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MicroRNA-196a-5p overexpression in Wharton’s jelly umbilical cord stem cells promotes their osteogenic differentiation and new bone formation in bone defects in the rat calvarium

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Abstract

The peri-tooth root alveolar loss often does not have sufficient space for repair material transplantation and plasticity. Mesenchymal stem cell (MSC) sheets have an advantage in providing more extracellular matrix (ECM) and may prove to be a new therapeutic consideration for this bone defect repair. The identification of key regulators that stimulate MSCs’ osteogenic potential and sheet-derived ECM deposition is the key to promoting its application. In this study, we found that inhibition or overexpression of miR-196a-5p led to a decline or enhancement, respectively, in the alkaline phosphatase (ALP) activity, mineralization, and the levels of osteogenic markers, Osteocalcin (OCN), Dentin Matrix Protein 1 (DMP1), Bone Sialoprotein (BSP), and Dentin Sialophosphoprotein (DSPP) of Wharton’s jelly of umbilical cord stem cells (WJCMSCs) in vitro. Moreover, the 5,6-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) analysis revealed inhibition of the WJCMSCs’ proliferative ability upon miR-196a-5p overexpression. Characterization of the sheet formation by picrosirius red and Masson staining indicated that miR-196a-5p overexpression significantly promoted the collagen content in whole WJCMSC sheet–derived ECM. Furthermore, micro-CT and histopathology results indicated that the miR-196a-5p-overexpressed WJCMSC sheets significantly promoted new bone regeneration and rat calvarial bone defect closure 12 weeks following transplantation. The mRNA microarray analysis of miR-196a-5p-overexpressed WJCMSCs revealed 959 differentially expressed genes (DEGs) (34 upregulated and 925 downregulated). Moreover, 241 genes targeted by miR-196a-5p were predicted by using miRNA function websites of which only 19 predicted genes were consistent with the microarray revealed DEGs. Hence, one unrevealed downregulated DEG Serpin Family B Member 2 (SERPINB2) was investigated. And the deletion of SERPINB2 enhanced the ALP activity and mineralization of WJCMSCs in vitro. In conclusion, our study found that miR-196a-5p, as a key regulator, could repress the proliferation tendency, while stimulating osteogenic ability and WJCMSC sheet–derived ECM deposition, thus promoting new bone formation and rat calvarial bone defect closure. Furthermore, SERPINB2 is a key downstream gene involved in the miR-196a-5p-promoted WJCMSC osteogenesis.

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Abbreviations

ADSCs:

Adipose-derived stem cells

ALP:

Alkaline phosphatase

BMSCs:

Bone marrow mesenchymal stem cells

BSP:

Bone Sialoprotein

CFSE:

5,6-Carboxyfluorescein Diacetate Succinimidyl Ester

CLDN11:

Claudin

DAPI:

4′,6‐Diamidino‐2‐phenylindole dihydrochloride

DEGs:

Differentially expressed genes

DPSCs:

Dental pulp stem cells

DSPP:

Dentin Sialophosphoprotein

DMP1:

Dentin Matrix Protein 1

ECM:

Extracellular matrix

ENPP1:

Ectonucleotide pyrophosphatase phosphodiesterase 1

EVs:

Extracellular vesicles

GNAS:

GNAS Complex Locus

H&E:

Hematoxylin-eosin

HOXC8 :

homeobox C8

IL6 :

interleukin 6

micro-CT:

micro-computed tomography

MiRNA:

microRNA

miR‐196a-5p:

microRNA-196a-5p

MSCs:

mesenchymal stem cells

OCN:

Osteocalcin

RT-PCR:

Reverse transcriptase polymerase chain reaction

SD:

Sprague Dawley

SERPINB2 :

Serpin Family B Member 2

VCAM1 :

Vascular cell adhesion molecule 1

WJCMSCs:

Wharton’s jelly of umbilical cord stem cells

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Funding

This work was supported by grants from the General Program (Key Program, Major Research Plan) of the National Natural Science Foundation of China (81670948 to Y. C.).

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Author notes

  1. Yantong Wang and Simin Zhang contributed equally.

Authors and Affiliations

  1. Department of General Dentistry, Capital Medical University School of Stomatology, Beijing, 100050, China

    Yantong Wang, Simin Zhang, Dianqin Yu, Yingchu Zhao & Yu Cao

  2. Laboratory of Molecular Signaling and Stem Cells TherapyKey Laboratory of Tooth Regeneration and Function ReconstructionDongcheng District, Capital Medical University School of Stomatology, 4 Tiantanxili, BeijingBeijing, 100050, China

    Yantong Wang, Haoqing Yang, Yangyang Cao & Yu Cao

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  1. Yantong Wang
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Contributions

WY and ZS performed the experiments. WY analyzed the data. YH, YD, and ZY were responsible for collection of data and technical expertise. CY supplied experimental materials and resources. YH, CYY, and CY conceived the study. WY and CYY drafted the manuscript. CYY reviewed this work. All the authors approved the final manuscript. WY and ZS are senior authors, contributed equally, and are co-correspondents of CY to this work.

Corresponding author

Correspondence to Yu Cao.

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Ethics approval

The animal study was approved by the animal care and use committee of Beijing Stomatological Hospital Animal laboratory (reference number: 81,670,948). The animal care and experimental procedure were performed following the guidelines of the Beijing Experimental Animal Management Ordinance. Human stem cell study complied with the ISSCR “guidelines for the Conduct of Human Embryonic Stem Cell Research.” Human WJCMSCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

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Wang, Y., Zhang, S., Yang, H. et al. MicroRNA-196a-5p overexpression in Wharton’s jelly umbilical cord stem cells promotes their osteogenic differentiation and new bone formation in bone defects in the rat calvarium. Cell Tissue Res 390, 245–260 (2022). https://doi.org/10.1007/s00441-022-03673-2

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