miR-217 Suppresses Osteogenic Differentiation of Human Dental Pulp Stem Cells by Down-Regulating Sirtuin 1

The paper is committed to uncovering the effect of miR-217 on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism. hDPSCs were separated from human dental pulp tissues for measurement of stemness. The osteogenic differentiation of hDPSCs was induced in an osteogenic induction medium. The hDPSCs were transfected with miR-217 mimic, miR-217 inhibitor and/or sh-SIRT1 accordingly. The expressions of miR-217 and SIRT1 were detected in hDPSCs after cell transfection and osteogenic differentiation. Calcium nodules were showed by alizarin red staining. Moreover, the expressions of osteogenic differentiation-related genes were also assessed. The binding of miR-217 to SIRT1 was predicted on starBase and further determined by dual-luciferase reporter assay. Down-regulated miR-217 and up-regulated SIRT1 were found during osteogenic differentiation of hDPSCs. The osteogenic differentiation of hDPSCs was suppressed after transfection of miR-217 mimic or sh-SIRT1 while promoted by miR-217 inhibition. Taken together, miR-217 can suppress osteogenic differentiation of hDPSCs by negatively regulating SIRT1.

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miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.759192 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ning Wang ◽

Xiao Han ◽

Haoqing Yang ◽

Dengsheng Xia ◽

Zhipeng Fan

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Binding Protein ◽

Dental Pulp Stem Cells ◽

Luciferase Reporter ◽

Control Group ◽

Alizarin Red ◽

Odontogenic Differentiation ◽

Human Dental Pulp ◽

Tooth Tissue

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation.Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A.Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs.Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

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Exosomal circLPAR1 Promoted Osteogenic Differentiation of Homotypic Dental Pulp Stem Cells by Competitively Binding to hsa-miR-31

BioMed Research International ◽

10.1155/2020/6319395 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Liangkun Xie ◽

Zheng Guan ◽

Mingzhu Zhang ◽

Sha Lyu ◽

Nattawut Thuaksuban ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Expression Profiles ◽

Circular Rna ◽

Inhibitory Effect ◽

Dental Pulp Stem Cells ◽

Great Promise ◽

Human Dental Pulp ◽

Osteogenic Induction

Human dental pulp stem cells (DPSCs) hold great promise in bone regeneration. However, the exact mechanism of osteogenic differentiation of DPSCs remains unknown, especially the role of exosomes played in. The DPSCs were cultured and received osteogenic induction; then, exosomes from osteogenic-induced DPSCs (OI-DPSC-Ex) at different time intervals were isolated and sequenced for circular RNA (circRNA) expression profiles. Gradually, increased circular lysophosphatidic acid receptor 1 (circLPAR1) expression was found in the OI-DPSC-Ex coincidentally with the degree of osteogenic differentiation. Meanwhile, results from osteogenic differentiation examinations showed that the OI-DPSC-Ex had osteogenic effect on the recipient homotypic DPSCs. To investigate the mechanism of exosomal circLPAR1 on osteogenic differentiation, we verified that circLPAR1 could competently bind to hsa-miR-31, by eliminating the inhibitory effect of hsa-miR-31 on osteogenesis, therefore promoting osteogenic differentiation of the recipient homotypic DPSCs. Our study showed that exosomal circRNA played an important role in osteogenic differentiation of DPSCs and provided a novel way of utilization of exosomes for the treatment of bone deficiencies.

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miR-20a-5p contributes to osteogenic differentiation of human dental pulp stem cells by regulating BAMBI and activating the phosphorylation of Smad5 and p38

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02501-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiao Cen ◽

Xuefeng Pan ◽

Bo Zhang ◽

Wei Huang ◽

Fang Pei ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Bone Tissue Regeneration ◽

Luciferase Reporter ◽

Ct Reconstruction ◽

Osteogenic Markers ◽

Human Dental Pulp

Abstract Background Human dental pulp stem cells (hDPSCs) are the preferable choice of seed cells for craniomaxillofacial bone tissue regeneration. As a member of the miR-17-92 cluster, miR-20a-5p functions as an important regulator during bone remodeling. This study aimed to investigate the roles and mechanisms of miR-20a-5p during osteogenesis of hDPSCs. Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to determine the expression of miR-20a-5p during osteogenesis of hDPSCs. We interfered with the expression of miR-20a-5p in hDPSCs to clarify the function of miR-20a-5p on osteogenesis both in vitro and vivo. Direct bind sites between miR-20a-5p and BAMBI were confirmed by dual-luciferase reporter assay, and the underlying mechanisms were investigated with cell co-transfections. Results The expression of miR-20a-5p was showed to be upregulated during osteogenesis of hDPSCs. Inhibition of miR-20a-5p could weaken the intensity of ALP/ARS staining and downregulate the expression of mRNAs and proteins of osteogenic markers, while overexpression of miR-20a-5p could enhance the intensity of ALP/ARS staining and the expression of osteogenic markers. Both micro-CT reconstruction images and histological results showed that miR-20a-5p could promote the regeneration of calvarial defects. miR-20a-5p directly targeted bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), and the latter one was an inhibitor of hDPSC osteogenesis. Silencing BAMBI partially reversed the suppression effect of miR-20a-5p knockdown on osteogenesis. Phosphorylation of Smad5 and p38 was decreased when miR-20a-5p was silenced, whereas p-Smad5 and p-p38 were upregulated when miR-20a-5p was overexpressed or BAMBI was silenced. Conclusions It is demonstrated that miR-20a-5p functioned as a regulator of BAMBI to activate the phosphorylation of Smad5 and p38 during osteogenic differentiation of hDPSCs.

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circAKT3 positively regulates osteogenic differentiation of human dental pulp stromal cells via miR-206/CX43 axis

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02058-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Bo Zhang ◽

Sibei Huo ◽

Xiao Cen ◽

Xuefeng Pan ◽

Xinqi Huang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Stromal Cells ◽

Dental Pulp ◽

Luciferase Reporter ◽

Circular Rnas ◽

Alizarin Red ◽

Cx43 Expression ◽

Human Dental Pulp

Abstract Background Human dental pulp stromal cells (hDPSCs) are promising sources of mesenchymal stem cells (MSCs) for bone tissue regeneration. Circular RNAs (circRNAs) have been demonstrated to play critical roles in stem cell osteogenic differentiation. Herein, we aimed to investigate the role of circAKT3 during osteogenesis of hDPSCs and the underlying mechanisms of its function. Methods We performed circRNA sequencing to investigate the expression profiles of circular RNAs during osteogenesis of hDPSCs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression pattern of circAKT3 and miR-206 in hDPSCs during osteogenesis. We knocked down circAKT3 and interfered the expression of miR-206 to verify their regulatory role in hDPSC osteogenesis. We detected hDPSCs mineralization by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining and used dual-luciferase reporter assay to validate the direct binding between circAKT3 and miR-206. To investigate in vivo mineralization, we performed subcutaneous transplantation in nude mice and used hematoxylin and eosin, Masson’s trichrome, and immunohistochemistry staining. Results Totally, 86 circRNAs were differentially expressed during hDPSC osteogenesis, in which 29 were downregulated while 57 were upregulated. circAKT3 was upregulated while miR-206 was downregulated during hDPSC osteogenesis. Knockdown of circAKT3 inhibited ALP/ARS staining and expression levels of osteogenic genes. circAKT3 directly interacted with miR-206, and the latter one suppressed osteogenesis of hDPSCs. Silencing miR-206 partially reversed the inhibitory effect of circAKT3 knockdown on osteogenesis. Connexin 43 (CX43), which positively regulates osteogenesis of stem cells, was predicted as a target of miR-206, and overexpression or knockdown of miR-206 could correspondingly decrease and increase the expression of CX43. In vivo study showed knockdown of circAKT3 suppressed the formation of mineralized nodules and expression of osteogenic proteins. Conclusion During osteogenesis of hDPSCs, circAKT3 could function as a positive regulator by directly sponging miR-206 and arresting the inhibitive effect of miR-206 on CX43 expression.

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SIRT1 Promotes Osteogenic Differentiation in Human Dental Pulp Stem Cells through Counteracting the Activation of STAT3

Coatings ◽

10.3390/coatings11111353 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1353

Author(s):

Dan Zhao ◽

Wen Kang ◽

Yiwen Wang ◽

Jiuyu Ge ◽

Jianfeng Huang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Histone Acetylation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Sirtuin 1 ◽

Laboratory Research ◽

Class Iii ◽

Western Blot Assay ◽

Human Dental Pulp

Human dental pulp stem cells (hDPSCs), which are characterized by self-renewal capacity and the ability of multilineage differentiation, have gained increased attention in regenerative medicine recently. Histone acetylation modulator proteins (HAMPs) are a protein family that mediates the modification and identification of histone acetylation and participates in various critical cellular processes. Here, we comprehensively surveyed the expression profile of HAMPs during osteoblast differentiation of hDPSCs and found that the HDAC class III pathway was upregulated, whereas the signal transducer and activator of transcription 3 (STAT3) signaling was downregulated during osteogenesis. Further laboratory research demonstrated that Sirtuin-1 (SIRT1), a class III HDAC, was upregulated and STAT3 activation was downregulated during osteogenic differentiation. SIRT1 counteracted the activation of STAT3 to promote osteogenic differentiation of hDPSCs at 7 and 21 days in both Western blot assay and chemical staining, which highlights the promising utility of SIRT1 activators in hDPSCs-based therapies for bone augmentation strategies and provides clinical insights that may lead to the development of osteogenic agents.

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Hyaluronan based gel promotes human Dental Pulp Stem Cells bone differentiation by activating YAP/TAZ pathway

10.21203/rs.3.rs-740744/v1 ◽

2021 ◽

Author(s):

Marcella La Noce ◽

Antonietta Stellavato ◽

Valentina Vassallo ◽

Marcella Cammarota ◽

Luigi Laino ◽

...

Keyword(s):

Stem Cells ◽

Molecular Weight ◽

Osteogenic Differentiation ◽

Tissue Regeneration ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Nodule Formation ◽

Alizarin Red ◽

Qrt Pcr ◽

Human Dental Pulp

Abstract Background. Hyaluronic acid (HA) is the major component of the extracellular matrix of human tissue, where it regulates processes such as osmotic pressure, water retention, cell migration, and differentiation. For these reasons, hyaluronans are currently used in regenerative medicine in different areas. Nevertheless, hyaluronans exist in different forms accordingly with molecular weight and degree of crosslinking, which can have a different and context-depended effects on cellular processes. Thus, picking the most appropriate form of hyaluronan turn out to be fundamental as it can make a huge difference in tissue regeneration. MSCs have attracted attention in tissue regeneration for their proliferation potential and ability to differentiate in several cytotypes. Among MSCs, human Dental Pulp Stem Cells (hDPSCs) were shown to be remarkably suitable for bone differentiation.In this study, we tested the capability to induce osteogenic differentiation in hDPSCs of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high (HHA), low molecular weight (LHA), and the recently stabilized hybrid cooperative complexes (HCC), containing both sizes.Methods. hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. CD44, the main receptor of the HA on cell surface and an upstream regulator of YAP/TAZ signaling, was analyzed by immunofluorescence. YAP/TAZ expression was measured by qRT-PCR. To confirm the involvement of YAP/TAZ pathway, YAP/TAZ inhibitor-1 was used and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence.Results. HCC was found to be the most impacting in inducing osteogenesis, with significant effects already at 7-14 days of treatment. HCC induced strong overexpression of osteocalcin, osteopontin, and bone sialoprotein, calcification nodule formation, and CD44 up-regulation.In addition, we showed that this biological process is associated to the activation of YAP/TAZ pathway and its target genes CTGF, ANKDR-1, RUNX-1, and RUNX-2.Conclusions. In conclusion, in this study we show that HA’s molecular weight can have a tremendous impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells.

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Chrysin induces osteogenic differentiation of human dental pulp stem cells

Experimental Cell Research ◽

10.1016/j.yexcr.2020.112466 ◽

2021 ◽

Vol 400 (2) ◽

pp. 112466

Author(s):

J.F. Huo ◽

M.L. Zhang ◽

X.X. Wang ◽

D.H. Zou

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Human Dental Pulp

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Chlorogenic acid promotes osteogenic differentiation of human dental pulp stem cells via Wnt signaling

Stem Cells and Development ◽

10.1089/scd.2020.0193 ◽

2021 ◽

Author(s):

Xiaoping Hu ◽

Li Wang ◽

Yuanqiao He ◽

Minli Wei ◽

Huilin Yan ◽

...

Keyword(s):

Stem Cells ◽

Wnt Signaling ◽

Chlorogenic Acid ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Human Dental Pulp

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Evaluation of Resin-Based Material Containing Copaiba Oleoresin (Copaifera Reticulata Ducke): Biological Effects on the Human Dental Pulp Stem Cells

Biomolecules ◽

10.3390/biom10070972 ◽

2020 ◽

Vol 10 (7) ◽

pp. 972

Author(s):

Roberta Souza D’Almeida Couto ◽

Maria Fernanda Setubal Destro Rodrigues ◽

Leila Soares Ferreira ◽

Ivana Márcia Alves Diniz ◽

Fernando de Sá Silva ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Biological Effects ◽

Dental Pulp Stem Cells ◽

Nodule Formation ◽

Alizarin Red ◽

Pulp Capping ◽

Hsp 27 ◽

Dental Pulp Capping ◽

Human Dental Pulp

The purpose of this study was to analyze in vitro the biological effects on human dental pulp stem cells triggered in response to substances leached or dissolved from two experimental cements for dental pulp capping. The experimental materials, based on extracts from Copaifera reticulata Ducke (COP), were compared to calcium hydroxide [Ca(OH)2] and mineral trioxide aggregate (MTA), materials commonly used for direct dental pulp capping in restorative dentistry. For this, human dental pulp stem cells were exposed to COP associated or not with Ca(OH)2 or MTA. Cell cytocompatibility, migration, and differentiation (mineralized nodule formation (Alizarin red assay) and gene expression (RT-qPCR) of OCN, DSPP, and HSP-27 (genes regulated in biomineralization events)) were evaluated. The results showed that the association of COP reduced the cytotoxicity of Ca(OH)2. Upregulations of the OCN, DSPP, and HSP-27 genes were observed in response to the association of COP to MTA, and the DSPP and HSP-27 genes were upregulated in the Ca(OH)2 + COP group. In up to 24 h, cell migration was significantly enhanced in the MTA + COP and Ca(OH)2 + COP groups. In conclusion, the combination of COP with the currently used materials for dental pulp capping [Ca(OH)2 and MTA] improved the cell activities related to pulp repair (i.e., cytocompatibility, differentiation, mineralization, and migration) including a protective effect against the cytotoxicity of Ca(OH)2.

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