Generation and characterization of cortical organoids from iPSC-derived dental pulp stem cells using traditional and innovative approaches.

Cortical organoids derived from human induced pluripotent stem cells (hiPSCs) represent a powerful in vitro experimental system to investigate human brain development and disease, often inaccessible to direct experimentation. However, despite steady progress in organoid technology, several limitations remain, including high cost and variability, use of hiPSCs derived from tissues harvested invasively, unexplored three-dimensional (3D) structural features and neuronal connectivity. Here, using a cost-effective and reproducible protocol as well as conventional two-dimensional (2D) immunostaining, we show that cortical organoids generated from hiPSCs obtained by reprogramming stem cells from human exfoliated deciduous teeth (SHED) recapitulate key aspects of human corticogenesis, such as polarized organization of neural progenitor zones with the presence of outer radial glial stem cells, and differentiation of superficial- and deep-layer cortical neurons and glial cells. We also show that 3D bioprinting and magnetic resonance imaging of intact cortical organoids are alternative and complementary approaches to unravel critical features of the 3D architecture of organoids. Finally, extracellular electrical recordings in whole organoids showed functional neuronal networks. Together, our findings suggest that SHED-derived cortical organoids constitute an attractive model of human neurodevelopment, and support the notion that a combination of 2D and 3D techniques to analyze organoid structure and function may help improve this promising technology.

The Exosomes of Stem Cells from Human Exfoliated Deciduous Teeth Suppress Inflammation in Osteoarthritis.

Hyaluronic acid injection is commonly used clinically to slow down the development of osteoarthritis (OA). A newly developed therapeutic method is to implant chondrocytes/stem cells to regenerate cartilage in the body. The curative effect of stem cell therapy has been proven to come from the paracrine of stem cells. In this study, exosomes secreted by stem cells from human exfoliated deciduous teeth (SHED) and hyaluronic acid were used individually to evaluate the therapeutic effect in slowing down OA. SHED was cultured in a serum-free medium for three days, and the supernatant was collected and then centrifuged with a speed difference to obtain exosomes containing CD9 and CD63 markers, with an average particle size of 154.1 nm. SW1353 cells were stimulated with IL-1ß to produce the inflammatory characteristics of OA and then treated with 40 µg/mL exosomes and hyaluronic acid individually. The results showed that the exosomes successfully inhibited the pro-inflammatory factors, including TNF-α, IL-6, iNOS, NO, COX-2 and PGE2, induced by IL-1ß and the degrading enzyme of the extrachondral matrix (MMP-13). Collagen II and ACAN, the main components of the extrachondral matrix, were also increased by 1.76-fold and 2.98-fold, respectively, after treatment, which were similar to that of the normal joints. The effect can be attributed to the partial mediation of SHED exosomes to the NF-κB pathway, and the ability of exosomes to inhibit OA is found not inferior to that of hyaluronic acid.

Activation of cellular antioxidative stress and migration activities by purified components from immortalized stem cells from human exfoliated deciduous teeth.

Although stem cell-based regenerative medicine has been extensively studied, it remains difficult to reconstruct three dimensional tissues and organs in combination with vascular systems in vitro. One clinically successful therapy is transplantation of mesenchymal stem cells (MSC) into patients with graft versus host disease. However, transplanted cells are immediately damaged and destroyed because of innate immune reactions provoked by thrombogenic inflammation, and patients need to take immunosuppressive drugs for the immunological regulation of allogeneic cells. This reduces the benefits of stem cell transplantation. Therefore, alternative therapies are more realistic options for clinical use. In this study, we aimed to take advantage of the therapeutic efficacy of MSC and use multiple cytokines released from MSC, that is, stem cells from human exfoliated deciduous teeth (SHEDs). Here, we purified components from conditioned media of immortalized SHED (IM-SHED-CM) and evaluated the activities of intracellular dehydrogenase, cell migration, and antioxidative stress by studying the cells. The immortalization of SHED could make the stable supply of CM possible. We found that the fractionated component of 50-100 kD from IM-SHED-CM had higher efficacy than the original IM-SHED-CM in terms of intracellular dehydrogenase and cell migration in which intracellular signal transduction was activated via receptor tyrosine kinases, and the glutathione peroxidase and reductase system was highly active. Although antioxidative stress activities in the fractionated component of 50-100 kD had slightly lower than that of original IM-SHE-CM, the fraction still had the activity. Thus, the use of fractionated components of 50-100 kD from IM-SHED-CM could be an alternative choice for MSC transplantation because the purified components from CM could maintain the effect of cytokines from SHED.

Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) hold promise in regenerative medicine owing to their multipotent capabilities resembling mesenchymal stem cells (MSCs). Despite their potential, SHED have not been extensively investigated because their limited lifespan and unavailability of cell-lines pose challenges for therapeutic applications. This study investigated the effect of ectopic human telomerase reverse transcriptase (hTERT) expression on SHEDs' proliferation while preserving stemness and genomic integrity. METHODS: Deciduous teeth were collected from children aged 6-10 years. After isolation and characterization, the SHED were transduced with pBabe-puro-hTERT retrovirus to establish SHED cell-line, which was evaluated and compared with pBabe-puro (mock control) for stemness, multipotency and growth attributes through flow cytometry, trilineage differentiation, and growth kinetics. We also estimated hTERT gene expression, genomic integrity, and validated cell-line through STR analysis. RESULTS: Following hTERT transduction, SHED displayed elevated hTERT gene expression while retaining fibroblast-like morphology and mesenchymal stem cell markers. Moreover, after hTERT transduction cellular shape remained same along with increased replicative lifespan and proliferation potential. SHED-hTERT cells exhibited multi-potency and maintained stemness, as evidenced by surface marker expression and multilineage differentiation. Furthermore, genomic integrity was not affected by hTERT integration, as confirmed by STR analysis and CDKN2A gene assessment. CONCLUSION: Ectopic hTERT expression in SHED successfully prolonged their replicative lifespan and improved their ability to proliferate and migrate, while preserving their stemness, multipotency and genomic integrity, suggesting minimal carcinogenic risk. Establishment of SHED cell-line holds potential in regenerative medicine applications, especially in cell-based drugs and tissue engineering experiments.

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

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

A Review of Stem Cell Attributes Derived from the Oral Cavity.

Oral cavity stem cells (OCSCs) have been the focus of intense scientific efforts due to their accessibility and stem cell properties. The present work aims to compare the different characteristics of 6 types of dental stem cells derived from the oral cavity: dental pulp stem cells (DPSC), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSC), stem cells from the apical papilla (SCAP), bone marrow mesenchymal stem cells (BMSC), and gingival mesenchymal stem cells (GMSC). Using immunofluorescence and real-time polymerase chain reaction techniques, we analysed the cells for stem cell, differentiation, adhesion, and extracellular matrix markers; the ability to proliferate in vitro; and multilineage differentiation potential. Markers such as vimentin, CD44, alkaline phosphatase, CD146, CD271, CD49f, Oct 3/4, Sox 9, FGF7, nestin, and BMP4 showed significant differences in expression levels, highlighting the heterogeneity and unique characteristics of each cell type. At the same time, we confirmed that all cell types successfully differentiated into osteogenic, chondrogenic, or adipose lineages, with different readiness. In conclusion, our study reveals the distinct properties and potential applications of various dental-derived stem cells. These findings contribute to a deeper understanding of OCSCs and their significance in future clinical applications.

Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Advancements in Spinal Cord Injury Repair: Insights from Dental-Derived Stem Cells.

Spinal cord injury (SCI), a prevalent and disabling neurological condition, prompts a growing interest in stem cell therapy as a promising avenue for treatment. Dental-derived stem cells, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells from the apical papilla (SCAP), dental follicle stem cells (DFSCs), are of interest due to their accessibility, minimally invasive extraction, and robust differentiating capabilities. Research indicates their potential to differentiate into neural cells and promote SCI repair in animal models at both tissue and functional levels. This review explores the potential applications of dental-derived stem cells in SCI neural repair, covering stem cell transplantation, conditioned culture medium injection, bioengineered delivery systems, exosomes, extracellular vesicle treatments, and combined therapies. Assessing the clinical effectiveness of dental-derived stem cells in the treatment of SCI, further research is necessary. This includes investigating potential biological mechanisms and conducting Large-animal studies and clinical trials. It is also important to undertake more comprehensive comparisons, optimize the selection of dental-derived stem cell types, and implement a functionalized delivery system. These efforts will enhance the therapeutic potential of dental-derived stem cells for repairing SCI.

Apoptotic Vesicles Modulate Endothelial Metabolism and Ameliorate Ischemic Retinopathy via PD1/PDL1 Axis.

Pathological angiogenesis with subsequent disturbed microvascular remodeling is a major cause of irreversible blindness in a number of ischemic retinal diseases. The current anti-vascular endothelial growth factor therapy can effectively inhibit angiogenesis, but it also brings significant side effects. The emergence of stem cell derived extracellular vesicles provides a new underlining strategy for ischemic retinopathy. Apoptotic vesicles (apoVs) are extracted from stem cells from human exfoliated deciduous teeth (SHED). SHED-apoVs are delivered into the eyeballs of oxygen-induced retinopathy (a most common model of angiogenic retinal dieseases) mice through intravitreal injection. The retinal neovascularization and nonperfusion area, vascular structure, and density changes are observed during the neovascularization phase (P17) and vascular remodeling phase (P21), and visual function is measured. The expression of extracellular acidification rate and lactic acid testing are used to detect endothelial cells (ECs) glycolytic activity. Furthermore, lentivirus and neutralizing antibody are used to block PD1-PDL1 axis, investigating the effects of SHED-apoVs on glycolysis and angiogenic activities. This work shows that SHED-apoVs are taken up by ECs and modulate the ECs glycolysis, leading to the decrease of abnormal neovessels and vascular remodeling. Furthermore, it is found that, at the molecular level, apoVs-carried PD1 interacts with PDL1 on hypoxic ECs to regulate the angiogenic activation. SHED-apoVs inhibit pathological angiogenesis and promote vascular remodeling in ischemic retinopathy partially by modulating ECs glycolysis through PD1/PDL1 axis. This study provides a new potential strategy for the clinical treatment of pathological retinal neovascularization.

Molecular insights into the proteomic composition of porcine treated dentin matrix.

Background: Human-treated dentin matrix (hTDM) has recently been studied as a natural extracellular matrix-based biomaterial for dentin pulp regeneration. However, porcine-treated dentin matrix (pTDM) is a potential alternative scaffold due to limited availability. However, there is a dearth of information regarding the protein composition and underlying molecular mechanisms of pTDM.Methods: hTDM and pTDM were fabricated using human and porcine teeth, respectively, and their morphological characteristics were examined using scanning electron microscopy. Stem cells derived from human exfoliated deciduous teeth (SHEDs) were isolated and characterized using flow cytometry and multilineage differentiation assays. SHEDs were cultured in three-dimensional environments with hTDM, pTDM, or biphasic hydroxyapatite/tricalcium phosphate. The expression of odontogenesis markers in SHEDs were assessed using real-time polymerase chain reaction and immunochemical staining. Subsequently, SHEDs/TDM and SHEDs/HA/TCP complexes were transplanted subcutaneously into nude mice. The protein composition of pTDM was analyzed using proteomics and compared to previously published data on hTDM.Results: pTDM and hTDM elicited comparable upregulation of odontogenesis-related genes and proteins in SHEDs. Furthermore, both demonstrated the capacity to stimulate root-related tissue regeneration in vivo. Proteomic analysis revealed the presence of 278 protein groups in pTDM, with collagens being the most abundant. Additionally, pTDM and hTDM shared 58 identical proteins, which may contribute to their similar abilities to induce odontogenesis. Conclusions: Both hTDM and pTDM exhibit comparable capabilities in inducing odontogenesis, potentially owing to their distinctive bioactive molecular networks.

Protective Mechanism of Stem Cells from Human Exfoliated Deciduous Teeth in Treating Spinal Cord Injury.

Spinal cord injury (SCI) induces devastating permanent deficits. Recently, cell transplantation therapy has become a notable treatment for SCI. Although stem cells from human exfoliated deciduous teeth (SHED) are an attractive therapy, their precise mechanism of action remains to be elucidated. In this study, we explored one of the neuroprotective mechanisms of SHED treatment at the subacute stage after SCI. We used a rat clip compression SCI model. The animals were randomly divided into three groups: SCI, SCI + phosphate-buffered saline (PBS), and SCI + SHED. The SHED or PBS intramedullary injection was administered immediately after SCI. After SCI, we explored the effects of SHED on motor function, as assessed by the Basso-Beattie-Bresnahan score and the inclined plane method, the signal transduction pathway, especially the Janus kinase (JAK) and the signal transducer and activator of transcription 3 (STAT3) pathway, the apoptotic pathway, and the expression of neurocan, one of the chondroitin sulfate proteoglycans. SHED treatment significantly improved functional recovery from Day 14 relative to the controls. Western blot analysis showed that SHED significantly reduced the expression of glial fibrillary acidic protein (GFAP) and phosphorylated STAT3 (p-STAT3) at Tyr705 on Day 10 but not on Day 5. However, SHED had no effect on the expression levels of Iba-1 on Days 5 or 10. Immunohistochemistry revealed that p-STAT3 at Tyr705 was mainly expressed in GFAP-positive astrocytes on Day 10 after SCI, and its expression was reduced by administration of SHED. Moreover, SHED treatment significantly induced expression of cleaved caspase 3 in GFAP-positive astrocytes only in the epicenter lesions on Day 10 after SCI but not on Day 5. The expression of neurocan was also significantly reduced by SHED injection on Day 10 after SCI. Our results show that SHED plays an important role in reducing astrogliosis and glial scar formation between Days 5 and 10 after SCI, possibly via apoptosis of astrocytes, ultimately resulting in improvement in neurological functions thereafter. Our data revealed one of the neuroprotective mechanisms of SHED at the subacute stage after SCI, which improved functional recovery after SCI, a serious condition.

SHED-derived exosomes attenuate trigeminal neuralgia after CCI of the infraorbital nerve in mice via the miR-24-3p/IL-1R1/p-p38 MAPK pathway.

BACKGROUND: Microglial activation in the spinal trigeminal nucleus (STN) plays a crucial role in the development of trigeminal neuralgia (TN). The involvement of adenosine monophosphate-activated protein kinase (AMPK) and N-methyl-D-aspartate receptor 1 (NMDAR1, NR1) in TN has been established. Initial evidence suggests that stem cells from human exfoliated deciduous teeth (SHED) have a potential therapeutic effect in attenuating TN. In this study, we propose that SHED-derived exosomes (SHED-Exos) may alleviate TN by inhibiting microglial activation. This study sought to assess the curative effect of SHED-Exos administrated through the tail vein on a unilateral infraorbital nerve chronic constriction injury (CCI-ION) model in mice to reveal the role of SHED-Exos in TN and further clarify the potential mechanism. RESULTS: Animals subjected to CCI-ION were administered SHED-Exos extracted by differential ultracentrifugation. SHED-Exos significantly alleviated TN in CCI mice (increasing the mechanical threshold and reducing p-NR1) and suppressed microglial activation (indicated by the levels of TNF-α, IL-1ß and IBA-1, as well as p-AMPK) in vivo and in vitro. Notably, SHED-Exos worked in a concentration dependent manner. Mechanistically, miR-24-3p-upregulated SHED-Exos exerted a more significant effect, while miR-24-3p-inhibited SHED-Exos had a weakened effect. Bioinformatics analysis and luciferase reporter assays were utilized for target gene prediction and verification between miR-24-3p and IL1R1. Moreover, miR-24-3p targeted the IL1R1/p-p38 MAPK pathway in microglia was increased in CCI mice, and participated in microglial activation in the STN. CONCLUSIONS: miR-24-3p-encapsulated SHED-Exos attenuated TN by suppressing microglial activation in the STN of CCI mice. Mechanistically, miR-24-3p blocked p-p38 MAPK signaling by targeting IL1R1. Theoretically, targeted delivery of miR-24-3p may offer a potential strategy for TN.

Effects of ethanol washing and storage duration on primary culture of stem cells from human exfoliated deciduous teeth.

Purpose: Since the oral environment harbors various microorganisms, the removal of contaminants during the primary culture process of stem cells from human exfoliated deciduous teeth (SHEDs) is very important. We investigated optimal methods for primary culture of SHEDs with minimal contamination rates. Materials and methods: Three different storage conditions for deciduous teeth were utilized:1) storing teeth in Hank's Balanced Salt Solution (HBSS) with 3% penicillin and streptomycin (P/S), 2) storing teeth in HBSS with 3% antibiotics and antimycotics (A-A), and 3) storing teeth in HBSS with A-A, and additional washing with 70% ethanol just before primary culture of dental pulp. In addition, the storage time from the extraction of teeth to the primary culture was measured. Results: The contamination rates were about 70% for HBSS with P/S, 40% for HBSS with A-A, and less than 10% for HBSS with A-A and additional washing with 70% ethanol. When the primary culture was conducted within 12 h after teeth extraction, the contamination rate was the lowest in all conditions. Furthermore, when the teeth were delivered in HBSS with A-A and an additional 70% ethanol washing was performed, the contamination rate was 0% until 48 h after teeth extraction. Ethanol washing had little effect on the cellular characteristics and stemness of SHEDs, including their morphology, growth rate, expression of surface markers, and differentiation potential. Conclusions: We suggested that both delivering teeth in HBSS with A-A and additional 70% ethanol washing are critical considerations for the successful culture of SHEDs without contamination.

The Importance of Stem Cells Isolated from Human Dental Pulp and Exfoliated Deciduous Teeth as Therapeutic Approach in Nervous System Pathologies.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Hsa-miR-27b-5p suppresses the osteogenic and odontogenic differentiation of stem cells from human exfoliated deciduous teeth via targeting BMPR1A: An ex vivo study.

AIM: Recently, miR-27b-5p was shown to be abundantly expressed in extracellular vehicles (EVs) from the inflammatory microenvironment. This study determined the role of miR-27b-5p in regulating osteogenic and odontogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs) and further examined the regulatory mechanism of bone morphogenetic protein receptor type-1A (BMPR1A). METHODOLOGY: Characteristics of SHEDs and SHEDs-EVs derived from SHEDs were evaluated respectively. The expression of miR-27b-5p in SHEDs and EVs was detected during osteo-induction. Mechanically, SHEDs were treated with miR-27b-5p mimics or an inhibitor, and the osteogenic/odontogenic differentiation and proliferation were assessed. Bioinformatic analysis and luciferase reporter were utilized for target gene prediction and verification. Finally, BMPR1A-overexpressed plasmids were transfected into SHEDs to investigate the participation of the BMPR1A/SMAD4 pathway. Data were analysed using Student's t-test, one-way analysis of variance and Chi-square test. RESULTS: MiR-27b-5p was expressed in both SHEDs and EVs and was significantly increased at the initial stage of differentiation and then decreased in a time-dependent manner (p < .01). Upregulation of miR-27b-5p significantly suppressed osteogenic/odontogenic differentiation of SHEDs and inhibited proliferation (p < .05), whereas inhibition of miR-27b-5p enhanced the differentiation (p < .05). Dual-luciferase reporter assay and pull-down assay confirmed the binding site between miR-27b-5p and BMPR1A (p < .05). The overexpression of BMPR1A rescued the effect of miR-27b-5p, while contributed to the decrease of pluripotency (p < .05). Additionally, miR-27b-5p maintained pluripotency in BMPR1A-overexpressed SHEDs (p < .05). CONCLUSIONS: MiR-27b-5p in SHEDs/EVs was inversely associated with differentiation and suppressed the osteogenic and odontogenic differentiation of SHEDs and maintained the pluripotency of SHEDs partly by shuttering BMPR1A-targeting BMP signalling. Theoretically, inhibition of miR-27b-5p represents a potential strategy to promote osteanagenesis and dentinogenesis. However, miR-27b-5p capsuled EVs might maintain cell pluripotency and self-renewal for non-cell-targeted therapy.

Amyloid-mediated remineralization for tooth hypoplasia of cleidocranial dysplasia.

Introduction: Cleidocranial dysplasia (CCD) is an autosomal-dominant, heritable skeletal and dental disease, involving hypoplastic clavicles, defective ossification of the anterior fontanelle, dentin and enamel hypoplasia, and supernumerary teeth, which can seriously affect the oral and mental health of patients. Amyloid-like protein aggregation, which is established by lysozyme conjugated with polyethylene glycol (Lyso-PEG), forms a mineralized nanofilm layer on a healthy enamel surface. However, whether it can form a remineralization layer in dental tissues from CCD remains unclear. Methods: This study evaluated deciduous teeth from healthy individuals and a patient with CCD. Because pulp and dentin are functionally closely related, stem cells from human exfoliated deciduous teeth (SHED) from CCD patients and healthy individuals were collected to compare their biological properties. Results: The results found that deciduous teeth from patients with CCD exhibited dentin hypoplasia. In addition, the proliferative ability and osteogenic potential of SHED from patients with CCD were lower than those of control individuals. Finally, Lyso-PEG was applied to dentin from the CCD and control groups, showing a similar remineralization-induced effect on the dentin surfaces of the two groups. Conclusion: These results extend our understanding of the dentin and SHED of patients with CCD, exhibiting good caries-preventive capacity and good biocompatibility of Lyso-PEG, thus providing a novel dental therapy for CCD and patients with tooth hypoplasia.

Bone Differentiation Ability of CD146-Positive Stem Cells from Human Exfoliated Deciduous Teeth.

Regenerative therapy for tissues by mesenchymal stem cell (MSCs) transplantation has received much attention. The cluster of differentiation (CD)146 marker, a surface-antigen of stem cells, is crucial for angiogenic and osseous differentiation abilities. Bone regeneration is accelerated by the transplantation of CD146-positive deciduous dental pulp-derived mesenchymal stem cells contained in stem cells from human exfoliated deciduous teeth (SHED) into a living donor. However, the role of CD146 in SHED remains unclear. This study aimed to compare the effects of CD146 on cell proliferative and substrate metabolic abilities in a population of SHED. SHED was isolated from deciduous teeth, and flow cytometry was used to analyze the expression of MSCs markers. Cell sorting was performed to recover the CD146-positive cell population (CD146+) and CD146-negative cell population (CD146-). CD146 + SHED without cell sorting and CD146-SHED were examined and compared among three groups. To investigate the effect of CD146 on cell proliferation ability, an analysis of cell proliferation ability was performed using BrdU assay and MTS assay. The bone differentiation ability was evaluated using an alkaline phosphatase (ALP) stain after inducing bone differentiation, and the quality of ALP protein expressed was examined. We also performed Alizarin red staining and evaluated the calcified deposits. The gene expression of ALP, bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) was analyzed using a real-time polymerase chain reaction. There was no significant difference in cell proliferation among the three groups. The expression of ALP stain, Alizarin red stain, ALP, BMP-2, and OCN was the highest in the CD146+ group. CD146 + SHED had higher osteogenic differentiation potential compared with SHED and CD146-SHED. CD146 contained in SHED may be a valuable population of cells for bone regeneration therapy.

Tracking of Stem Cells from Human Exfoliated Deciduous Teeth Labeled with Molday ION Rhodamine-B during Periodontal Bone Regeneration in Rats.

Background and Objectives: Chronic periodontitis can lead to alveolar bone resorption and eventually tooth loss. Stem cells from exfoliated deciduous teeth (SHED) are appropriate bone regeneration seed cells. To track the survival, migration, and differentiation of the transplanted SHED, we used super paramagnetic iron oxide particles (SPIO) Molday ION Rhodamine-B (MIRB) to label and monitor the transplanted cells while repairing periodontal bone defects. Methods and Results: We determined an appropriate dose of MIRB for labeling SHED by examining the growth and osteogenic differentiation of labeled SHED. Finally, SHED was labeled with 25 µg Fe/ml MIRB before being transplanted into rats. Magnetic resonance imaging was used to track SHED survival and migration in vivo due to a low-intensity signal artifact caused by MIRB. HE and immunohistochemical analyses revealed that both MIRB-labeled and unlabeled SHED could promote periodontal bone regeneration. The colocalization of hNUC and MIRB demonstrated that SHED transplanted into rats could survive in vivo. Furthermore, some MIRB-positive cells expressed the osteoblast and osteocyte markers OCN and DMP1, respectively. Enzyme-linked immunosorbent assay revealed that SHED could secrete protein factors, such as IGF-1, OCN, ALP, IL-4, VEGF, and bFGF, which promote bone regeneration. Immunofluorescence staining revealed that the transplanted SHED was surrounded by a large number of host-derived Runx2- and Col II-positive cells that played important roles in the bone healing process. Conclusions: SHED could promote periodontal bone regeneration in rats, and the survival of SHED could be tracked in vivo by labeling them with MIRB. SHED are likely to promote bone healing through both direct differentiation and paracrine mechanisms.

Enhanced bone tissue regeneration with hydrogel-based scaffolds by embedding parathyroid hormone in mesoporous bioactive glass.

OBJECTIVES: To evaluate hydrogel-based scaffolds embedded with parathyroid hormone (PTH)-loaded mesoporous bioactive glass (MBG) on the enhancement of bone tissue regeneration in vitro. MATERIALS AND METHODS: MBG was produced via sol-gel technique followed by PTH solution imbibition. PTH-loaded MBG was blended into the hydrogels and submitted to a lyophilisation process associated with a chemical crosslinking reaction to the production of the scaffolds. Characterisation of the MBG and PTH-loaded MBG scaffolds, including the scanning electron microscope (SEM) connected with an X-ray detector (EDX), Fourier transform infrared (FTIR), compression strength, rheological measurements, swelling and degradation rates, and PTH release analysis, were performed. Also, bioactivity using simulated-body fluid (SBF), biocompatibility (MTT), and osteogenic differentiation analyses (von Kossa and Alizarin Red stainings, and µ-computed tomography, µCT) of the scaffolds were carried out. RESULTS: SEM images demonstrated MBG particles dispersed into the hydrogel-based scaffold structure, which was homogeneously porous and well interconnected. EDX and FTIR revealed large amounts of carbon, oxygen, sodium, and silica in the scaffold composition. Bioactivity experiments revealed changes on sample surfaces over the analysed period, indicating the formation of carbonated hydroxyapatite; however, the chemical composition remained stable. PTH-loaded hydrogel-based scaffolds were biocompatible for stem cells from human-exfoliated deciduous teeth (SHED). A high quantity of calcium deposits on the extracellular matrix of SHED was found for PTH-loaded hydrogel-based scaffolds. µCT images showed MBG particles dispersed into the scaffolds' structure, and a porous, lamellar, and interconnected hydrogel architecture. CONCLUSIONS: PTH-loaded hydrogel-based scaffolds demonstrated consistent morphology and physicochemical properties for bone tissue regeneration, as well as bioactivity, biocompatibility, and osteoinductivity in vitro. Thus, the scaffolds presented here are recommended for future studies on 3D printing. CLINICAL RELEVANCE: Bone tissue regeneration is still a challenge for several approaches to oral and maxillofacial surgeries, though tissue engineering applying SHED, scaffolds, and osteoinductive mediators might help to overcome this clinical issue.

Effect of CD146+ SHED on bone regeneration in a mouse calvaria defect model.

OBJECTIVE: Stem cells from human exfoliated deciduous teeth (SHED) have bone regeneration ability and potential therapeutic applications. CD146, a cell adhesion protein expressed by vascular endothelial cells, is involved in osteoblastic differentiation of stem cells. The effect of CD146 on SHED-mediated bone regeneration in vivo remains unknown. We aimed to establish efficient conditions for SHED transplantation. MATERIALS AND METHODS: SHED were isolated from the pulp of an extracted deciduous tooth and cultured; CD146-positive (CD146+ ) and CD146-negative (CD146- ) populations were sorted. Heterogeneous populations of SHED and CD146+ and CD146- cells were transplanted into bone defects generated in the skulls of immunodeficient mice. Micro-computed tomography was performed immediately and 4 and 8 weeks later. Histological and immunohistochemical assessments were performed 8 weeks later. RESULTS: Bone regeneration was observed upon transplantation with CD146+ and heterogeneous populations of SHED, with significantly higher bone regeneration observed with CD146+ cells. Bone regeneration was higher in the CD146- group than in the control group, but significantly lower than that in the other transplant groups at 4 and 8 weeks. Histological and immunohistochemical assessments revealed that CD146+ cells promoted bone regeneration and angiogenesis. CONCLUSION: Transplantation of CD146+ SHED into bone defects may be useful for bone regeneration.