ABSTRACT
Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-ß and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.
Subject(s)
Mice, Knockout , Neurofibromin 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Pseudarthrosis , Schwann Cells , Animals , Female , Humans , Male , Mice , Cell Differentiation/drug effects , Fibrosis , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neurofibromatosis 1/pathology , Neurofibromatosis 1/metabolism , Neurofibromatosis 1/complications , Neurofibromin 1/metabolism , Neurofibromin 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pseudarthrosis/pathology , Pseudarthrosis/metabolism , Pseudarthrosis/congenital , Schwann Cells/metabolism , Schwann Cells/drug effects , Schwann Cells/pathology , Stem Cells/metabolism , Stem Cells/drug effects , Tibia/pathologyABSTRACT
The periosteum covering the outer surface of bone contains skeletal stem/progenitor cells that can efficiently form cartilage and bone during bone repair. Several methods have been described to isolate periosteal cells based on bone scraping and/or enzymatic digestion. Here, we describe an explant culture method to isolate periosteum-derived stem/progenitor cells for subsequent in vitro and in vivo analyses. Periosteal cells (PCs) isolated using this protocol express mesenchymal markers, can be expanded in vitro, and exhibit high regenerative potential after in vivo transplantation at a fracture site, suggesting that this protocol can be employed for PC production to use in new cell-based therapies.
