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

Abstract

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.

Effect of lentivirus‐mediated BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells

“…OPN is also a sign of early differentiation of osteoblasts, which can promote the reconstruction of mineralized tissues 40 . The level of COL‐I content and the osteogenic capacity of cells are complementary to each other and can be used as an important indicator of the differentiation ability of osteoblasts 41 . OC and BSP play a very important regulatory role in the process of cell mineralization and are mainly expressed in the later stages of osteoblasts.…”

“…40 The level of COL-I content and the osteogenic capacity of cells are complementary to each other and can be used as an important indicator of the differentiation ability of osteoblasts. 41 OC and BSP play a very important regulatory role in the process of cell mineralization and are mainly expressed in the later stages of osteoblasts. They are important indicators of osteoblast maturation and mineralization.…”

Comprehensive transcriptome analysis of sika deer antler using PacBio and Illumina sequencing

“…Wergedal discovered that the periosteal bone formation rate and mineral apposition rate were reduced in Wnt16 knockout vs wild-type mice 31 . Ying Jin revealed that Wnt16 promotes osteoblast differentiation of periosteal-derived cells in vitro and in vivo with a potential application in improving bone regeneration 32 . Together, it is speculated Wnt16 signaling pathway can regulate ossification rather than chondrogenesis of antler.…”

Wnt16 signaling in bone homeostasis and osteoarthristis

“…Among these studies, two found that the overexpression of Wnt16 in osteoblasts or osteocytes primarily increased trabecular bone mass rather than cortical bone mass, suggesting that trabecular bone may be more sensitive to altered Wnt16 expression ( 58 , 59 ). In addition, in a mouse calvarial defect model, local transplantation of periosteal derived cells (PDCs), which were genetically engineered to express Wnt16, significantly increased trabecular bone volume/tissue volume and trabecular number ( 74 ). These findings are in consistent with human GWAS studies performed by different groups demonstrating the association between Wnt16 and spine/heel (predominantly trabecular bone) BMD ( 54 , 67 , 75 ).…”

“…Wnt16 is capable of inducing perivascular stem cell (PSC) proliferation and enhancing PSC differentiation towards osteoblasts in a c-Jun N-terminal kinase (JNK) pathway-dependent fashion ( 80 , 81 ). Similarly, Wnt16 promotes osteoblast differentiation from periosteal derived cells (PDCs) ( 74 ) as well as the differentiation of osteoblast progenitors into bone matrix-synthesizing osteoblasts by inhibiting canonical Wnt activity and upregulating Runx2 expression ( 82 ). However, it is worth noting that Wnt16 can also promote canonical Wnt activity to inhibit Runx2 expression and repress the differentiation of pre-osteoblasts ( 60 ).…”