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NOTCH SIGNALING IN THE OSTEOINDUCTION OF ADIPOSE-DERIVED STEM CELLS: A POSSIBLE THERAPEUTIC ROLE IN THE VIABILITY OF NON-VASCULARIZED BONE GRAFTS
Presenter: Shaun D Mendenhall, MD
Co-Authors: Lough D; Chambers CB; Bueno RA; Germann G; Reichensperger JD; Neumeister MW
Southern Illinois University School of Medicine

Background: Adipose derived stem cells (ADSCs) have been touted as an easily obtainable population to use in such projects; however, little is known regarding the molecular mechanisms that regulate ADSC physiology. This study investigated the impact of inhibiting endogenous Notch signals on ADSC proliferation and osteogenic differentiation. Understanding this process could lead to advancements in tissue engineering of ex-vivo bone grafts as well as augmentation/enhancement of bone matrices in composite tissue allografts.

Methods: ADSCs were isolated from the inguinal fat pads of male Lewis rats and treated with either vehicle or Notch inhibitor, which prevents a critical proteolytic step during Notch activation. Cells were grown in growth media containing either 0.1% DMSO (vehicle) or 1microM DAPT. Proliferation was assessed by the MTT assay. For differentiation analysis, ADSCs were grown in osteoinduction media and matrix deposition was assessed by Alizarin Red staining. Focused gene arrays targeting Notch and osteoinduction pathways were utilized in order to validate transcript suppression and relative levels of bone formation.

Results: DAPT treatment of ADSCs resulted in a significant reduction in cell proliferation. RT-PCR evaluation of the Notch pathway trascriptome validated a 2x103 and 1x102 fold decrease in both Notch1 and Notch3 respectively. DAPT treatment of osteoinduced ADSCs resulted in a significant reduction in extracellular matrix deposition. Gene array analysis further validated that an intact Notch signaling pathway led to significant upregulation of Collagen-1, BMP-3, BMP-4, BMP-5 and BMP-7, suggesting a role in osteoblast differentiation and matrix synthesis.

Conclusions: Endogenous Notch signals are important for ADSC proliferation and differentiation. Notch inhibition effectively reduced ADSC proliferation and negatively regulated extracellular matrix production. Understanding this, we seek to utilize Notch ligand therapy in tissue engineering of ex-vivo bone grafts for bony defects as well as with augmentation/enhancement of bone matrices in composite tissue allografts.


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