The regulation and interplay between the Notch and BMP pathways in the periosteum and its implications in bone tissue engineering

摘要

Introduction: The Notch and BMP pathway are highly conserved cell signalling pathway that plays a key role in cell proliferation, fate and differentiation. In the following study we report on the interaction and cross-talk between these pathways in the periosteum. The periosteum is a specialised connective tissue that lines bone and contains an osteoprogenitor niche that plays a key role in bone maintenance and repair. The study explores how the Notch, Wnt and BMP pathways regulate this niche in normal bone and in a BMP-2 based tissue engineered construct in a large segmental sheep defect model. Materials and Methods: Four sample groups were utilised in the study; PCL scaffold only (n=6), PCL scaffold with alginate hydrogel (n=6), PCL scaffold with alginate hydrogel loaded with 2mg of rhBMP-2 (n=6) and normal contralateral bone (n = 5). Tissue engineered constructs were evaluated in a large segmenta! defect model using Merino sheep (6 years) as previously described[3]. In brief, a 3cm defect was created in mid diaphyseal of the tibia and a custom PCL tubular scaffold was placed around the defect and filled with alginate, alginate with BMP-2 or left empty. The defects were fixed then with a broad dynamic compression plate. Animals were allowed unrestricted weight bearing after the surgery and sacrificed after 6 months. The samples were evaluated using immunohistochemistry, proximity ligand assay and western blot analysis. Results and Discussion: In normal control bone a distinct Notch-1 was expressed throughout the periosteum, whilst the Notch ligand Jagged-1 was exclusively expressed in cells directly adjacent to the bone. The downstream effector protein of the Notch pathway Hey-1 was highly expressed at the interface where Notch-1 and Jag-1 intersect and overlap indicating potential activation of the notch pathway via Notch-1/Jagged-1 binding. Expression of Notch-1 was upregulated throughout the periosteum of the tissue engineered constructs in all conditions, whilst jagged-1 expression was mainly localised in the distal and proximal ends of the constructs near the host bone and was only expressed in the mid-section of BMP-2 treated group. Notch-1 expression in the tissue engineered construct was co-localised with periostin, an ECM protein exclusively expressed in periostin that has been shown to stabilise precursor Notch-1 and may facilitate its expression in the newly formed bone. Whilst the factor that influence jagged-1 expression remain unclear, the data suggests that its expression in periosteum is influenced by BMP-2. BMP2/4 was constitutively expressed in the periosteum of normal bone and upregulated in the periosteum of all conditions in tissue engineered constructs, as was the BMP antagonist, Noggin. Phosphorylation of SMAD 1/5/8 and the downstream effector RUNX2 indicates that the co-localisation of Noggin does not inhibit BMP receptor signalling. Mature osteoblast markers ALP and osteocalcin was localised only in the cells directly lining the bone in the periosteum indicating a potential cell hierachy within the periosteum.