Novel Bioactive Adhesive Monomer CMET Promotes Odontogenic Differentiation and Dentin Regeneration

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.

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Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02085-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ping Zhou ◽

Jia-Min Shi ◽

Jing-E Song ◽

Yu Han ◽

Hong-Jiao Li ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Human Pluripotent Stem Cells ◽

Cell Counting ◽

Cell Type ◽

Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

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Novel Molecule Nell-1 Promotes the Angiogenic Differentiation of Dental Pulp Stem Cells

Frontiers in Physiology ◽

10.3389/fphys.2021.703593 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mengyue Li ◽

Qiang Wang ◽

Qi Han ◽

Jiameng Wu ◽

Hongfan Zhu ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Umbilical Vein ◽

Dental Pulp Stem Cells ◽

Exposure Model ◽

Dental Tissues ◽

Normal Rat ◽

Pulp Exposure ◽

Angiogenic Markers

IntroductionThis work aimed to reveal the crucial role of Nell-1 in the angiogenic differentiation of human dental pulp stem cells (DPSCs) alone or co-cultured with human umbilical vein endothelial cell (HUVECs) in vitro and whether this molecule is involved in the pulp exposure model in vivo.MethodsImmunofluorescence was conducted to ascertain the location of Nell-1 on DPSCs, HUVECs, and normal rat dental tissues. RT-PCR, Western blot, and ELISA were performed to observe the expression levels of angiogenic markers and determine the angiogenic differentiation of Nell-1 on DPSCs alone or co-cultured with HUVECs, as well as in vitro tube formation assay. Blood vessel number for all groups was observed and compared using immunohistochemistry by establishing a rat pulp exposure model.ResultsNell-1 is highly expressed in the nucleus of DPSCs and HUVECs and is co-expressed with angiogenic markers in normal rat pulp tissues. Hence, Nell-1 can promote the angiogenic marker expression in DPSCs alone and co-cultured with other cells and can enhance angiogenesis in vitro as well as in the pulp exposure model.ConclusionNell-1 may play a positive role in the angiogenic differentiation of DPSCs.

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PD-1 Suppresses the Osteogenic and Odontogenic Differentiation of Stem Cells From Dental Apical Papilla via Targeting SHP2/NF-κB axis

10.21203/rs.3.rs-927273/v1 ◽

2021 ◽

Author(s):

Na Li ◽

Zehan Li ◽

Lin Fu ◽

Ming Yan ◽

Yanqiu Wang ◽

...

Keyword(s):

Vital Role ◽

Cell Counting ◽

Alizarin Red ◽

Odontogenic Differentiation ◽

Root Dentin ◽

Apical Papilla ◽

Effect Of Pd ◽

First Time ◽

The Relationship

Abstract BackgroundStem cells from the apical papilla (SCAPs) are important for tooth root development and regeneration of root dentin. Here, we examined the expression of programmed cell death protein-1 (PD-1) in SCAPs and investigated the effect of PD-1 on odontogenic and osteogenic differentiation and the relationship between PD-1 and cell differentiation and SHP2/NF-κB signals.MethodsSCAPs were obtained culture in the related medium. The proliferation ability was evaluated by cell counting kit 8 and 5‐ethynyl‐20‐deoxyuridine (EdU) assay. Alkaline phosphatase (ALP) activity assay, ALP staining, western blot, real-time RT-PCR, Alizarin Red S staining, and immunofluorescence staining were performed to explore the osteo/odontogenic potential and the involvement of SHP2/NF-κB pathways. Besides, we transplanted SCAPs component into mouse calvaria defects to evaluate osteogenesis in vivo. ResultsWe found that human SCAPs expressed PD-1 for the first time. PD-1 knockdown enhanced the osteo/odontogenic differentiation of SCAPs by suppressing SHP2 pathway and activating NF-κB pathway. Overexpression of PD-1 inhibited the osteogenesis and odontogenesis of SCAPs via activation of SHP2 signal and inhibition of NF-κB pathway. ConclusionPD-1 activated SHP2 signal to block NF-κB signal and then played a vital role in osteo/odontogenic differentiation of SCAPs.

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Effect of Different Terpene-Containing Essential Oils on the Proliferation ofEchinococcus granulosusLarval Cells

Interdisciplinary Perspectives on Infectious Diseases ◽

10.1155/2014/746931 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Clara María Albani ◽

Guillermo María Denegri ◽

María Celina Elissondo

Keyword(s):

Essential Oils ◽

Effective Means ◽

Control Cell ◽

Larval Growth ◽

Public Health Problem ◽

Mentha Piperita ◽

Major Public Health Problem ◽

Isolated Cells ◽

Vitro Effect

Human cystic echinococcosis remains a major public health problem on several countries and the treatment strategies are not solved. The aim of the present work was to determine the in vitro effect of thymol andMentha piperita,M. pulegium,andRosmarinus officinalisessential oils on the proliferation ofE. granulosuslarval cells. Isolated cells and cellular aggregates were obtained from hydatid cyst’s germinal layer and exposed to 1, 5, and 10 μg/ml of thymol and the different essential oils for 7 days. Drug effect was evaluated using test viability and scanning electron microscopy. Control cell culture viability was 2.1 x 106(100%) after 7 days of incubation. At day 7, thymol 5 μg/ml caused a reduction in cell viability of 63% and the essential oils ofM. piperita10 μg/ml,M. pulegium10 μg/ml, andR. officinalis10 μg/ml produced a reduction in the viability of 77, 82, and 71%, respectively. Moreover essential oils caused reduction in cell number, collapsed cells, and loss of normal tridimensional composition of the aggregates. Due to the inhibitory effect caused by essential oils onE. granulosuscells we suggested that it would be an effective means for suppression of larval growth.

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LINC00152 down-regulated miR-193a-3p to enhance MCL1 expression and promote gastric cancer cells proliferation

Bioscience Reports ◽

10.1042/bsr20171607 ◽

2018 ◽

Vol 38 (3) ◽

Cited By ~ 17

Author(s):

Yong Huang ◽

Hui Luo ◽

Fang Li ◽

Yun’e Yang ◽

Guangsheng Ou ◽

...

Keyword(s):

Gastric Cancer ◽

Xenograft Model ◽

Cell Counting ◽

Gastric Cancer Cells ◽

Normal Tissues ◽

Non Coding Rna ◽

Mouse Xenograft Model ◽

Vitro Effect

The present work aimed to probe into the effect of long non-coding RNA (lncRNA) LINC00152 on gastric cancer (GC) cells proliferation by regulating miR-193a-3p and its target gene MCL1. Transfected si-LINC00152 was used to down-regulate LINC00152, and cells proliferation was measured by the cell counting kit-8 (CCK-8) assay. Cell apoptosis and cell cycle were analyzed by flow cytometry (FCM). Besides, we also detected the potential functional effects of differential expression of LINC00152 in vivo using nude mouse xenograft model. We overexpressed and downexpressed miR-193a-3p to study the in vitro effect of miR-193a-3p on GC cells proliferation and vitality. And MCL1 was silenced by shRNA to investigate the effect of MCL1 on proliferation of GC cells. In this research, LINC00152 was proven to have a higher expression level in GC tissues than in the adjacent normal tissues. GC cells proliferation was inhibited after LINC00152 was down-regulated. LINC00152 inhibited the expression of miR-193a-3p, which negatively regulated MCL1. In addition, GC cells proliferation was inhibited by cell transfection with shRNA-MCL1, and enhanced by transfection with miR-193a-3p mimics. Our study suggested that LINC00152 was overexpressed in GC tissues, and it down-regulated miR-193a-3p to enhance MCL1 expression thereby promoting GC cells proliferation.

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Activation of Mesenchymal Stem Cells Promotes New Bone Formation Within Dentigerous Cyst

10.21203/rs.3.rs-63524/v1 ◽

2020 ◽

Author(s):

Yejia Yu ◽

Mengyu Li ◽

Yuqiong Zhou ◽

Yueqi Shi ◽

Wenjie Zhang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Regeneration ◽

Bone Defect ◽

Dentigerous Cyst ◽

Healing Process ◽

Cell Counting ◽

Cranial Bone ◽

Alizarin Red

Abstract Background: Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables bone to regenerate with capsules maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC derive from odontogenic epithelium remnants at embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization.Methods: Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues were analyzed by morphology, surface marker and multi-differentiation assays. Comparison of proliferation ability between Am-DCSCs and Bm-DCSCs were evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F) and 5’‐ethynyl‐2’‐deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by Alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with Real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice were performed to detect their bone regeneration and bone defect repair ability.Results: Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony forming cells expressed MSC markers (CD44+, CD90+, CD31-, CD34-, CD45-), and they could differentiate into osteoblast-, adipocyte- and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. Conclusions: There are MSCs residing in capsules of DC, and the cell viability as well as osteogenic capacity of them are largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.

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Activation of mesenchymal stem cells promotes new bone formation within dentigerous cyst

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01999-8 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Yejia Yu ◽

Mengyu Li ◽

Yuqiong Zhou ◽

Yueqi Shi ◽

Wenjie Zhang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Regeneration ◽

Bone Defect ◽

Dentigerous Cyst ◽

Healing Process ◽

Cell Counting ◽

Cranial Bone ◽

Alizarin Red

Abstract Background Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables the bone to regenerate with capsule maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC are derived from odontogenic epithelium remnants at the embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. Methods Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues was analyzed by morphology, surface marker, and multi-differentiation assays. Comparison of proliferation ability between MSCs isolated from DC capsules before (Bm-DCSCs) and after (Am-DCSCs) marsupialization was evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F), and 5′-ethynyl-2′-deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice was performed to detect their bone regeneration and bone defect repairability. Results Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony-forming cells expressed MSC markers (CD44+, CD90+, CD31−, CD34−, CD45−), and they could differentiate into osteoblast-, adipocyte-, and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed a greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. Conclusions There are MSCs residing in capsules of DC, and the cell viability as well as the osteogenic capacity of them is largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.

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A Novel Fragment Derived from Laminin-411 Facilitates Proliferation and Differentiation of Odontoblast-Like Cells

BioMed Research International ◽

10.1155/2018/9465383 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Jia Tang ◽

Takashi Saito

Keyword(s):

Proliferative Activity ◽

Mrna Level ◽

Cell Counting ◽

Rt Pcr ◽

Alizarin Red ◽

Alp Activity ◽

Proliferation And Differentiation ◽

Post Hoc ◽

Laminin 411

The aim for the present study was to evaluate the in vitro effects of iMatrix-411 in odontoblast-like cells. To that end, iMatrix-411 was coated to both nontissue culture treated- (Non-PS) and tissue culture treated-polystyrene (TCPS) multiwells. MDPC-23 cells were seeded into noncoated (control) or coated wells. Optimal coating density and cell proliferation were assessed by cell counting kit-8 (CCK-8) at day two, day three, and day five. Osteo/odontogenic differentiation was evaluated by real-time RT-PCR and alkaline phosphatase (ALP) activity at days seven and eight, respectively. Calcific deposition of cells was visualized by alizarin red staining. Data were analyzed with post hoc Tukey HSD test (p<0.05). Optimal coating density for iMatrix-411 was 8 μg/cm2. Exposure of MDPC-23 cells to iMatrix-411 in either non-PS or TCPS significantly enhanced proliferative activity. iMatrix-411 elevated ALP activity in both types of culture plates. iMatrix-411 significantly increased the mRNA level of OCN, BSP, OPN, ALP, and DMP-1. Meanwhile, it enhanced the expression of several integrin subunits: ITGA1, ITGA5, ITGAV, ITGB1, and ITGB5. Finally, iMatrix-411 also accelerated the mineralization at day eight in Non-PS. The results indicated iMatrix-411 stimulates proliferation and favours differentiation of odontoblast-like cells.

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Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

10.21203/rs.3.rs-45401/v3 ◽

2020 ◽

Author(s):

Ping Zhou ◽

Jia-Min Shi ◽

Jing-E Song ◽

Yu Han ◽

Hong-Jiao Li ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Human Pluripotent Stem Cells ◽

Cell Counting ◽

Cell Type ◽

Alizarin Red

Abstract Background: Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed.Methods: Monolayer cultured human embryonic stem cells and human induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week.Results: The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrated the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded.Conclusions: Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a step-wise induction system in the future.

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Extracellular IL-37 Enhances Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells via Autophagy Pathway

10.21203/rs.3.rs-291517/v1 ◽

2021 ◽

Author(s):

Na Li ◽

Yan Chen ◽

Ming Yan ◽

Yanqiu Wang ◽

Jintao Wu ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Inflammatory Factor ◽

Alizarin Red ◽

Dental Tissues ◽

Alp Activity ◽

Odontogenic Differentiation ◽

Marrow Mesenchymal Stem Cells

Abstract BackgroundThe osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) contributes to the restoration and regeneration of dental tissues. Previous study indicated that IL-37 has often been identified as an anti-inflammatory factor that affects other pro-inflammatory signals. It is known to be a factor capable of inducing in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). The aims of this study were to explore the effects of IL-37 on the differentiation of DPSCs.MethodsDPSCs were cultured in growth medium with different concentration of IL-37, ALP activity was done to detect the optimal concentration for the following experiments. CCK-8 were conducted to assess the effect of IL-37 on proliferation of DPSCs. To assess differentiation, alkaline phosphatase activity, ALP staining, alizarin red S staining and real‐time RT‐PCR of DSPP, Runx2, ALP, and OSX were measured. Western blot was conducted to examine the levels of autophagy related markers (Beclin1, P62, LC3). ResultsCells cultured with 1 ng/mL IL-37 owned the highest ALP activity. IL-37 enhanced the osteogenic and odontogenic differentiation of DPSCs following upregulated the expression of Beclin1, downregulated the expression of P62, and reduced the ratio of LC3II/I, whereas depletion of autophagy suppressed DPSCs osteogenic and odontogenic differentiation. ConclusionIL-37 increased osteogenic and odontogenic differentiation via autophagy.

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