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Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

BACKGROUND: Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover w...

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Autores principales: Jin, Ying, Sun, Xiaoyan, Pei, Fang, Zhao, Zhihe, Mao, Jeremy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694570/
https://www.ncbi.nlm.nih.gov/pubmed/33282557
http://dx.doi.org/10.7717/peerj.10374
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author Jin, Ying
Sun, Xiaoyan
Pei, Fang
Zhao, Zhihe
Mao, Jeremy
author_facet Jin, Ying
Sun, Xiaoyan
Pei, Fang
Zhao, Zhihe
Mao, Jeremy
author_sort Jin, Ying
collection PubMed
description BACKGROUND: Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. METHODS: We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. RESULTS: Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. CONCLUSION: Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.
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spelling pubmed-76945702020-12-04 Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo Jin, Ying Sun, Xiaoyan Pei, Fang Zhao, Zhihe Mao, Jeremy PeerJ Biochemistry BACKGROUND: Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. METHODS: We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. RESULTS: Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. CONCLUSION: Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration. PeerJ Inc. 2020-11-24 /pmc/articles/PMC7694570/ /pubmed/33282557 http://dx.doi.org/10.7717/peerj.10374 Text en ©2020 Jin et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biochemistry
Jin, Ying
Sun, Xiaoyan
Pei, Fang
Zhao, Zhihe
Mao, Jeremy
Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
title Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
title_full Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
title_fullStr Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
title_full_unstemmed Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
title_short Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
title_sort wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo
topic Biochemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694570/
https://www.ncbi.nlm.nih.gov/pubmed/33282557
http://dx.doi.org/10.7717/peerj.10374
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