The Impact of the VEGF/VEGFR2/PI3K/AKT Signaling Axis on the Proliferation and Migration Abilities of Human Dental Pulp Stem Cells.

The potential therapeutic benefits of human dental pulp stem cells (HDPSCs) in dental regenerative medicine have been demonstrated. However, little is known about the molecular mechanisms regulating the biological characteristics of HDPSCs. The experiment aims to explore whether VEGF activates signaling pathways such as FAK, PI3K, Akt, and p38 in HDPSCs, and to investigate the molecular mechanisms by which VEGF influences proliferation and migration of HDPSCs. Normal and inflamed human dental pulp (HDP) samples were collected, and the levels of VEGF in HDP were assessed. HDPSCs were cultured and purified. HDPSCs were stimulated with lipopolysaccharide (LPS) at gradient concentrations, and real-time quantitative polymerase chain reaction (qPCR) was used to assess changes in VEGF mRNA. Gradient concentrations of VEGF were used to stimulate HDPSCs, and cell migration ability was evaluated through scratch assays and Transwell chamber experiments. Phosphorylation levels of FAK, AKT, and P38 were assessed using Western blotting. Inhibitors of VEGFR2, FAK, AKT, P38, and VEGF were separately applied to HDPSCs, and cell migration ability and phosphorylation levels of FAK, AKT, and P38 were determined. The results indicated significant differences in VEGF levels between normal and inflamed HDP tissues, with levels in the inflamed state reaching 435% of normal levels (normal: 87.91 ng/mL, inflamed: 382.76 ng/mL, P < 0.05). LPS stimulation of HDPSCs showed a significant increase in VEGF mRNA expression with increasing LPS concentrations (LPS concentrations of 0.01, 0.1, 1, and 10 µg/mL resulted in VEGF mRNA expressions of 181.2%, 274.2%, 345.8%, and 460.9%, respectively, P < 0.05). VEGF treatment significantly enhanced the migration ability of HDPSCs in Transwell chamber experiments, with migration rates increasing with VEGF concentrations (VEGF concentrations of 0, 1, 10, 20, 50, and 100 ng/mL resulted in migration rates of 8.41%, 9.34%, 21.33%, 28.41%, 42.87%, and 63.15%, respectively, P < 0.05). Inhibitors of VEGFR2, FAK, AKT, P38, and combined VEGF stimulation demonstrated significant migration inhibition, with migration rates decreasing to 8.31%, 12.64%, 13.43%, 18.32%, and 74.17%, respectively. The migration rate with combined VEGF stimulation showed a significant difference (P < 0.05). The analysis of phosphorylation levels revealed that VEGF stimulation significantly activated phosphorylation of FAK, AKT, and P38, with phosphorylation levels increasing with VEGF concentrations (P < 0.05). The VEGF/VEGFR2 signaling axis regulated the migration ability of HDPSCs through the FAK/PI3K/AKT and P38MAPK pathways. This finding highlighted not only the crucial role of VEGF in injury repair of HDPSCs but also provided important clues for a comprehensive understanding of the potential applications of this signaling axis in dental regenerative medicine.

Sphingolipidomic profiling of human Dental Pulp Stem Cells undergoing osteogenic differentiation.

It is now recognized that sphingolipids are involved in the regulation and pathophysiology of several cellular processes such as proliferation, migration, and survival. Growing evidence also implicates them in regulating the behaviour of stem cells, the use of which is increasingly finding application in regenerative medicine. A shotgun lipidomic study was undertaken to determine whether sphingolipid biomarkers exist that can regulate the proliferation and osteogenic differentiation of human Dental Pulp Stem Cells (hDPSCs). Sphingolipids were extracted and identified by direct infusion into an electrospray mass spectrometer. By using cells cultured in osteogenic medium and in medium free of osteogenic stimuli, as a control, we analyzed and compared the SPLs profiles. Both cellular systems were treated at different times (72hours, 7 days, and 14 days) to highlight any changes in the sphingolipidomic profiles in the subsequent phases of the differentiation process. Signals from sphingolipid species demonstrating clear differences were selected, their relative abundance was determined, and statistical differences were analyzed. Thus, our work suggests a connection between sphingolipid metabolism and hDPSC osteogenic differentiation and provides new biomarkers for improving hDPSC-based orthopaedic regenerative medicine.

Enhancing the Physical, Antimicrobial, and Osteo/Odontogenic Properties of a Sol-Gel-Derived Tricalcium Silicate by Graphene Oxide for Vital Pulp Therapies.

OBJECTIVES: This study developed a sol-gel tricalcium silicate/graphene oxide (TCS-GO) composite and examined its physicochemical properties, antimicrobial activity, and osteo/odontogenic effect on dental pulp stem cells. METHODS: Tricalcium silicate was synthesized and combined with graphene oxide at three different concentrations, namely 0.02%, 0.04%, and 0.08% w/w, while tricalcium silicate and mineral trioxide aggregate served as controls. The setting time, compressive strength, pH, and calcium ion release of the composites were evaluated, as well as antimicrobial properties against Streptococcus mutans and Lactobacillus acidophilus. Additionally, the viability of dental pulp stem cells; apatite forming ability; and the gene expression of Alkaline phosphatase, Dentin sialophosphoprotein, and Runt-related transcription factor 2 were assessed. RESULTS: TCS-GO (0.08%) showed a significantly shorter setting time and higher compressive strength when compared to MTA (p < 0.05). Additionally, tricalcium silicate and TCS-GO groups showed a higher release of Ca ions than MTA, with no significant difference in pH values among the different groups. TCS-GO (0.08%) also demonstrated a significantly stronger antimicrobial effect against Lactobacillus acidophilus compared to MTA (p < 0.05). ALP expression was higher in TCS-GO (0.08%) than MTA on days 3 and 7, while DSPP expression was higher in TCS-GO (0.08%) than MTA on day 3 but reversed on day 7. There was no significant difference in RUNX2 expression between TCS-GO (0.08%) and MTA on days 3 and 7. CONCLUSIONS: The TCS-GO (0.08%) composite demonstrated superior physicochemical characteristics and antimicrobial properties compared to MTA. Moreover, the early upregulation of ALP and DSPP markers in TCS-GO (0.08%) indicates that it has the potential to promote and enhance the osteo/odontogenic differentiation of DPSCs.

Yeast ß-glucan-based nanoparticles loading methotrexate promotes osteogenesis of hDPSCs and periodontal bone regeneration under the inflammatory microenvironment.

The regeneration of absorbed alveolar bone and reconstruction of periodontal support tissue are huge challenges in the clinical treatment of periodontitis due to the limited regenerative capacity of alveolar bone. It is essential to regulate inflammatory reaction and periodontal cell differentiation. Based on the anti-inflammatory effect of baker's yeast ß-glucan (BYG) with biosafety by targeting macrophages, the BYG-based nanoparticles loading methotrexate (cBPM) were fabricated from polyethylene glycol-grafted BYG through chemical crosslinking for treatment of periodontitis. In our findings, cBPM promoted osteogenesis of human dental pulp stem cells (hDPSCs) under inflammatory microenvironment, characterized by the enhanced expression of osteogenesis-related Runx2 and activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/Erk) pathway in vitro. Animal experiments further demonstrate that cBPM effectively promoted periodontal bone regeneration and achieved in a better effect of recovery indicated by 19.2 % increase in tissue volume, 7.1 % decrease in trabecular separation, and a significant increase in percent bone volume and trabecular thickness, compared with the model group. Additionally, cBPM inhibited inflammation and repaired alveolar bone by transforming macrophage phenotype from inflammatory M1 to anti-inflammatory M2. This work provides an alternative strategy for the clinical treatment of periodontitis through BYG-based delivery nanoplatform of anti-inflammatory drugs.

Enhancement of the chondrogenic differentiation capacity of human dental pulp stem cells via chondroitin sulfate-coated polycaprolactone-MWCNT nanofibers.

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases.

Insulin promotes the bone formation capability of human dental pulp stem cells through attenuating the IIS/PI3K/AKT/mTOR pathway axis.

BACKGROUND: Insulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms. METHODS: Cell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining. RESULTS: Insulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10- 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10- 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10- 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation. CONCLUSIONS: Insulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states.

Odontoblasts or odontocytes, expression of stem cells markers and differentiation markers among human adult odontoblasts.

Despite that, the odontoblasts of the dental pulp are considered a terminally differentiated type of cell. We were interested in investigating if they express any embryonic, mesenchymal, or neural stem cell markers, along with other differentiation markers they were reported to express previously. Methods: An immunohistochemistry study was performed on wisdom teeth extracted from healthy donors aged between 17 and 19 for dental reasons. Nine markers were tested: c-Myc, SOX2, MCAM, CD73, NCAM1, STRO1, osteocalcin, S100, and Thy1. Results: Odontoblasts expressed the following markers: embryonic stem cell markers SOX2, c-Myc, mesenchymal stem cell marker MCAM, the neural differentiation marker S100, and the osteogenic differentiation marker osteocalcin. Odontoblasts did not express the following markers: mesenchymal stem cell markers CD73, STRO1, Thy1, and neural stem cell marker NCAM1. Conclusion: These findings suggest that odontoblasts' expression of these stem cell markers may enable them to dedifferentiate under certain conditions. Further investigation is needed into whether dental materials could induce such dedifferentiation for functional dentin regeneration.

Evaluation of Ilex guayusa and Piper marginatum Extract Cytotoxicity on Human Dental Pulp Mesenchymal Stem Cells.

BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

Intracranial graft of bioresorbable polymer scaffolds loaded with human Dental Pulp Stem Cells in stab wound murine injury model.

The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization.

LIM homeobox 8 reduced apoptosis and promoted periodontal tissue regeneration function of dental pulp stem cells.

Stem cell-mediated tissue regeneration is a promising strategy for repairing tissue defects and functional reconstruction in periodontitis, a common disease that leads to the loss of alveolar bone and teeth. However, stem cell apoptosis, widely observed during tissue regeneration, impairs its efficiency. Therefore, the regulation of stem cell apoptosis is critical for improving regeneration efficiency. The LIM homeobox 8 gene LHX8, belongs to the LIM homeobox family, which was involved in tooth morphogenesis. Here, we found that LHX8 was significantly expressed in dental pulp. LHX8 knockdown significantly increased dental pulp mesenchymal stem cells (DPSCs) apoptosis, as confirmed by RT-PCR, western blotting, flow cytometry, and transmission electron microscopy. Additionally, LHX8 overexpression inhibited apoptosis and enhanced the osteo/odontogenic differentiation potential of hDPSCs in vitro. Furthermore, LHX8-overexpression could enhance the periodontal tissue regeneration efficiency of hDPSCs in mice with periodontitis. In conclusion, the present study indicates that LHX8 inhibits stem cell apoptosis and promotes functional tissue formation in stem cell-based tissue regeneration engineering, suggesting a new therapeutic target to increase the efficacy of periodontal tissue regeneration.

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.

Evaluation of Human Platelet Lysate as an Alternative to Fetal Bovine Serum for Potential Clinical Applications of Stem Cells from Human Exfoliated Deciduous Teeth.

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization.

BACKGROUND: The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine. Stem cells can self-organise into microsized organ units, partially modelling tissue function and regeneration. Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases. However, the lack of vasculature limits the utility of dental pulp organoids. AIM: To improve survival and aid in recovery after stem cell transplantation, we demonstrated the three-dimensional (3D) self-assembly of adult stem cell-human dental pulp stem cells (hDPSCs) and endothelial cells (ECs) into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium (CM). METHODS: During culture, primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM. The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids. The biological characteristics of the organoids were analysed, and the regulatory pathways associated with angiogenesis were studied. RESULTS: The combination of these two agents resulted in prevascularized human dental pulp organoids (Vorganoids) that more closely resembled dental pulp tissue in terms of morphology and function. Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis. The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids. CONCLUSION: In this innovative study, we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration, facilitating the development of clinical treatment strategies.

Dental Pulp Stem Cell-Derived Exosomes Promote Sciatic Nerve Regeneration via Optimizing Schwann Cell Function.

Repair strategies for injured peripheral nerve have achieved great progresses in recent years. However, the clinical outcomes remain unsatisfactory. Recent studies have found that exosomes secreted by dental pulp stem cells (DPSC-exos) have great potential for applications in nerve repair. In this study, we evaluated the effects of human DPSC-exos on improving peripheral nerve regeneration. Initially, we established a coculture system between DPSCs and Schwann cells (SCs) in vitro to assess the effect of DPSC-exos on the activity of embryonic dorsal root ganglion neurons (DRGs) growth in SCs. We extracted and labeled human DPSC-exos, which were subsequently utilized in uptake experiments in DRGs and SCs. Subsequently, we established a rat sciatic nerve injury model to evaluate the therapeutic potential of DPSC-exos in repairing sciatic nerve damage. Our findings revealed that DPSC-exos significantly promoted neurite elongation by enhancing the proliferation, migration, and secretion of neurotrophic factors by SCs. In vivo, DPSC-exos administration significantly improved the walking behavior, axon regeneration, and myelination in rats with sciatic nerve injuries. Our study underscores the vast potential of DPSC-exos as a therapeutic tool for tissue-engineered nerve construction.

How to make full use of dental pulp stem cells: an optimized cell culture method based on explant technology.

Introduction: Dental pulp stem cells from humans possess self-renewal and versatile differentiation abilities. These cells, known as DPSC, are promising for tissue engineering due to their outstanding biological characteristics and ease of access without significant donor site trauma. Existing methods for isolating DPSC mainly include enzyme digestion and explant techniques. Compared with the enzymatic digestion technique, the outgrowth method is less prone to cell damage and loss during the operation, which is essential for DPSC with fewer tissue sources. Methods: In order to maximize the amount of stem cells harvested while reducing the cost of DPSC culture, the feasibility of the optimized explant technique was evaluated in this experiment. Cell morphology, minimum cell emergence time, the total amount of cells harvested, cell survival, and proliferative and differentiation capacity of DPSC obtained with different numbers of explant attachments (A1-A5) were evaluated. Results: There was a reduction in the survival rate of the cells in groups A2-A5, and the amount of harvested DPSC decreased in A3-A5 groups, but the DPSC harvested in groups A1-A4 had similar proliferative and differentiation abilities. However, starting from group A5, the survival rate, proliferation and differentiation ability of DPSC decreased significantly, and the adipogenic trend of the cells became more apparent, indicating that the cells had begun to enter the senescence state. Discussion: The results of our study demonstrated that the DPSC obtained by the optimized explant method up to 4 times had reliable biological properties and is available for tissue engineering.

Antimicrobial Peptide- and Dentin Matrix-Functionalized Hydrogel for Vital Pulp Therapy via Synergistic Bacteriostasis, Immunomodulation, and Dentinogenesis.

The preservation of vital pulps is crucial for maintaining the physiological functions of teeth; however, vital pulp therapy (VPT) of pulpitis teeth remains a substantial challenge due to uncontrolled infection, excessive inflammation, and limited regenerative potential. Current pulp capping agents have restricted effects in the infectious and inflammatory microenvironment. To address this, a multifunctional hydrogel (TGH/DM) with antibacterial, immunomodulatory, and mineralization-promoting effects is designed. The antimicrobial peptide (AMP) and demineralized dentin matrix are incorporated into the hydrogel, achieving sustainable delivery of AMP and a cocktail of growth factors. In vitro results show that TGH/DM could kill endodontic microbiota, ameliorate inflammatory responses of human dental pulp stem cells (hDPSCs), and prompt odontogenic differentiation of inflammatory hDPSCs via activation of peroxisome proliferator-activated receptor gamma. In vivo results suggest that TGH/DM is capable of inducing M2 phenotype transformation of macrophages in mice and fostering the regeneration of the dentin-pulp complex in inflamed pulps of beagle dogs. Overall, this study first proposes the synergistic regulation of AMP and tissue-specific extracellular matrix for the treatment of pulpitis, and the advanced hydrogel provides a facile and effective way for VPT.

Osteoblastic differentiation and changes in the redox state in pulp stem cells by laser treatment.

The aim of this study was to determine the effect of low-level laser therapy (LLLT) on cell proliferation, mitochondrial membrane potential changes (∆Ψm), reactive oxygen species (ROS), and osteoblast differentiation of human dental pulp stem cells (hDPSCs). These cells were irradiated with 660- and 940-nm lasers for 5 s, 50 s, and 180 s. Cell proliferation was assessed using the resazurin assay, cell differentiation by RUNX2 and BMP2 expression, and the presence of calcification nodules using alizarin-red S staining. ROS was determined by the dichlorofluorescein-diacetate technique and changes in ∆Ψm by the tetramethylrhodamine-ester assay. Data were analyzed by a Student's t-test and Mann-Whitney U test. The 940-nm wavelength for 5 and 50 s increased proliferation at 4 days postirradiation. After 8 days, a significant decrease in proliferation was observed in all groups. Calcification nodules were evident in all groups, with a greater staining intensity in cells treated with a 940-nm laser for 50 s, an effect that correlated with increased RUNX2 and BMP2 expression. ROS production and Δψm increased independently of irradiation time. In conclusion, photobiomodulation (PBM) with LLLT induced morphological changes and reduced cell proliferation rate, which was associated with osteoblastic differentiation and increased ROS and Δψm, independent of wavelength and time.

Amino acid-based supramolecular chiral hydrogels promote osteogenesis of human dental pulp stem cells via the MAPK pathway.

Critical-size defects (CSDs) of the craniofacial bones cause aesthetic and functional complications that seriously impact the quality of life. The transplantation of human dental pulp stem cells (hDPSCs) is a promising strategy for bone tissue engineering. Chirality is commonly observed in natural biomolecules, yet its effect on stem cell differentiation is seldom studied, and little is known about the underlying mechanism. In this study, supramolecular chiral hydrogels were constructed using L/d-phenylalanine (L/D-Phe) derivatives. The results of alkaline phosphatase expression analysis, alizarin red S assay, as well as quantitative real-time polymerase chain reaction and western blot analyses suggest that right-handed D-Phe hydrogel fibers significantly promoted osteogenic differentiation of hDPSCs. A rat model of calvarial defects was created to investigate the regulation of chiral nanofibers on the osteogenic differentiation of hDPSCs in vivo. The results of the animal experiment demonstrated that the D-Phe group exhibited greater and faster bone formation on hDPSCs. The results of RNA sequencing, vinculin immunofluorescence staining, a calcium fluorescence probe assay, and western blot analysis indicated that L-Phe significantly promoted adhesion of hDPSCs, while D-Phe nanofibers enhanced osteogenic differentiation of hDPSCs by facilitating calcium entry into cells and activate the MAPK pathway. These results of chirality-dependent osteogenic differentiation offer a novel therapeutic strategy for the treatment of CSDs by optimising the differentiation of hDPSCs into chiral nanofibers.

Lysine demethylase 3A promotes chondrogenic differentiation of aged human dental pulp stem cells.

Background/purpose: Aging severely impairs the beneficial effects of human dental pulp stem cells (hDPSCs) on cartilage regeneration. Lysine demethylase 3A (KDM3A) is involved in regulating mesenchymal stem cells (MSCs) senescence and bone aging. In this study, we investigated the role of KDM3A in hDPSCs aging and whether KDM3A could rejuvenate aged hDPSCs to enhance their chondrogenic differentiation capacity. Materials and methods: The cellular aging of hDPSCs was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. Protein levels were determined using Western blot analysis. KDM3A was overexpressed in aged hDPSCs by lentivirus infection. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to determine the mRNA levels of stemness markers. Toluidine blue staining was used to evaluate the effect of KDM3A overexpression on the chondrogenic differentiation of aged hDPSCs. Results: hDPSCs at passage 12 or treated with etoposide exhibited augmented cellular senescence as evidenced by increased SA-ß-gal activity. KDM3A was significantly increased during senescence of hDPSCs. Overexpression of KDM3A did not affect the stemness properties but significantly promoted the chondrogenic differentiation of aged hDPSCs. Conclusion: Our findings indicate that KDM3A plays an important role in the maintenance of the chondrogenic differentiation capacity of aged hDPSCs and suggest that therapies targeting KDM3A may be a novel strategy to rejuvenate aged hDPSCs.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 µg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.