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Mechanism of Cyclic Tensile Stress in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

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Abstract

Human periodontal ligament stem cells (hPDLSCs) can undergo osteogenic differentiation under induction conditions. Cyclic tensile stress (CTS) can stimulate stem cell osteogenic differentiation. The present study explored the mechanism of CTS in hPDLSC osteogenic differentiation. The hPDLSCs of the 4th passage were selected. hPDLSCs were subjected to CTS with deformation of 10% elongation at 0.5 Hz for 1, 4, 8, 12 and 24 h. ALP activity and staining, ARS staining and detection of expressions of osteogenesis-related genes (RUNX2, OPN, Sp7 and OCN) were used to assess hPDLSC osteogenic differentiation ability. microRNA (miR)-129-5p and BMP2 expression and p-Smad1/5 level were detected under CTS stimulation. The binding relationship between miR-129-5p and BMP2 was predicted and verified. The osteogenic differentiation ability of CTS-treated hPDLSCs was evaluated after intervention of miR-129-5p and BMP2. CTS induced hPDLSC osteogenic differentiation, as manifested by increased ALP activities, osteogenesis-related gene expressions and mineralized nodules, together with positive ALP staining. CTS inhibited miR-129-5p expression, and promoted BMP2 expression and p-Smad1/5 level in hPDLSCs. miR-129-5p targeted BMP2. Overexpressed miR-129-5p or silenced BMP2 prevented hPDLSC osteogenic differentiation ability. We demonstrated that CTS inhibited miR-129-5p expression, and then activated the BMP2/Smad pathway, thereby showing stimulative effects on hPDLSC osteogenic differentiation.

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Abbreviations

α-MEM:

Alpha-minimal essential medium

DMEM:

Dulbecco’s modified Eagle’s medium

PBS:

Phosphate buffer solution

FBS:

Fetal bovine serum

BSA:

Bovine serum albumin

BCA:

Bicinchoninic acid

PDLCs:

Human periodontal ligament cells

LSD:

Least significant difference

PDL:

Periodontal ligament

MSCs:

Mesenchymal stem cells

hPDLSCs:

Human periodontal ligament stem cells

miR:

MicroRNA

IgG:

Immunoglobulin G

ANOVA:

Analysis of variance

wt:

Wild-type

mut:

Mutant

ADSCs:

Adipose-derived stem cells

CTS:

Cyclic tensile stress

ALP:

Alkalin phospatase

ARS:

Alizarin red S

RUNX2:

Runt-related transcription factor-2

OPN:

Osteopontin

OCN:

Osteocalcin

BMP2:

Bone morphogenetic protein 2

Smad:

Drosophila mothers against decapentaplegic

STRO-1:

Stromal precursor antigen-1

CD:

Cluster of differentiation

si-RNAs:

Small-interfering RNAs

NC:

Negative control

cDNA:

Complementary DNA

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

qRT-PCR:

Quantitative reverse transcription-polymerase chain reaction

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Funding

This work was supported by the Guangdong Basic and Applied Basic Research Foundation (2019A1515110854), National Undergraduate Training Program for Innovation and Entrepreneurship of Sun Yat-sen University funded by Ministry of Education (201901113).

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Author notes

  1. Xiayi Wu and Yi Li have contributed equally to this work.

Authors and Affiliations

  1. Zhujiang Newtown Dental Clinic, Guanghua School of Stomatology, Hospital of Stomatology, Institute of Stomatological Research, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, No.49 Huaxia Road, Guangzhou, 510627, Guangdong, People’s Republic of China

    Xiayi Wu

  2. Guanghua School of Stomatology, Hospital of Stomatology, Institute of Stomatological Research, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, No.56 Lingyuan West Rd, Guangzhou, 510080, People’s Republic of China

    Yi Li, Zeyuan Cao, Yunyi Xie, Chuanqiang Fu & Huan Chen

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  1. Xiayi Wu
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Correspondence to Xiayi Wu.

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This study was approved by the the Ethics Committee of Sun Yat-sen University (ERC-[2016]-46). I certify that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

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Wu, X., Li, Y., Cao, Z. et al. Mechanism of Cyclic Tensile Stress in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells. Calcif Tissue Int 108, 640–653 (2021). https://doi.org/10.1007/s00223-020-00789-x

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