Human Dental Pulp Stem Cells Research Articles

Effect of tetracycline hydrochloride application on dental pulp stem cell metabolism-booster or obstacle for tissue engineering?

Introduction: Stem cells and scaffolds are an important foundation and starting point for tissue engineering. Human dental pulp stem cells (DPSC) are mesenchymal stem cells with self-renewal and multi-directional differentiation potential, and are ideal candidates for tissue engineering due to their excellent biological properties and accessibility without causing major trauma at the donor site. Tetracycline hydrochloride (TCH), a broad-spectrum antibiotic, has been widely used in recent years for the synthesis of cellular scaffolds to reduce the incidence of postoperative infections. Methods: In order to evaluate the effects of TCH on DPSC, the metabolism of DPSC in different concentrations of TCH environment was tested. Moreover, cell morphology, survival rates, proliferation rates, cell migration rates and differentiation abilities of DPSC at TCH concentrations of 0-500μg/ml were measured. Phalloidin staining, live-dead staining, MTS assay, cell scratch assay and real-time PCR techniques were used to detect the changes in DPSC under varies TCH concentrations. Results: At TCH concentrations higher than 250μg/ml, DPSC cells were sequestered, the proportion of dead cells increased, and the cell proliferation capacity and cell migration capacity decreased. The osteogenic and adipogenic differentiation abilities of DPSC, however, were already inhibited at TCH con-centrations higher than 50μg/ml. Here, the expression of the osteogenic genes, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN), the lipogenic genes lipase (LPL), as well as the peroxisome proliferator-activated receptor-γ (PPAR-γ) expression were found to be down-regulated. Discussion: The results of the study indicated that TCH in concentrations above 50µg/ml negatively affects the differentiation capability of DPSC. In addition, TCH at concentrations above 250µg/ml adversely affects the growth status, percentage of living cells, proliferation and migration ability of cells.

Human Dental Pulp Stem Cells Are Subjected to Metabolic Reprogramming and Repressed Proliferation and Migration by the Sympathetic Nervous System via α1B-Adrenergic Receptor

IntroductionHuman dental pulp stem cells (hDPSCs) reside in specialized microenvironments in the dental pulp, termed “niches,” which are composed of diverse cellular components including nerves. Sensory nerves can positively regulate the expansion and differentiation of pulp cells, while the biological effects of the sympathetic nervous system (SNS) on hDPSCs remain elusive. This study is devoted to investigating the effects and underlying mechanisms of the SNS on the proliferation and migration of hDPSCs. MethodsThe distribution of sympathetic nerve fibers in human dental pulp was examined by immunofluorescence staining of tyrosine hydroxylase. The concentration of norepinephrine in healthy and carious human dental pulp tissues was detected using enzyme-linked immunosorbent assay. RNA-sequencing was applied to identify the dominant sympathetic neurotransmitter receptor in hDPSCs. Seahorse metabolic assay, adenosine triphosphate assay, lactate assay, and mitochondrial DNA copy number were performed to determine the level of glycometabolism. Transwell assay, wound healing assay, 5-ethynyl-2'-deoxyuridine staining assay, cell cycle assay, and Cell Counting Kit-8 assay were conducted to analyze the migratory and proliferative capacities of hDPSCs. ResultsSprouting of sympathetic nerve fibers and an increased concentration of norepinephrine were observed in inflammatory pulp tissues. Sympathetic nerve fibers were mainly distributed along blood vessels, and aldehyde dehydrogenase 1–positive hDPSCs resided in close proximity to neurovascular bundles. ADRA1B was identified as the major sympathetic neurotransmitter receptor expressed in hDPSCs, and its expression was enhanced in inflammatory pulp tissues. In addition, the SNS inhibited the proliferation and migration of hDPSCs through metabolic reprogramming via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways. ConclusionsThis study demonstrates that the SNS can shift the metabolism of hDPSCs from oxidative phosphorylation to anaerobic glycolysis via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways, thereby inhibiting the proliferative and migratory abilities of hDPSCs. This metabolic shift may facilitate the maintenance of the quiescent state of hDPSCs.

Conditioned medium from human dental pulp stem cells treats spinal cord injury by inhibiting microglial pyroptosis.

Human dental pulp stem cell transplantation has been shown to be an effective therapeutic strategy for spinal cord injury. However, whether the human dental pulp stem cell secretome can contribute to functional recovery after spinal cord injury remains unclear. In the present study, we established a rat model of spinal cord injury based on impact injury froma dropped weight and then intraperitoneally injected the rats with conditioned medium from human dental pulp stem cells. We found that the conditioned medium effectively promoted the recovery of sensory and motor functions in rats with spinal cord injury, decreased expression of the microglial pyroptosis markers NLRP3, GSDMD, caspase-1, and interleukin-1β, promoted axonal and myelin regeneration, and inhibited the formation of glial scars. In addition, in a lipopolysaccharide-induced BV2 microglia model, conditioned medium from human dental pulp stem cells protected cells from pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1β pathway. These results indicate that conditioned medium from human dental pulp stem cells can reduce microglial pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1β pathway, thereby promoting the recovery of neurological function after spinal cord injury. Therefore, conditioned medium from human dental pulp stem cells may become an alternative therapy for spinal cord injury.

Effects of sEV derived from SHED and DPSC on the proliferation, migration and osteogenesis of PDLSC

IntroductionPeriodontitis is a highly prevalent oral disease characterized by irreversible bone resorption and tooth loss. The proliferation, migration and osteogenic differentiation of periodontal ligament stem cell (PDLSC) are crucial to the regeneration of periodontal bone defects. There is increasing evidence that small extracellular vesicle (sEV) derived from pulp stem cell, including human exfoliated deciduous teeth stem cell (SHED) and human dental pulp stem cell (DPSC), is a potential mediator for bone tissue regeneration. However, which one is more suitable for periodontal bone formation still remains to be studied. MethodsIn this study, NTA and BCA were performed to compare the productivity of sEV derived from SHED (SHED-sEV) and sEV derived from DPSC (DPSC-sEV). CCK-8, transwell assay, alkaline phosphatase staining and activity assay, alizarin red staining, qRT-PCR, and western blotting were conducted to detect the proliferation, migration, and osteogenesis of PDLSCs coculturing with SHED-sEV or DPSC-sEV. ResultsThe secretory efficiency of SHED-sEV was much higher than that of DPSC-sEV. The cellular uptake of sEVs could promote the proliferation, migration and osteogenesis of DPLSCs. Compared with DPSC-sEV, SHED-sEV showed better ability in such promotion. ConclusionsSHED-sEV showed higher productivity and better osteogenic induction ability than DPSC-sEV. Thus, SHED-sEV may be a more promising candidate for periodontal bone regeneration.

Extracellular Vesicles Carrying RUNX3 Promote Differentiation of Dental Pulp Stem Cells.

This study aims to clarify the mechanism underlying dental pulp cells-extracellular vesicles (DPC-EVs) carrying runt-related transcription factor 3 (RUNX3) in mediating odontogenic differentiation of dental pulp stem cells (DPSCs) with the involvement of miR-30a-5p-regulated NOTCH1. Extracellular vesicles (EVs) were isolated from human DPSCs, and identified using transmission electron microscopy, and nanoparticle tracking analysis. PBS, EVs, or EV inhibitor GW4869 was added to DPSCs for co-culture, whilst odontogenic differentiation was assessed in terms of ratio of mineralized nodules and expression odontoblast differentiation markers. Dual luciferase reporter gene assay and chromatin immunoprecipitation for binding relation among RUNX3, miR-30a-5p and NOTCH1were employed to evaluate their roles in odontogenic differentiation was determined. Animal experiment was established to confirm the effect of DPC-EVs-loaded RUNX3 on dental pulp. In vitro finding demonstrated that EVs delivered RUNX3 to DPSCs, thereby activated miR-30a-5p expression and inhibited NOTCH1 expression, which was reversed by addition of GW4869. RUNX3 upregulation promoted miR-30a-5p while miR-30a-5p targeted and inhibited NOTCH1. Silencing of RUNX3 in EVs decreased expression of those differentiation markers, downregulated miR-30a-5p and upregulated NOTCH1. DPSC-EVs can carry RUNX3 to the DPSCs, promote the transcription of miR-30a-5p, and then inhibit the expression of NOTCH1, and finally promote the odontogenic differentiation of DPSCs.

Multifunctional Lithium-Doped Mesoporous Nanoparticles for Effective Dentin Regeneration in vivo.

Effective infection control without irritating the pulp tissue is the key to successful vital pulp therapy. Developing a novel antibacterial biomaterial that promotes dentin regeneration for pulp capping is thus a promising strategy for enhancing vital pulp therapy. Lithium-doped mesoporous nanoparticles (Li-MNPs) were synthesized using an alkali-catalyzed sol-gel method. The particle size, elemental distribution, surface morphology, pore structure, and ion release from Li-MNPs were measured. Human dental pulp stem cells (hDPSCs) and Streptococcus mutans (S. mutans) were used to evaluate the biological effects of Li-MNPs. In addition, a dental pulp exposure mouse model was used to evaluate the regenerative effects of Li-MNPs. Li-MNPs had a larger surface area (221.18 m2/g), a larger pore volume (0.25 cm3/g), and a smaller particle size (520.92 ± 35.21 nm) than MNPs. The in vitro investigation demonstrated that Li-MNPs greatly enhanced the biomineralization and odontogenic differentiation of hDPSCs through the Wnt/β-catenin signaling pathway. Li-MNPs showed a strong antibacterial effect on S. mutans. As expected, Li-MNPs significantly promoted dentin regeneration in situ and in vivo. Li-MNPs promoted dentin regeneration and inhibited S. mutans growth, implying a possible application as a pulp capping agent in vital pulp therapy.

Biological Function and Potential Applications of Garcinol in Human Dental Pulp Stem Cells

IntroductionThe regeneration of pulp tissue is crucial for true regenerative endodontic treatment, which requires a reduction in osteogenic differentiation. Garcinol, a histone acetyltransferase inhibitor, is a natural regulator that is known to suppress the osteogenic differentiation of dental pulp stem cells. In this study, the inhibitory effect of garcinol on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) was evaluated using three-dimensional culture under in vitro and in vivo conditions. MethodshDPSCs were obtained from caries-free third molars and cultured with 10 μM garcinol for 7 days in an ultra-low attachment plate. The cell stemness and expression of osteogenic differentiation-related genes were analyzed using reverse transcription-polymerase chain reaction and single-cell analysis. A transplantation experiment was performed in mice to investigate whether garcinol-treated hDPSCs showed restrained osteogenic differentiation. ResultshDPSCs cultured in the U-shaped ultra-low attachment plate showed the highest expression of stemness-related genes. Garcinol-treated hDPSCs demonstrated downregulation of osteogenic differentiation, with lower expression of bone sialoprotein, which is related to bone formation, and higher expression of dentin sialophosphoprotein, which is related to dentin formation. However, the garcinol-treated hDPSCs did not show any alterations in their stemness. Consistent results were observed in the transplantation experiment in mice. ConclusionsGarcinol reduced the osteogenic differentiation of hDPSCs, which can contribute to true regenerative endodontic treatment.

Development of cobalt-incorporated chitosan scaffold for regenerative potential in human dental pulp stem cells: An in vitro study

The aim of the study was to comparatively evaluate chitosan and Cobalt incorporated chitosan (CoCH) scaffold at varying concentrations in terms of their material characteristics, cytotoxicity and cell adhesion potential. In the present study, cobalt incorporated chitosan scaffolds at varying concentrations were prepared and dried. The synthesised scaffolds were characterised using XRD, FTIR, SEM-EDX and BET which revealed amorphous, porous surface of CoCH scaffolds and FTIR analysis showed the complexation confirming the chelation of cobalt with chitosan. The experimental scaffolds proved to be non-cytotoxic when compared to chitosan scaffolds on XTT analysis. Cell-seeding assay revealed enhanced adherence of hDPSCs to CoCH scaffold at 1:1 ratio in the concentration of 100 mL of 100 μmol/L cobalt chloride solution in 100mL of 2% chitosan solution, when compared to other groups. The results highlighted that 100 μmol/L concentration of cobalt chloride when incorporated in 1:1 ratio into 2 % CH solution yields a promising porous, biocompatible scaffold with enhanced cellular adhesion for dentin-pulp regeneration.

Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair

BackgroundRedox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling.MethodshDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs.ResultsIn differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction.ConclusionsAltogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

Ruxolitinib altered IFN-β induced necroptosis of human dental pulp stem cells during osteoblast differentiation

ObjectiveThis study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-β) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). DesignhDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-β protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. ResultsIn the IFN-β-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-β-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. ConclusionRuxolitinib interfered with the IFN-β-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.

Novel Bioactive Nanocomposites Containing Calcium Fluoride and Calcium Phosphate with Antibacterial and Low-Shrinkage-Stress Capabilities to Inhibit Dental Caries.

Composites are commonly used for tooth restorations, but recurrent caries often lead to restoration failures due to polymerization shrinkage-stress-induced marginal leakage. The aims of this research were to: (1) develop novel low-shrinkage-stress (L.S.S.) nanocomposites containing dimethylaminododecyl methacrylate (DMADDM) with nanoparticles of calcium fluoride (nCaF2) or amorphous calcium phosphate (NACP) for remineralization; (2) investigate antibacterial and cytocompatibility properties. Nanocomposites were made by mixing triethylene glycol divinylbenzyl ether with urethane dimethacrylate containing 3% DMADDM, 20% nCaF2, and 20% NACP. Flexural strength, elastic modulus, antibacterial properties against Streptococcus mutans biofilms, and cytotoxicity against human gingival fibroblasts and dental pulp stem cells were tested. Nanocomposites with DMADDM and nCaF2 or NACP had flexural strengths matching commercial composite control without bioactivity. The new nanocomposite provided potent antibacterial properties, reducing biofilm CFU by 6 logs, and reducing lactic acid synthesis and metabolic function of biofilms by 90%, compared to controls (p < 0.05). The new nanocomposites produced excellent cell viability matching commercial control (p > 0.05). Bioactive L.S.S. antibacterial nanocomposites with nCaF2 and NACP had excellent bioactivity without compromising mechanical and cytocompatible properties. The new nanocomposites are promising for a wide range of dental restorations by improving marginal integrity by reducing shrinkage stress, defending tooth structures, and minimizing cariogenic biofilms.

In vitro response of dental pulp stem cells to dural substitute grafts: Analysis of cytocompatibility and bioactivity.

The objective of this study was to assess and compare the cytocompatibility of decellularized porcine small intestine submucosal dural graft from Biodesign (BD) and polyester urethane-based Neuro-Patch (NP) dural substitute with the mineral trioxide aggregate (MTA) and the cyanoacrylate-based Histoacryl surgical adhesive. Furthermore, the study evaluated the inflammatory response and osteogenic differentiation of human dental pulp stem cells (hDPSCs) when cultured in direct contact with the dural substitutes in comparison with MTA. The viability of hDPSCs in direct contact with the tested materials was investigated in vitro by a CCK-8 assay. Additionally, the effects of dural substitutes and MTA on the expression of the inflammatory mediator interleukin-6 (IL-6) was investigated via enzyme-linked immunosorbent assay (ELISA), whilst effects on the differentiation were evaluated using alizarin red staining, alkaline phosphatase staining, ELISA and energy-dispersive X-ray elemental mapping. The dural substitutes were cytocompatible and promoted cellular adhesion. The Histoacryl and MTA demonstrated cytotoxicity in fresh preparations but showed a more favourable cellular reaction when set. Investigations of biological activity indicated that dural substitute membranes did not induce an inflammatory response or osteogenic differentiation of hDPSCs. In contrast, MTA induced the expression of IL-6 and alkaline phosphatase activity contributing to enhanced differentiation and mineralization. The dural substitute membranes showed cytocompatibility, did not provoke an inflammatory response and maintained the stemness of hDPSCs better than MTA. Additionally, the set Histoacryl surgical adhesive demonstrated good biocompatibility. Taken together, these results highlight the potential use of dural substitutes in regenerative endodontic procedures as coronal barriers alternative to MTA to reduce the incidence of intracanal calcifications.

Micropattern‐Based Stem Cell Gym: Mechanical Memory Enhanced Stemness Maintenance of Human Dental Pulp Stem Cells and Nerve Regeneration

AbstractDental pulp stem cells (DPSCs) which are derived from the neural crest are promising tools in stem cell therapy in the clinic. However, they tend to lose their multi‐lineage potency spontaneously during long‐term expansion in vitro. A micropattern‐based system is developed to train DPSCs with 1024 µm2 and aspect ratios of 1:2 and 1:4, which enhances their stemness and differentiation potential. This mechanical memory induced by micropattern would also rescue the pluripotency of long‐term expansion DPSCs. Micropatterns remodel the cytoskeleton and nuclear morphology, soften the cells, and induce the mitochondria into an immature state. Micropattern also activates extracellular related kinase, signal transducer, and activator of transcription 3, and inhibits Yes‐associated protein nuclear localization to enhance the stemness of DPSCs. In vivo implantation proves that DPSCs mechanically trained by micropattern could promote nerve regeneration in rats with peripheral nerve injury. A micropattern‐based approach is provided as a stem cell gym that absents exogenous growth factors to enhance the stemness and regenerative properties of DPSCs. It may shed light on future stem cell therapy.

Integration of blue light with near-infrared irradiation accelerates the osteogenic differentiation of human dental pulp stem cells.

Blue light is used less in photobiomodulation than red or near-infrared light because of concerns about its high energy. However, some reports have suggested that blue light releases NO from nitrosated proteins, affects cell signal regulation, and promotes stem cell differentiation. Because blue and red lights could have different mechanisms of action, their combination is expected to have new consequences. In this study, human dental pulp stem cells (hDPSCs) were sequentially exposed to blue and near-infrared light to study their effects on proliferation, osteogenic differentiation, and immunomodulation. We found that NIR irradiation applied after blue light can reduce blue light toxicity improving the cell viabiltiy. Delayed luminescence and transmission electron microscopy studies showed that this combination excited hDPSCs and activated mitochondrial biogenesis. Those modulations accelerated hDPSC differentiation, as shown by an increase of about 1.3-fold in alkaline phosphatase activity in vitro and an about 1.5-fold increase in the osteocalcin-positive regions in cells implanted in nude mice compared with mice exposed to near-infrared alone.

Evaluation of in vitro biocompatibility of human pulp stem cells with allogeneic, alloplastic, and xenogeneic grafts under the influence of extracellular vesicles

Therapies using dental pulp stem cells (DPSCs) or stem cell-derived extracellular vesicles (EVs) have shown promising applications for bone tissue engineering. This in vitro experiment evaluated the joint osteogenic capability of DPSCs and EVs on alloplastic (maxresorp), allogeneic (maxgraft), and xenogeneic (cerabone) bone grafts. We hypothesize that osteogenic differentiation and the proliferation of human DPSCs vary between bone grafts and are favorable under the influence of EVs. DPSCs were obtained from human wisdom teeth, and EVs derived from DPSCs were isolated from cell culture medium. DPSCs were seeded on alloplastic, allogeneic, and xenogeneic bone graft substitutes for control, and the same scaffolds were administered with EVs in further groups. The cellular uptake of EVs into DPSC cells was assessed by confocal laser scanning microscopy. Cell vitality staining and calcein acetoxymethyl ester staining were used to evaluate cell attachment and proliferation. Cell morphology was determined using scanning electron microscopy, and osteogenic differentiation was explored by alkaline phosphatase and Alizarin red staining. Within the limitations of an in vitro study without pathologies, the results suggest that especially the use of xenogeneic bone graft substitutes with DPSCS and EVs may represent a promising treatment approach for alveolar bone defects.

The Secretome of Human Dental Pulp Stem Cells and Its Components GDF15 and HB-EGF Protect Amyotrophic Lateral Sclerosis Motoneurons against Death.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.

Dextrin hydrogel loaded with a macroporous Bonelike® scaffold and dental pulp stem cells for critical-sized defect repair

Regeneration of severe bone defects remains a challenge. A formulation of synthetic glass-reinforced hydroxyapatite bone substitute, Bonelike® Poro (BL®P), 250–500 µm-diameter, with a dextrin-based hydrogel (HG), further loaded with human dental pulp stem cells (hDPSCs) with osteogenic differentiation ability, was tested for the management of critical-sized defects in an ovine model.Morphology, calcium release, and mechanical strength of HG + BL®P were analyzed. Then, BL®P, HG + BL®P, and 106 hDPSCs-loaded HG + BL®P were implanted in ovine critical-sized 14 mm-diameter calvaria defects. Bone samples were collected after 3 and 6 weeks for histological and micro-CT analysis.BL®P exhibits a suitable porous size for cell ingrowth, from the nm (>200 nm) to the µm (5 µm) range. The addition of BL®P granules to the HG resulted in increased compressive elastic modulus and ultimate tensile strength. The mildly acidic nature of the HG contributed to a faster dissolution of granules. In vivo results confirmed the HG suitability as a carrier, providing better defect filling, easy handling, and injectability of BL®P without compromising new bone formation nor biocompatibility. The HG + BL®P formulations can successfully regenerate critical-sized defects; however, addition of hDPSCs did not significantly enhance new bone formation under these conditions.Granular BL®P provides an effective alternative to autologous grafts. The HG acts as a biocompatible carrier of granular bone substitutes and cells, conferring injectability and cohesivity.

HnRNP A1 Suppresses the Odontogenic Differentiation and the Inclusion of RUNX2 Exon 5 of Dental Mesenchymal Cells.

RUNX2 (Runt-related transcription factor 2) acts as a key regulator in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). Moreover, the inclusion of exon 5 is important for RUNX2 function. Our previous study showed that Y-Box Binding Protein 1 (YBX1) promoted RUNX2 exon 5 inclusion and mineralization of hDPSCs. However, the regulatory mechanism of RUNX2 exon 5 alternative splicing needed further exploration. The expression level of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) during the odontogenic differentiation of hDPSCs was analyzed by RT-PCR and Western blot. The roles of hnRNP A1 in the alternative splicing of RUNX2 exon 5 and the odontogenic differentiation of dental mesenchymal cells were analyzed by gain- and loss-of-function experiments. Surprisingly, we found an alternative splicing factor, hnRNP A1, which had an opposite role to YBX1 in regulating RUNX2 exon 5 inclusion and odontogenic differentiation of hDPSCs. Through gain- and loss-of-function assay, we found that hnRNP A1 suppressed the inclusion of RUNX2 exon 5, resulting in the inhibition of odontoblastic differentiation. The overexpression of hnRNP A1 can inhibit the expression of ALP (alkaline phosphatase) and OCN (osteocalcin), and the formation of mineralized nodules during the odontogenic differentiation of both hDPSCs and mouse dental papilla cells (mDPCs), whereas the opposite results were obtained with an hnRNP A1 knockdown preparation. The present study indicated that hnRNP A1 suppressed RUNX2 exon 5 inclusion and reduced the odontogenic differentiation ability of hDPSCs and mDPCs.

BDNF/TrkB Is a Crucial Regulator in the Inflammation-Mediated Odontoblastic Differentiation of Dental Pulp Stem Cells.

The odontoblastic differentiation of dental pulp stem cells (DPSCs) associated with caries injury happens in an inflammatory context. We recently demonstrated that there is a link between inflammation and dental tissue regeneration, identified via enhanced DPSC-mediated dentinogenesis in vitro. Brain-derived neurotrophic factor (BDNF) is a nerve growth factor-related gene family molecule which functions through tropomyosin receptor kinase B (TrkB). While the roles of BDNF in neural tissue repair and other regeneration processes are well identified, its role in dentinogenesis has not been explored. Furthermore, the role of BDNF receptor-TrkB in inflammation-induced dentinogenesis remains unknown. The role of BDNF/TrkB was examined during a 17-day odontogenic differentiation of DPSCs. Human DPSCs were subjected to odontogenic differentiation in dentinogenic media treated with inflammation inducers (LTA or TNFα), BDNF, and a TrkB agonist (LM22A-4) and/or antagonist (CTX-B). Our data show that BDNF and TrkB receptors affect the early and late stages of the odontogenic differentiation of DPSCs. Immunofluorescent data confirmed the expression of BDNF and TrkB in DPSCs. Our ELISA and qPCR data demonstrate that TrkB agonist treatment increased the expression of dentin matrix protein-1 (DMP-1) during early DPSC odontoblastic differentiation. Coherently, the expression levels of runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN) were increased. TNFα, which is responsible for a diverse range of inflammation signaling, increased the levels of expression of dentin sialophosphoprotein (DSPP) and DMP1. Furthermore, BDNF significantly potentiated its effect. The application of CTX-B reversed this effect, suggesting TrkB`s critical role in TNFα-mediated dentinogenesis. Our studies provide novel findings on the role of BDNF-TrkB in the inflammation-induced odontoblastic differentiation of DPSCs. This finding will address a novel regulatory pathway and a therapeutic approach in dentin tissue engineering using DPSCs.

Efficacy of redox nanoparticles for improving survival of transplanted cells in a mouse model of ischemic stroke.

The success of cell transplantation therapy for ischemic stroke is hindered by the low cell survival rate in poststroke brain, due in part to high free radical production and ensuing oxidative stress. We have developed redox nanoparticles to eliminate reactive oxygen species. In this study, we tested the protective efficacy of these redox nanoparticles in cell culture and a mouse model of ischemic stroke. Induced human dental pulp stem cells were subjected to oxygen-glucose deprivation and reoxygenation to recapitulate ischemia and reperfusion in the penumbra surrounding a cerebral infarct. Cell viability using WST-8 assay, apoptosis using TUNEL, free radicals using MitoSOX, and inflammatory cytokines using ELISA kit were measured in the presence and absence of redox nanoparticles after oxygen-glucose deprivation and reoxygenation. The scavenging activity of redox nanoparticles against reactive oxygen species was detected by electron spin resonance. Moreover, induced cells were transplanted intracerebrally into to the distal middle cerebral artery occlusion model with and without redox nanoparticles, and the survival rate measured. Cell viability was enhanced, while apoptosis, free radical generation, and inflammatory cytokine expression levels were reduced in cultures with redox nanoparticles. Further, reduced redox nanoparticles were detected in the cytoplasm, indicating free radical scavenging. Addition of redox nanoparticles also improved the survival rate of transplanted cells after 6 weeks in vivo. These redox nanoparticles may increase the applicability and success of induced stem cell therapy for ischemic stroke patents by promoting long-term survival.