Effects of ethylenediaminetetraacetic acid on stem cells from the apical papilla: In vitro study.

Background/purpose: Ethylenediaminetetraacetic acid (EDTA) is used as an irrigant in regenerative endodontic treatment. The present study aimed to investigate the effects of EDTA on stem cells from apical papilla (SCAPs) in vitro. Materials and methods: Human SCAPs were isolated and characterised. The cells were treated with media supplemented with EDTA at concentrations ranging from 1.25% to 17%. Cell proliferation and apoptosis were examined using MTT assay and annexin V/propidium iodide staining. Cell migration was determined by a scratch assay. Gene expression was evaluated using a real-time polymerase chain reaction. Mineral deposition, a hallmark of osteogenesis in vitro, was determined using alizarin red s staining. Results: Overall, SCAPs exhibited mesenchymal stem cell characteristics. EDTA treatment at 2.50% and 1.25% did not significantly exhibit cytotoxicity and alter cell morphology. However, EDTA attenuated cell proliferation and reduced MKI67 mRNA expression in SCAPs. Further, EDTA significantly induced early cell apoptosis at 48 h. Cell migration was delayed with EDTA treatment. After maintaining SCAPs in an osteogenic induction medium, EDTA diminished mineral deposition by SCAPs on day 14. Conclusion: EDTA treatment exhibits adverse effects on SCAPs in vitro. Hence, EDTA exposure to periapical tissues should be avoided to minimise the negative impacts on SCAPs cells in regenerative processes.

Application of shear stress for enhanced osteogenic differentiation of mouse induced pluripotent stem cells.

The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy.

Responses of canine periodontal ligament cells to bubaline blood derived platelet rich fibrin in vitro.

Platelet-rich fibrin (PRF) promotes wound healing by providing the release of growth factors. Here, the influence of Thai and Murrah bubaline blood derived PRF on canine periodontal ligament cells (cPDLs) was investigated. PRF was prepared from Thai and Murrah buffaloes with single centrifugation. Results demonstrated that Thai bubaline blood derived PRF exhibited fiber-mesh like morphology and contained more platelet entrapment than Murrah bubaline blood derived PRF. Both bubaline PRFs were able to degrade in vitro under condition with trypsin. Thai but not Murrah bubaline blood derived PRF promoted cPDLs proliferation in serum free and 2% serum culture conditions. Correspondingly, the significant upregulation of KI67 mRNA expression was observed in those cells treated with Thai bubaline blood derived PRF. However, both Thai and Murrah bubaline blood derived PRF accelerated cell migration in an in vitro wound healing assay and facilitated cell spreading. Further, cPDLs cultured in osteogenic induction medium supplemented with Thai bubaline blood derived PRF exhibited the increased mineral deposition in vitro. Frozen Thai bubaline blood derived PRF also promoted cell proliferation, KI67 mRNA expression, cell migration, and cell spreading in cPDLs. Taken these evidence together, bubaline blood derived PRF could provide potential benefits for canine periodontal tissue healing.

Extracellular Matrix Derived From Dental Pulp Stem Cells Promotes Mineralization.

Background: Extracellular matrix (ECM) plays a pivotal role in many physiological processes. ECM macromolecules and associated factors differ according to tissues, impact cell differentiation, and tissue homeostasis. Dental pulp ECM may differ from other oral tissues and impact mineralization. Thus, the present study aimed to identify the matrisome of ECM proteins derived from human dental pulp stem cells (DPSCs) and its ability to regulate mineralization even in cells which do not respond to assaults by mineralization, the human gingival fibroblasts (GF). Methods: ECM were extracted from DPSCs cultured in normal growth medium supplemented with L-ascorbic acid (N-ECM) or in osteogenic induction medium (OM-ECM). ECM decellularization (dECM) was performed using 0.5% triton X-100 in 20 mM ammonium hydroxide after 21 days. Mass spectrometry and proteomic analysis identified and quantified matrisome proteins. Results: The dECM contained ECM proteins but lacked cellular components and mineralization. Interestingly, collagens (COL6A1, COL6A2, and COL6A3) and elastic fibers (FBN1, FBLN2, FN1, and HSPG2) were significantly represented in N-ECM, while annexins (ANXA1, ANXA4, ANXA5, ANXA6, ANXA7, and ANXA11) were significantly overdetected in OM-ECM. GF were reseeded on N-dECM and OM-dECM and cultured in normal or osteogenic medium. GF were able to attach and proliferate on N-dECM and OM-dECM. Both dECM enhanced mineralization of GF at day 14 compared to tissue culture plate (TCP). In addition, OM-dECM promoted higher mineralization of GF than N-dECM although cultured in growth medium. Conclusions: ECM derived from DPSCs proved to be osteoinductive, and this knowledge supported cell-derived ECM can be further utilized for tissue engineering of mineralized tissues.

Interleukin 6 promotes an in vitro mineral deposition by stem cells isolated from human exfoliated deciduous teeth.

Interleukin 6 (IL-6) plays various roles including stem cell regulation. The present study investigated the effect of IL-6 on cell proliferation, colony forming unit ability, stem cell marker expression and differentiation ability in stem cells isolated from human exfoliated deciduous teeth (SHEDs). We reported that the isolated cells from dental pulp tissues for deciduous teeth expressed CD44, CD90 and CD105 but not CD45. These cells were able to differentiate into osteoblasts, adipocytes and neuronal-like cells. IL-6 treatment resulted in the significant increase of NANOG, SOX2 and REX1 mRNA expression. However, IL-6 had no effect on cell proliferation and colony forming unit ability. IL-6 did not alter adipogenic and neurogenic differentiation potency. IL-6 supplementation in osteogenic medium led to a significant increase of mineralization. Furthermore, IL-6 upregulated ALP, ANKH and PIT1 mRNA levels. In conclusion, IL-6 participates in the regulation of pluripotent marker expression and is also involved in mineralization process of SHEDs. Hence, IL-6 could be employed as a supplementary substance in culture medium to maintain stemness and to induce osteogenic induction in SHEDs for future regenerative cell therapy.

Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth.

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior.

Hypoxia enhances osteogenic differentiation in retinoic acid-treated murine-induced pluripotent stem cells.

Hypoxic condition influences biological responses in various cell types. However, a hypoxic regulating osteogenic differentiation remains controversy. Here, an influence of short-term culture in hypoxic condition on osteogenic marker gene expression by retinoic acid-treated murine gingival fibroblast-derived induced pluripotent stem cells (RA-miPS) was investigated. Results demonstrated that hypoxic condition significantly upregulated Vegf, Runx2, Osx, and Ocn mRNA expression by RA-miPS in normal culture medium at day 3. Further, desferrioxamine significantly downregulated pluripotent marker (Nanog and Oct4) and enhanced osteogenic marker (Runx2, Osx, Dlx5, and Ocn) gene expression as well as promoted in vitro mineral deposition. However, the effect of cobalt chloride on osteogenic differentiation of RA-miPS was not robust. In summary, the results imply that hypoxic condition may be useful in the enhancement of osteogenic differentiation in RA-miPS. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s13770-016-9127-9 and is accessible for authorized users.

Cobalt chloride supplementation induces stem-cell marker expression and inhibits osteoblastic differentiation in human periodontal ligament cells.

OBJECTIVE: Low oxygen tension is one of the crucial factors of the stem-cell niche. However, the long-term hypoxic culture of stem cells is difficult and requires special equipment. In this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl2) could maintain human periodontal ligament (HPDL) cell stemness. METHODS: HPDL cells were treated with either 50 or 100 µM CoCl2. Cell proliferation was determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation was determined by assaying alkaline phosphatase activity and in vitro mineralization. RESULTS: The results showed that the CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1α inhibitor, indicated that CoCl2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism. CONCLUSION: This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory.

bFGF and JAGGED1 regulate alkaline phosphatase expression and mineralization in dental tissue-derived mesenchymal stem cells.

Basic fibroblast growth factor (bFGF) and Notch signaling play critical roles in various cell behaviors. Here, we investigated the influence of bFGF and Notch signaling in alkaline phosphatase (ALP) expression and mineralization process in human periodontal ligament-derived mesenchymal stem cells (PDLSCs) and stem cells isolated from human exfoliated deciduous teeth (SHEDs). PDLSCs and SHEDs were cultured in osteogenic medium supplemented with bFGF or on the immobilized Notch ligands, JAGGED1. The ALP mRNA and protein expression were measured by quantitative reverse transcriptase polymerase chain reaction and enzymatic activity assay, respectively. Mineral deposition was determined using alizarin red S staining. The results showed that the addition of bFGF resulted in the decrease of ALP mRNA expression and enzymatic activity. In addition, the attenuation of mineralization was noted. These phenomenons were blocked by the addition of a fibroblast growth factor receptor inhibitor (SU5402) or a MEK inhibitor (PD98059). Interestingly, bFGF supplementation also decreased the Notch signaling component mRNA levels. Thus, to evaluate effect of Notch signaling in mineralization process, PDLSCs and SHEDs were exposed to JAGGED1 modified surface. The ALP mRNA and protein expression were significantly upregulated and the mineral deposition was markedly increased. These results could be reversed by the addition of a γ-secretase inhibitor. In addition, bFGF could attenuate the Notch-signaling-induced mineralization in both PDLSCs and SHEDs. These results suggest that mineralization was enhanced by Notch signaling but attenuated by bFGF signaling. This knowledge can be further utilized to control PDLSCs and SHEDs mineralization for tissue regeneration purpose.

Asiaticoside induces type I collagen synthesis and osteogenic differentiation in human periodontal ligament cells.

Asiaticoside, an active ingredient extracted from Centella asiatica, has been widely used to promote wound healing. In this study, the effects of asiaticoside on proliferation, protein synthesis, and osteogenic differentiation in human periodontal ligament cells (HPDLs) were investigated. HPDLs were treated with asiaticoside at concentrations of 25, 50, and 100 µg/mL. Cell number was determined by MTT assay. The mRNA expression was analyzed by reverse transcription-polymerase chain reaction. Western blot analysis and immunocytochemistry were used to confirm protein synthesis. Osteogenic differentiation was determined by alkaline phosphatase activity, osteoblast marker gene expression, and in vitro mineralization. The results showed that asiaticoside treatment, ranging from 25 to 100 mg/mL, had no effect on cytotoxicity or cell proliferation. When HPDLs were treated with asiaticoside in serum-free medium, dose-dependent increases in the levels of fibronectin and collagen type I mRNA and protein were observed at 72 h. Moreover, asiaticoside attenuated matrix metalloproteinase-1 but enhanced tissue inhibitor of metalloproteinase-1 mRNA expression. The addition of asiaticoside to osteogenic medium resulted in an increase in alkaline phosphatase enzymatic activity, up-regulation of osteoblast marker gene mRNA expression, and enhancement of mineralization by HPDLs. These results suggest the potential application of asiaticoside for enhancing periodontal tissue healing.

Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering.

Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PP(i)), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (P(i)), an inducer of hydroxyapatite formation. P(i) is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with beta-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.