Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-ß1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-ß1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

Preliminary study on the mechanism by which exosomes derived from human exfoliated deciduous teeth improve the proliferation and osteogenic inhibitory effect of glucocorticoid-induced BMSCs.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a disease characterized by a collapsed femoral head caused by the overuse of glucocorticoids. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is an important pathological feature of SONFH. In this study, we investigated whether exosomes from SHEDs (stem cells from human exfoliated deciduous teeth) have a therapeutic effect on glucocorticoid-induced inhibition of proliferation and osteogenesis in BMSCs, and elucidated the underlying mechanisms involved. METHODS: Primary dental pulp cells were isolated and cultured from human deciduous tooth pulp, SHEDs were isolated and purified by the limiting dilution method and exosomes were isolated from the supernatants of SHEDs by ultracentrifugation. The cell surface markers CD31, CD34, CD45, CD73, CD90 and CD105 were detected by flow cytometry. A Cell-Counting-Kit-8 assay was used to detect cell activity. ALP and Alizarin Red staining were used to identify osteogenic differentiation ability, and exosomes were identified using transmission electron microscopy, NanoFCM and Western blotting. PKH67 fluorescence was used to track the uptake of exosomes by BMSCs. Transcriptome analysis combined with quantitative real-time PCR was used to explore the underlying mechanism involved. RESULTS: Exosomes secreted by SHEDs can be endocytosed by BMSCs, and can partially reverse the inhibitory effects of glucocorticoids on the viability and osteogenic differentiation of BMSCs. Transcriptome sequencing analysis revealed that the differentially expressed mRNAs regulated by SHED-derived exosomes were enriched mainly in signaling pathways such as the apoptosis pathway, the PI3K-Akt signaling pathway, the Hippo signaling pathway and the p53 signaling pathway. qPCR showed that SHED-derived exosomes reversed the dexamethasone-induced upregulation of HGF and ITGB8 expression and the inhibition of EFNA1 expression, but further increased the dexamethasone-induced downregulation of IL7 expression. In conclusion, SHED-derived exosomes partially reversed the inhibitory effects of glucocorticoids on BMSC proliferation and osteogenesis by inhibiting the expression of HGF, ITGB8 and IL7, and upregulating the expression of EFNA1.

Permanent and decidua dentition as chronological biomarkers of heavy metal contamination: A review of the forensic literature.

STATEMENT OF PROBLEM: Contamination with heavy metals (HM) has great environmental consequences in the environment due to lack of biodegradation, in addition, accumulation in living beings causes defects in tissues and organs, deteriorating their function and inducing a wide spectrum of diseases. Human biomonitoring consists of the periodic measurement of a certain chemical substance or metabolite in a particular population, using matrices that can be acute or chronic. Teeth are chronic matrices that have great characteristics of resistance and chronological storage of information. This review aims to identify the mechanisms, spatial location, and affinity of HM within teeth, along with understanding its applicability as a chronological record matrix in the face of HM contamination. MATERIAL AND METHODS: A systematic search review was performed using the PubMed/Medline, Web of Science, and Scopus metasearch engines, and the terms "teeth" OR "dental" OR "tooth" AND "heavy metals" were intersected. Complete articles are included in Spanish, English and Portuguese without time restrictions, involving studies in humans or in vitro; Letters to the editor, editorials and those that did not refer to information on the incorporation and relationship of HM with the teeth were excluded. RESULTS: 837 published articles were detected, 91 were adjusted to the search objective, and 6 were manually included. Teeth are structures with a great capacity for information retention in the face of HM contamination due to low physiological turnover and their long processes of marked formations by developmental biorhythm milestones such as the neonatal line (temporal reference indicator). The contamination mechanisms inside the tooth are linked to the affinity of hydroxyapatite for HM; this incorporation can be in the soft matrix during the apposition phase or as part of the chemical exchanges between hydroxyapatite and the elements of the environment. CONCLUSION: The teeth present unique characteristics of great resistance and affinity for HM, as well as a chronological biomarker for human biomonitoring, so they can be used as means of expertise or evidence to confirm or rule out a fact of environmental characteristics in the legal field.

Spatiotemporal Expression Patterns of Critical Genes Involved in FGF Signaling During Morphogenesis and Odontogenesis of Deciduous Molars in Miniature Pigs.

The fibroblast growth factor (FGF) pathway plays an important role in epithelial-mesenchymal interactions during tooth development. Nevertheless, how the ligands, receptors, and antagonists of the FGF pathway are involved in epithelial-mesenchymal interactions remains largely unknown. Miniature pigs exhibit tooth anatomy and replacement patterns like those in humans and hence can serve as large animal models. The present study investigated the spatiotemporal expression patterns of critical genes encoding FGF ligands (FGF3, FGF4, FGF7, and FGF9), antagonists (SPRY2 and SPRY4) and receptors (FGFR1, FGFR2, and FGFR3) in the third deciduous molars of miniature pigs at the cap (embryonic day 40, E40), early bell (E50), and late bell (E60) stages. The results of in situ hybridization (ISH) with tyramide signal amplification and of qRT-PCR analysis revealed increased expression of FGF7, FGFR1, FGFR2, and SPRY4 in dental epithelium and of FGF7 and FGFR1 in mesenchyme from E40 to E50. In contrast, the results revealed decreased expression of FGF3, FGF4, FGF9, and FGFR3 in dental epithelium and of FGF4, FGF9, FGFR2, and FGFR3 in the mesenchyme from E40 to E60. Mesenchyme signals of FGF3, FGF4, FGF7, SPRY2, FGFR2, and FGFR3 were concentrated in the odontoblast layer from E50 to E60. The distinct expression patterns of these molecules indicated elaborate regulation during dental morphogenesis. Our results provide a foundation for further investigation into fine-tuning dental morphogenesis and odontogenesis by controlling interactions between dental epithelium and mesenchyme, thus promoting tooth regeneration in large mammals.

Stem Cells from Human Exfoliated Deciduous teeth Promote Hair Regeneration in Mouse.

Stem cells in different types may interact with each other to maintain homeostasis or growth and the interactions are complicated and extensive. There is increasing evidence that mesenchymal-epithelial interactions in early morphogenesis stages of both tooth and hair follicles show many similarities. In order to explore whether stem cells from one tissue could interact with cells from another tissue, a series of experiments were carried out. Here we successfully extracted and identified stem cells from human exfoliated deciduous teeth (SHED) of 8-12 years old kids, and then found that SHED could promote hair regeneration in a mouse model. In vitro, SHED shortened the hair regeneration cycle and promoted the proliferation and aggregation of dermal cells. In vivo, when SHED and skin cells of C57 mice were subcutaneously co-transplanted to nude mice, more hair was formed than skin cells without SHED. To further explore the molecular mechanism, epidermal and dermal cells were freshly extracted and co-cultured with SHED. Then several signaling molecules in hair follicle regeneration were detected and we found that the expression of Sonic Hedgehog (Shh) and Glioma-associated oncogene 1 (Gli1) was up-regulated. It seems that SHED may boost the prosperity of hairs by increase Shh/Gli1 pathway, which brings new perspectives in tissue engineering and damaged tissue repairing.

Multipotent stem cells from apical pulp of human deciduous teeth with immature apex.

Isolation of high-quality human postnatal stem cells from accessible sources is an important goal for dental tissue engineering. Stem cells from developing organs are a better cell source but are hard to obtain. With extensive caries that are difficult to restore, the extracted deciduous tooth with an immature apex is a developing organ for investigation. In the present study, a cell population from the tip of apical pulp of human deciduous teeth with an immature apex was isolated and termed apical pulp-derived cells of deciduous teeth (De-APDCs). De-APDCs expressed STRO-1, CD44, CD90 and CD105 but not CD34 or CD45. Furthermore, De-APDCs demonstrated a significantly higher clonogenic and proliferative ability and osteo/dentinogenic differentiation capacity than dental pulp cells from exfoliated deciduous teeth (De-DPCs) (P < 0.05). Differentiation potential toward adipogenic, neurogenic and chondrogenic lineages was also observed in induced De-APDCs. In addition, after De-APDCs were seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds and transplanted into nude mice, they were able to regenerate dentin/pulp-like structures aligned with human odontoblast-like cells. In conclusion, De-APDCs, which are derived from a developing tissue, represent an accessible and prospective cell source for tooth regeneration.

Amniotic membrane matrix effects on calcineurin-NFAT-related gene expressions of SHED treated with VEGF for endothelial differentiation.

The nuclear factor of activated T-cell (NFAT) signaling pathway is involved in angiogenesis following initiation by vascular endothelial growth factor (VEGF). A number of angiogenic genes have been associated with calcineurin in the NFAT pathway, forming a calcineurin-NFAT pathway. This study aims to investigate the involvement of four angiogenic genes within the calcineurin-NFAT pathway in the endothelial-like differentiation of stem cells from human exfoliated deciduous teeth (SHED) cultured on a human amniotic membrane (HAM) induced by VEGF. SHED were induced with VEGF for 24 h, then cultured on the stromal side of HAM. The cells were then further induced with VEGF until days 1 and 14. To understand the role of calcineurin, its potent inhibitor, cyclosporin A (CsA), was added into the culture. Results from SEM and H&E analyses showed SHED grew on HAM surface. Gene expression study of Cox-2 showed a drastically reduced expression with CsA treatment indicating Cox-2 involvement in the calcineurin-NFAT pathway. Meanwhile, IL-8 was probably controlled by another pathway as it showed no CsA inhibition. In contrast, high expression of ICAM-1 and RCAN1.4 by VEGF and CsA implied that these genes were not controlled by the calcineurin-NFAT-dependent pathway. In conclusion, the results of this study suggest the involvement of Cox-2 in the calcineurin-NFAT-dependent pathway while RCAN1.4 was controlled by NFAT molecule in endothelial-like differentiation of SHED cultured on HAM with VEGF induction.

Modulating stemness of mesenchymal stem cells from exfoliated deciduous and permanent teeth by IL-17 and bFGF.

Mesenchymal stem cells (MSCs) have been identified within dental pulp tissues of exfoliated deciduous (SHEDs) and permanent (DPSCs) teeth. Although differences in their proliferative and differentiation properties were revealed, variability in SHEDs and DPSCs responsiveness to growth factors and cytokines have not been studied before. Here, we investigated the influence of interleukin-17 (IL-17) and basic fibroblast growth factor (bFGF) on stemness features of SHEDs and DPSCs by analyzing their proliferation, clonogenicity, cell cycle progression, pluripotency markers expression and differentiation after 7-day treatment. Results indicated that IL-17 and bFGF differently affected SHEDs and DPSCs proliferation and clonogenicity, since bFGF increased proliferative and clonogenic potential of both cell types, while IL-17 similarly affected SHEDs, exerting no effects on adult counterparts DPSCs. In addition, both factors stimulated NANOG, OCT4, and SOX2 pluripotency markers expression in SHEDs and DPSCs showing diverse intracellular expression patterns dependent on MSCs type. As for the differentiation capacity, both factors displayed comparable effects on SHEDs and DPSCs, including stimulatory effect of IL-17 on early osteogenesis in contrast to the strong inhibitory effect showed for bFGF, while having no impact on SHEDs and DPSCs chondrogenesis. Moreover, bFGF combined with IL-17 reduced CD90 and stimulated CD73 expression on both types of MSCs, whereas each factor induced IL-6 expression indicating its' role in IL-17/bFGF-modulated properties of SHEDs and DPSCs. All these data demonstrated that dental pulp MSCs from primary and permanent teeth exert intrinsic features, providing novel evidence on how IL-17 and bFGF affect stem cell properties important for regeneration of dental pulp at different ages.

Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth.

In in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and ß-glycerophosphate (ßGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1-SHED + Dulbecco's Modified Eagles' Medium (DMEM) + fetal bovine serum (FBS); G2-SHED + DMEM + FBS + DEX; G3-SHED + DMEM + FBS + ASC + ßGLY; G4-SHED + DMEM + FBS + ASC + ßGLY + DEX; G5-MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + ßGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and ß-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

Stem cells from human exfoliated deciduous teeth transmit microRNA-26a to protect rats with experimental intracerebral hemorrhage from cerebral injury via suppressing CTGF.

OBJECTIVE: A large number of studies have shown that stem cells from human exfoliated deciduous teeth (SHED) has a protective effect on brain damage, but its specific mechanism is unclear. This research focused on the effect of microRNA (miR)-26a that transmitted by SHED in intracerebral hemorrhage (ICH). METHODS: SHED were extracted from deciduous teeth of healthy children and miR-26a expression in SHED was altered through transfection, and then the SHED were conducted with neuron differentiated induction, expression of ß3 tubulin, MAP-2 and glial fibrillary acidic protein (GFAP), number of dendritic spines and cell proliferation were detected. ICH rat models were established by stereotactic injection of collagenase VII into the left striatum and the modeled rats were injected with miR-26a mimic or inhibitor-transfected SHED suspension. Then, the brain water content, blood-brain barrier permeability, pathological changes, and injury and apoptosis in the nervous cells in brain were assessed. The expression of miR-26a and CTGF in SHED and rats' brain tissues was evaluated and the target relation between miR-26a and CTGF was detected. RESULTS: In SHED after induction, upregulated miR-26a could increase number of dendritic spines, cell proliferation, and expression of ß3 tubulin, MAP-2 and GFAP, and restrain CTGF expression. In rat models, SHED engineered to overexpress miR-26a could attenuate brain water content, Evans blue content, apoptosis, pathological injury and expression of CTGF and Bax, while promoted number of Nissl bodies and expression of Bcl-2 in the nervous cells in brain in ICH rats. Furthermore, miR-26a competitively bound to CTGF. CONCLUSION: Our findings provided the evidence that SHED could transmit miR-26a to protect ICH rats from cerebral injury by repressing CTGF, which may contribute to ICH therapy.

Overexpression of microRNA-221 promotes the differentiation of stem cells from human exfoliated deciduous teeth to neurons through activation of Wnt/ß-catenin pathway via inhibition of CHD8.

microRNAs have been proved to function in some processes of differentiation and the effect is favorable. At present, the differentiation of stem cells is not so ideal because of the high expenses and inaccessibility. Therefore, we explored the possibility that microRNA-221 (miR-221) affects differentiation from stem cells from human deciduous tooth (SHEDs) to neurons through Wnt/ß-catenin pathway via binding to CHD8. After collection of SHEDs, differentiation from SHEDs to neurons was conducted by neurotrophic factor induction method in vitro, followed by gain- and loss-of-function experiments. Expression of neuron-related genes in SHEDs was examined by immunohistochemistry. The relationship between CHD8 and miR-221 was detected by dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to determine miR-221 expression, and the mRNA and protein expression of CHD8, Wnt/ß-catenin pathway- and neuron-related genes. Cell viability, and cell cycle and apoptosis were investigated by MTT assay and flow cytometry respectively. Dual luciferase reporter assay displayed that miR-221 targeted CHD8 and then affected the differentiation progression. Results of RT-qPCR and Western blot analysis showed that expression of Wnt/ß-catenin pathway-related genes increased significantly, CHD8 expression decreased in neuron-induced SHEDs after miR-221 overexpression or CHD8 silencing. In response to miR-221 overexpression and CHD8 silencing, cell viability and cell cycle entry were increased, and apoptosis was reduced. Moreover, overexpression of miR-221 or silencing of CHD8 elevated the expression of neuron-related genes in neuron-induced SHEDs. Taken together, upregulation of miR-221 promotes differentiation from SHEDs to neuron cells through activation of Wnt/ß-catenin pathway by binding to CHD8.

Protective effects of stem cells from human exfoliated deciduous teeth derived conditioned medium on osteoarthritic chondrocytes.

Treatment of osteoarthritis (OA) is still a major clinical challenge due to the limited inherent healing capacity of cartilage. Recent studies utilising stem cells suggest that the therapeutic benefits of these cells are mediated through the paracrine mechanism of bioactive molecules. The present study evaluates the regenerative effect of stem cells from human exfoliated deciduous teeth (SHED) conditioned medium (CM) on OA chondrocytes. The CM was collected after the SHED were cultured in serum-free medium (SFM) for 48 or 72 h and the cells were characterised by the expression of MSC and pluripotency markers. Chondrocytes were stimulated with interleukin-1ß and treated with the CM. Subsequently, the expression of aggrecan, collagen type 2 (COL 2), matrix metalloproteinase-13 (MMP-13), nuclear factor-kB (NF-kB) and the level of inflammatory and anti-inflammatory markers were evaluated. SHED expressed mesenchymal stromal cell surface proteins but were negative for haematopoietic markers. SHED also showed protein expression of NANOG, OCT4 and SOX2 with differential subcellular localisation. Treatment of OA chondrocytes with CM enhanced anti-inflammation compared to control cells treated with SFM. Furthermore, the expression of MMP-13 and NF-kB was significantly downregulated in stimulated chondrocytes incubated in CM. The study also revealed that CM increased the expression of aggrecan and COL 2 in OA chondrocytes compared to SFM control. Both CM regenerate extracellular matrix proteins and mitigate increased MMP-13 expression through inhibition of NF-kB in OA chondrocytes due to the presence of bioactive molecules. The study underscores the potential of CM for OA treatment.

Exosomes derived from human exfoliated deciduous teeth ameliorate adult bone loss in mice through promoting osteogenesis.

Cell-free based therapy is an effective strategy in regenerative medicine as it avoids controversial issues, such as immunomodulation and stability. Recently, exosomes have been explored as a favorable substitution for stem cell therapy as they exhibit multiple advantages, such as the ability to be endocytosed and innate biocompatibility. This study aimed to investigate the effects of stem cells from human exfoliated deciduous teeth (SHED)-derived exosomes (SHED-Exo) on bone marrow stromal cells (BMSCs) osteogenesis and bone recovery. SHED-Exo were isolated, characterized, and applied to the bone loss area caused by periodontitis in a mouse model. We found that the injection of SHED-Exo restored bone loss to the same extent as original stem cells. Without affecting BMSCs proliferation, SHED-Exo mildly inhibited apoptosis. Moreover, SHED-Exo specifically promoted BMSCs osteogenesis and inhibited adipogenesis compared with SHED-derived conditioned medium. The expression of osteogenic marker genes, alkaline phosphatase activity, and Alizarin Red S staining of BMSCs was significantly increased by co-culturing with SHED-Exo. Moreover, Western blot analysis showed that Runx2, a key transcriptional factor in osteogenic differentiation, and p-Smad5 were upregulated upon SHED-Exo stimulation. Expression of the adipogenic marker PPARγ and the amount of lipid droplets decreased when exosomes were present. Low doses of exosomes inhibited the expression of the inflammatory cytokines IL-6 and TNF-α. In conclusion, SHED-Exo directly promoted BMSCs osteogenesis, differentiation, and bone formation. Therefore, exosomes have the potential to be utilized in the treatment of periodontitis and other bone diseases.

Peripheral Nerve Regeneration in a Novel Rat Model of Dysphagia.

The superior laryngeal nerve (SLN) is known to play an essential role in the laryngeal reflex and swallowing. Damage to the SLN causes difficulty swallowing, that is, dysphagia. We successfully developed a novel rat model of dysphagia by SLN injury, in which we could evaluate the neuroregenerative capacity of stem cell from human exfoliated deciduous teeth (SHED). The dysphagic rats exhibit weight loss and altered drinking patterns. Furthermore, SLN injury induces a delayed onset of the swallowing reflex and accumulation of laryngeal debris in the pharynx. This rat model was used to evaluate the systemic application of SHED-conditioned medium (SHED-CM) as a therapeutic candidate for dysphagia. We found that SHED-CM promoted functional recovery and significant axonal regeneration in SLNs through the polarization shift of macrophages from activated inflammatory macrophages (M1) to anti-inflammatory macrophages (M2) and angiogenesis. This chapter describes the establishment of SLN-injury induced dysphagia rat model and the preparation and application of SHED-CM.

A Biobank of Stem Cells of Human Exfoliated Deciduous Teeth: Overview of Applications and Developments in Brazil.

A biobank is an organized collection of biological human material and its associated information stored for research according to regulations under institutional responsibility, without commercial purposes, being a mandatory and strategical activity for research, regenerative medicine, and innovation. Stem cells have largely been employed in research and frequently stored in biobanks, which have been used as an essential source of biological materials. Stem cells of human exfoliated deciduous teeth (SHED) are stem cells which have a high multipotency and can be easily obtained. Besides, this extremely accessible tissue has advantages with respect to storage, as the SHED obtained in childhood can be used in later life, which implies the necessity for the creation and regulation of biobanks. The proper planning for the creation of a biobank includes knowledge of the material types to be stored, requirements regarding handling and storage conditions, storage time, and room for the number of samples. Thus, this study aimed to establish an overview of the development of a SHED biobank. Ethical and legal standardization, current applications, specific orientations, and challenges for the implementation of a SHED biobank were discussed. Through this overview, we hope to encourage further studies to use SHED biobanks.

A study of the differentiation of stem cells from human exfoliated deciduous teeth on 3D silk fibroin scaffolds using static and dynamic culture paradigms.

Stem cells from human exfoliated deciduous teeth (SHED) are considered the best current source of human stem cells due to their ability to differentiate into multiple cell lineages. Dynamic co-culture systems can improve the culture environment, as they provide cells with signaling factors, extracellular matrixes, and cellular shear force, as well as enable the formation of heterotypic clusters. We seeded SHED in 3D silk fibroin porous scaffolds under static and dynamic cultures for 28 days, using the NIH3T3 cultivated medium as an induction agent. Many hepatospheres formed in these porous scaffolds, and cellular viability was shown to continually increase by MTT assays. Hepatic AFP and ALB gene expression, as well as glycogen storage, albumin secretion, and urea synthesis, were greater in cells in the 3D porous scaffold under a dynamic culture than in those cultured under 3D static culture and petri dish conditions. However, the 3D static culture is still superior to the traditional petri dish culture. The NIH3T3 cultivated medium can significantly induce hepatic differentiation of SHED, while the 3D dynamic culture system significantly enhances hepatic differentiation of SHED. This study provides alternative sources of hepatocytes for liver disease treatment.

Amniotic Membrane Enhance the Effect of Vascular Endothelial Growth Factor on the Angiogenic Marker Expression of Stem Cells from Human Exfoliated Deciduous Teeth.

Previously, it was reported that human amniotic membrane (AM) induced stem cells from human deciduous exfoliated teeth (SHED) endothelial-like-cell differentiation. This interesting effect of AM matrix on SHED demands further elucidation. Objective of this in vitro work was to study the effect of 24-h VEGF induced on SHED endothelial differentiation when seeded on acellular stromal side (SS) of AM matrix. Stemness of SHED was identified by flow cytometry. Cell attachment and morphological changes towards the matrix was observed by scanning electron microscopy. Protein expression of endothelial marker was examined by Western blot. The expression of stem cells and endothelial-specific gene markers of VEGF-induced SHED cultured on human AM was inspected via reverse transcriptase-polymerase chain reaction. Results showed SHED at both passages retain stemness property. Ang-1 protein was expressed in SHED. Cells treated with VEGF and cultured on AM transformed attached well to AM. VEGF-induced SHED expressed both stem cell and endothelial-specific markers throughout the treatments and timeline. Interestingly, prolonged VEGF treatment increased the expression of Cox-2 and VE-Cadherin genes in all treated groups when compared to SHED. It was concluded that the VEGF-induced SHED showed better expression of endothelial-specific markers when cultured on SS of AM, with prolonged VEGF treatment.

SHED-derived conditioned exosomes enhance the osteogenic differentiation of PDLSCs via Wnt and BMP signaling in vitro.

The exosomes from human exfoliated deciduous teeth (SHED-Exos) have exhibited potential therapeutic role in dental and oral disorders. The biological effects of exosomes largely depend on cellular origin and physiological status of donor cell. In the present study, we explored the influence of conditioned exosomes from SHED with osteogenic induction on periodontal ligament stem cells (PDLSCs) in vitro. Conditioned SHED-Exos from a 3-day osteogenic supernatant were applied during PDLSCs osteogenic differentiation. We found that conditioned SHED-Exos had no cytotoxicity on PDLSCs viability assessed by CCK-8 assay. These SHED-Exos promoted PDLSCs osteogenic differentiation with deep Alizarin red staining, high alkaline phosphatase (ALP) activity and upregulated osteogenic gene expression (RUNX2, OPN and OCN). We further found BMP/Smad signaling and Wnt/ß-catenin were activated by enhanced Smad1/5/8 phosphorylation and increased nuclear ß-catenin protein expression. Inhibiting these two signaling pathways with specific inhibitors (cardamonin and LDN193189) remarkably weakened the enhanced osteogenic differentiation. Furthermore, Wnt3a and BMP2 were upregulated in SHED and SHED-Exos. Silencing Wnt3a and BMP2 in SHED-Exos partially counteracts the enhanced osteogenic differentiation. Our findings indicate that conditioned SHED-Exos-enhanced PDLSCs osteogenic differentiation was partly due to its carrying Wnt3a and BMP2. These data provide new insights into the use of SHED-Exos in periodontitis-induced bone defects therapy.

Coordination of WNT signaling and ciliogenesis during odontogenesis by piezo type mechanosensitive ion channel component 1.

Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation.