Transplantation of Neural Progenitor Cells Derived from Stem Cells from Apical Papilla Through Small-Molecule Induction in a Rat Model of Sciatic Nerve Injury.

BACKGROUND: Stem cell-based transplantation therapy holds promise for peripheral nerve injury treatment, but adult availability is limited. A cell culture protocol utilizing a small-molecule cocktail effectively reprogrammed stem cells from apical papilla (SCAPs) into neural progenitor cells, subsequently differentiating into neuron-like cells. This study aims to evaluate neural-induced SCAPs, with and without small-molecule cocktail, for sciatic nerve repair potential. METHODS: A scaffold-free cell sheet technique was used to construct a three-dimensional cell sheet. Subsequently, this cell sheet was carefully rolled into a tube and seamlessly inserted into a collagen conduit, which was then transplanted into a 5 mm sciatic nerve injury rat model. Functional sciatic nerve regeneration was evaluated via toe spread test, walking track analysis and gastrocnemius muscle weight. Additionally, degree of sciatic nerve regeneration was determined based on total amount of myelinated fibers. RESULTS: Small-molecule cocktail induced SCAPs enhanced motor function recovery, evident in improved sciatic function index and gastrocnemius muscle retention. We also observed better host myelinated fiber retention than undifferentiated SCAPs or neural-induced SCAPs without small-molecule cocktail. However, clusters of neuron-like cell bodies (surrounded by sparse myelinated fibers) were found in all cell sheet-implanted groups in the implantation region. This suggests that while the implanted cells likely survived transplantation, integration was poor and would likely hinder long-term recovery by occupying the space needed for host nerve fibers to project through. CONCLUSION: Neural-induced SCAPs with small-molecule cocktail demonstrated promising benefits for nerve repair; further research is needed to improve its integration and optimize its potential for long-term recovery.

An Injectable Hydrogel Loaded with GMSCs-Derived Neural Lineage Cells Promotes Recovery after Stroke.

Ischemic stroke is a devastating medical condition with poor prognosis due to the lack of effective treatment modalities. Transplantation of human neural stem cells or primary neural cells is a promising treatment approach, but this is hindered by limited suitable cell sources and low in vitro expansion capacity. This study aimed (1) use small molecules (SM) to reprogram gingival mesenchymal stem cells (GMSCs) commitment to the neural lineage cells in vitro, and (2) use hyaluronic acid (HA) hydrogel scaffolds seeded with GMSCs-derived neural lineage cells to treat ischemic stroke in vivo. Neural induction was carried out with a SM cocktail-based one-step culture protocol over a period of 24 h. The induced cells were analyzed for expression of neural markers with immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The Sprague-Dawley (SD) rats (n = 100) were subjected to the middle cerebral artery occlusion (MCAO) reperfusion ischemic stroke model. Then, after 8 days post-MCAO, the modeled rats were randomly assigned to six study groups (n = 12 per group): (1) GMSCs, (2) GMSCs-derived neural lineage cells, (3) HA and GMSCs-derived neural lineage cells, (4) HA, (5) PBS, and (6) sham transplantation control, and received their respective transplantation. Evaluation of post-stroke recovery were performed by behavioral tests and histological assessments. The morphologically altered nature of neural lineages has been observed of the GMSCs treated with SMs compared to the untreated controls. As shown by the qRT-PCR and immunocytochemistry, SMs further significantly enhanced the expression level of neural markers of GMSCs as compared with the untreated controls (all p < 0.05). Intracerebral injection of self-assembling HA hydrogel carrying GMSCs-derived neural lineage cells promoted the recovery of neural function and reduced ischemic damage in rats with ischemic stroke, as demonstrated by histological examination and behavioral assessments (all p < 0.05). In conclusion, the SM cocktail significantly enhanced the differentiation of GMSCs into neural lineage cells. The HA hydrogel was found to facilitate the proliferation and differentiation of GMSCs-derived neural lineage cells. Furthermore, HA hydrogel seeded with GMSCs-derived neural lineage cells could promote tissue repair and functional recovery in rats with ischemic stroke and may be a promising alternative treatment modality for stroke.

Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis.

INTRODUCTION: Stem cell-based dental pulp regeneration has been extensively studied, mainly focusing on exploiting dental stem cells' osteogenic and angiogenic potentials. Dental stem cells' neurogenic role is often overlooked. Stem cells from apical papilla (SCAPs), originating from the neural crest and capable of sphere formation, display potent neurogenic capacity. This study aimed to investigate the interactions of neuronally induced stem cells from apical papilla (iSCAP) spheres, SCAPs, and human umbilical vascular endothelial cells (HUVECs) on vasculogenesis and neurogenesis. METHODS: SCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small molecule neural induction medium, and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP sphere interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with ß-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. RESULTS: Neural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, ß-tubulin III, microtubule-associated protein 2, neuron-specific enolase, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared with iSCAPs. iSCAP sphere, HUVEC, and SCAP direct coculture significantly enhanced vessel formation along with up-regulated VEGF (P < .001) and multiple neural markers, such as Nestin (P < .01), microtubule-associated protein 2 (P < .001), S100 (P < .001), and NG2 (P < .001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and alpha-smooth muscle actin-positive and CD31-positive cells assembling for the vascular constitution. CONCLUSIONS: iSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.

Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-ß1 secretion.

AIM: Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell-cell interactions between MSCs, mural cells and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vein ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model. METHODOLOGY: Human umbilical vascular ECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media-2 (EBM-2) supplemented with 5% FBS for 6 days. Expression of SMC-specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor-beta 1 (TGF-ß1) in conditioned media (CM) of HUVECs monoculture (E-CM), DPSCs monoculture (D-CM) and HUVEC+DPSC cocultures (E+D-CM) were analysed by enzyme-linked immunosorbent assay. TGF-ß RI kinase inhibitor VI, SB431542, was used to block TGF-ß1/ALK5 signalling in DPSCs. RESULTS: The expression of SMC-specific markers, α-SMA, SM22α and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D-CM significantly upregulated the expression of SMC-specific markers in DPSCs compared to E-CM and D-CM. Activin A and TGF-ß1 were considerably higher in E+D-CM than that in D-CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC-specific markers in DPSCs, while treatment with TGF-ß1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF-ß1/ALK5 signalling inhibited the expression of α-SMA, SM22α and Calponin in DPSCs. CONCLUSIONS: TGF-ß1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-ß1/ALK5 signalling pathway played a vital role in this process.

One-Pot Preparation of Cetylpyridinium Chloride-Containing Nanoparticles for Biofilm Eradication.

Quaternary ammonium compounds (QACs) have been widely used due to their excellent antimicrobial activity. However, using the technology where nanomaterials are employed as drug carriers to deliver QAC drugs has not been fully explored. In this study, mesoporous silica nanoparticles (MSNs) with short rod morphology were synthesized in a one-pot reaction using an antiseptic drug cetylpyridinium chloride (CPC). CPC-MSN were characterized via various methods and tested against three bacterial species (Streptococcus mutans, Actinomyces naeslundii, and Enterococcus faecalis), which are associated with oral infections, caries, and endodontic pathology. The nanoparticle delivery system used in this study prolonged the release of CPC. The manufactured CPC-MSN effectively killed the tested bacteria within the biofilm, and their size allowed them to penetrate into dentinal tubules. This CPC-MSN nanoparticle delivery system demonstrates potential for applications in dental materials.

Formation of Three-Dimensional Spheres Enhances the Neurogenic Potential of Stem Cells from Apical Papilla.

Cell-based neural regeneration is challenging due to the difficulty in obtaining sufficient neural stem cells with clinical applicability. Stem cells from apical papilla (SCAPs) originating from embryonic neural crests with high neurogenic potential could be a promising cell source for neural regeneration. This study aimed to investigate whether the formation of 3D spheres can promote SCAPs' neurogenic potential. MATERIAL AND METHODS: Three-dimensional SCAP spheres were first generated in a 256-well agarose microtissue mold. The spheres and single cells were individually cultured on collagen I-coated µ-slides. Cell morphological changes, neural marker expression, and neurite outgrowth were evaluated by confocal microscope, ELISA, and RT-qPCR. RESULTS: Pronounced morphological changes were noticed in a time-dependent manner. The migrating cells' morphology changed from fibroblast-like cells to neuron-like cells. Compared to the 2D culture, neurite length, number, and the expression of multiple progenitors, immature and mature neural markers were significantly higher in the 3D spheres. BDNF and NGF-ß may play a significant role in the neural differentiation of SCAP spheres. CONCLUSION: The formation of 3D spheres enhanced the neurogenic potential of SCAPs, suggesting the advantage of using the 3D spheres of SCAPs for treating neural diseases.