was screened by RNA-Pulldown, protein profiling and bioinformatics. The target gene ALPL of lincRNA-ASAO/PTBP1 was identified by RNA-seq, bioinformatics technology and DNA agarose gel electrophoresis. FISH, IF, PAR-CLIP and bioinformatics techniques were used to determine the roles of lincRNA-ASAO, PTBP1 and ALPL pre-mRNA in the odontoblast differentiation of hDPSCs.

We identified a novel lincRNA-ASAO that could promote the odontogenic differentiation of human Dental Pulp Stem Cells (hDPSCs). And, the interaction between lincRNA-ASAO and alternative splicing factor PTBP1 promoted the odontoblast differentiation of hDPSCs. In addition, lincRNA-ASAO forms duplexes with ALPL pre-mRNA, targeting PTBP1 to exonic splicing silencer (ESS) of ALPL and regulating exon 2 skipping. Notably, lincRNA-ASAO/PTBP1 regulated ALPL production to increase the type 2 splice variant, which promoted the odontoblast differentiation of hDPSCs.

We have identified the novel lincRNA-ASAO, which can promote the odontoblast differentiation of hDPSCs. The mechanism study found that lincRNA-ASAO/PTBP1 mediated the exon 2 skipping of ALPL pre-mRNA, resulting in the type 2 splice variant of ALPL. Our results enrich the understanding of lncRNAs and alternative splicing in regulating the odontoblast differentiation of hDPSCs, and provide clues to improve the clinical therapeutic potential of hDPSCs for dental pulp restoration.

Exosomes-loaded hydrogels have potential value in wound treatment. Current studies focus on improving hydrogels' biocompatibility and optimizing different stem cell-derived exosomes for better therapeutic effect. Herein, we present a novel biocompatible recombinant human collagen (RHC) hydrogel loading with different MSCs-derived exosomes for promoting wound healing. We modify the RHC with methacrylate anhydride (MA) at optimal concentration, generating collagen hydrogel (RHCMA) with ideal physiochemical properties for exosome delivery (MSC-exos@RHCMA). Exosomes derived from human adipose-derived MSCs (ADSC-exos), bone marrow-derived MSCs (BMSC-exos) and umbilical cord MSCs (ucMSC-exos) are harvested from the culture supernatants and are loaded into RHCMA, respectively. These three hydrogel systems exhibit desired sustained release features, and can significantly improve cell proliferation and migration. In addition, these MSC-exos@RHCMAs show excellent therapeutic performance in treating the wounds of rats. Notably, we have demonstrated that the healing effect occurs best under the treatment of ucMSC-exos@RHCMA, following inflammatory resolution, angiogenesis, and collagen formation. These results would supply important value for the clinical application of MSC-exos in wound treatment in the future.

Regenerative endodontic procedures (REPs) offer an alternative to apexification in necrotic immature permanent teeth, promoting continued root development and dentinal wall thickening. Success in REPs requires effective disinfection and the survival of dental-derived mesenchymal stem cells (DMSCs), such as dental pulp stem cells (DPSCs), stem cells from the apical papilla (SCAPs), and periodontal ligament stem cells (PDLSCs). This review investigates the biocompatibility of irrigation solutions, including sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), and chlorhexidine (CHX), on DMSCs. Following PRISMA guidelines, a comprehensive search was conducted in PubMed, Scopus, Web of Science, Cochrane, and SciELO, with the last update on March 4, 2024. Studies from January 2008 to April 2024 assessing viability, proliferation, migration, differentiation, and mineralization of DMSCs treated with NaOCl, EDTA, and CHX were included. The papers were selected using PICOS criteria and quality was assessed using the PRILE checklist and risk of bias with the Quality Assessment Tool for In Vitro Studies. Of 738 studies identified, 15 met inclusion criteria. The findings suggest that NaOCl and CHX exhibit lower biocompatibility towards DMSCs compared to EDTA. NaOCl and CHX are cytotoxic to DMSCs, while EDTA demonstrates favorable biocompatibility, promoting osteogenic differentiation and mineralization. This highlights potential implications for irrigant selection in regenerative procedures, as appropriate irrigants may enhance cellular survival and improve clinical outcomes.

Hydroxyapatite nanoparticle (HANPs) utilization has recently been notable in bone tissue engineering. This surge owes itself to the biocompatibility of HANPs and their striking resemblance to the minerals found in natural bone. Furthermore, dental pulp-derived stem cells (DPSCs) have garnered attention due to their remarkable differentiation potential into multilineages, thus positioning them as a pivotal cell reservoir for regenerative medicine. This study aims to investigate the impact of HANPs on DPSCs cellular processes. The HANPs have been synthesized using the wet chemical precipitation method followed by freeze-drying and characterization using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The size of HANPs was reported to be in the range of 55-67 nm. Our dataset divulges that DPSCs can endure concentrations of HANPs up to ≤ 0.81 mg/mL without incurring any conspicuous alterations in their morphology or the pace of proliferation. Furthermore, the self-renewal potency of HANPs was upheld at concentrations ≤ 0.20 mg/mL. Flow cytometric analysis affirms a significant divergence in cell distribution across all cell cycle phases in DPSCs treated with 0.81 mg/mL HANPs. Intriguingly, no variance surfaced in the migratory capacity of DPSCs exposed to HANPs of ≤ 0.40 mg/mL. For osteogenic differentiation, HANPs at concentrations of ≤ 0.40 mg/mL demonstrated the aptitude to incite osteogenic differentiation within DPSCs, facilitating the formation of calcium deposits. In conclusion, combining HANPs and DPSCs shows promise for restoring damaged hard tissues, like bone and teeth, and enhancing regenerative therapies.

Spinal cord injury (SCI) is a serious neurological disorder that causes loss of mobility, pain, and autonomic dysfunction, resulting in altered sensation and devastating loss of function. Current treatments for SCI mainly focus on surgery and drug therapy to promote neurological recovery. However, there are virtually no effective remedies for irreversible nerve damage that result in a victim's loss of motor function and sensory changes that occur after an injury. With the continuous development of medical technology, stem-cell-based regenerative medicine provides researchers with new treatment ideas. The effectiveness of mesenchymal stem cells and their derivatives from different sources in treating SCI varies. Recent studies have highlighted that dental pulp stem cells (DPSCs) may contribute to anti-inflammatory regulation, anti-apoptotic regulation, and axonal regeneration in the treatment of SCI patients. In addition, the combination of new biomaterials and dental pulp stem cells is promising in the treatment of SCI. This article reviews the role of DPSCs in SCI treatment in recent years, discusses the advantages of DPSCs, explores potential development directions, and looks forward to providing new insights for future research in this critical field.

Mesenchymal stromal cells (MSCs) with high expression of CD146 have superior properties for tissue regeneration. However, high variability in the rate of CD146+ cells among donors is observed. In this study, the possible reasons behind this variability in human periodontal ligament MSCs (hPDL-MSCs) were explored.

hPDL-MSCs were isolated from 22 different donors, and rates of CD146+ cells were analyzed by flow cytometry. Furthermore, populations with various rates of CD146+ cells were isolated with magnetic separation. The dependency of cell proliferation, viability, cell cycle, and osteogenic differentiation on the rates of CD146+ cells was investigated. Besides, the effects of various factors, like cell density, confluence, and inflammatory environment on the CD146+ rate and expression were analyzed.

The rate of CD146+ cells exhibited high variability between donors, with the percentage of CD146+ cells ranging from 3% to 67%. Higher percentage of CD146+ cells was associated with higher proliferation, presumably due to the higher percentage of cells in the S-phase, and higher osteogenic differentiation potential. Prolonged cell confluence and higher cell seeding density led to the decline in the rate of CD146+ cells. The surface rate of CD146 in hPDL-MSCs was stimulated by the treatment with interleukin-1β and tumor necrosis factor-α, and inhibited by the treatment with interferon-γ.

These results suggest that hPDL-MSCs with high rate of CD146+ cells are a promising subpopulation for enhancing the effectiveness of MSC-based regenerative therapies, however the rate of CD146 is affected by various factors, which must be considered for cell propagation and their potential application in vivo.

Tumors and injuries often lead to large mandibular defects. Accelerating the osteogenesis of large bone defect areas is a major concern in current research. In this study, dental pulp mesenchymal stem cells (DPSCs) were used as seed cells, and the local pulsatile parathyroid hormone (PTH) delivery system was used as an osteogenic-inducing active ingredient to act on DPSCs and osteoblasts, which were applied to the jaw defect area to evaluate its therapeutic effect on bone regeneration.

Pulsatile delivery systems, both with and without PTH, were developed following the protocols outlined in our previous study. In vitro, the biocompatibility of the pulsatile delivery system with DPSCs was assessed using the Cell Counting Kit-8 (CCK8) assay and live/dead cell staining. Osteogenic differentiation was evaluated through alkaline phosphatase staining and alizarin red staining. In vivo, critical bone defects with a diameter of 10 mm were created in the mandibles of white rabbits. The osteogenic effect was further assessed through gross observation, X-ray imaging, and histological examination.

In vitro experiments using CCK8 assays and live/dead cell staining demonstrated that DPSCs successfully adhered to the surface of the PTH pulsatile delivery system, showing no significant difference compared to the control group. Furthermore, alkaline phosphatase staining and Alizarin Red staining confirmed that the localized pulsatile parathyroid hormone delivery system effectively induced the differentiation of DPSCs into osteoblasts, leading to the secretion of abundant calcium nodules. Animal studies further revealed that the PTH pulsatile delivery system promoted the osteogenic differentiation of DPSCs, facilitating the repair of critical mandibular bone defects.

The rhythmic release of PTH from the pulsatile delivery system effectively induces the osteogenic differentiation of DPSCs. By leveraging the synergistic interaction between PTH and DPSCs, this approach facilitates the repair of extensive mandibular bone defects.

Based on the potential of DPSCs as the most promising candidates for bone tissue engineering, we comprehensively investigated the time-dependent cellular and molecular changes that occur during their osteodifferentiation. To analyze this area in-depth, we used both cellular and molecular approaches. Morphological changes were monitored using bright-field microscopy, while the production of mineral deposits was quantified spectrophotometrically. The expression of a key mesenchymal stem cell marker, CD90, was assessed via flow cytometry. Finally, protein-level changes in whole cells were examined by fluorescence microscopy. Our results show successful long-term osteodifferentiation of the patient's DPSCs within 25 days. In differentiated cells, mineralized extracellular matrix production gradually increased; in contrast, the expression of the specific stem cell marker CD90 significantly decreased. We observed dynamic changes in intracellular and extracellular proteins when collagen1 A1 and osteopontin appeared as earlier markers of osteogenesis, while apolipoprotein A2, bone morphogenetic protein 9, dentin sialophosphoprotein, and matrix metalloproteinase 8 were produced mainly in the late stages of this process. A decrease in actin microfilament expression indicated a reduction in cell proliferation, which could be used as another marker of osteogenic initiation. Our results suggest a coordinated process in vitro in which cells synthesize the necessary proteins and matrix components to regulate the growth of hydroxyapatite crystals and form the bone matrix.