Immobilization of VEGF in biomaterials in order to induce neovascularization for tissue repair
Introduction: Growth factors (GF) are cytokines known to be involved in the regulation of several cellular processes such as migration, differentiation, proliferation or healing making their use extremely appellative for tissue engineering and regenerative medicine therapies. The easiest and simplest mechanism to delivery GF is in a soluble form, however due to the low stability of GF in the body and diffusion-related problems, supra-physiological concentrations and systematic administration are required to achieve the desired effect. To overcome these problems GF have been immobilized into biomaterials in order to avoid diffusion-related issues and achieve co-localization of the growth factor in the implantation site. We have recently developed a system where we can immobilize growth factors (TGF-β1 and BMP-2) in biomaterials through the affinity of the growth factor towards synthetic peptides. We confirmed the affinity of the synthetic peptides towards the GFs, showed that the immobilized GFs are bioactive and activate the signaling pathways on cells and finally that they induce the synthesis of components known to be involved in the healing/regeneration process (ex: collagen synthesis and bone formation). Inadequate vascularization remains one of the major challenges facing tissue engineering. Angiogenesis is the morphogenic process of forming new blood vessels from pre-existing ones. This event plays an important, normal physiological role in wound healing, tissue repair, pregnancy, and exercise and requires the activity of soluble growth factors such as Vascular Endothelial Growth Factor (VEGF). In this work the student will synthetize a peptide that has affinity towards VEGF. Next, the a biomaterial will be functionalized in order to immobilize the peptide on it. The affinity of the peptide towards VEGF will be confirmed and the bioactivity of the VEGF to activate the signaling pathway will be assessed. Finally the advantage of using immobilized VEGF in experiments relevant for tissue engineering proposes will be tested.
Techniques to be used by the student: Peptide synthesis, High Performance Liquid Chromatography, Mass Spectroscopy, Water Contact Angle, Oxygen Plasma, Polymer handling and functionalization, Immunofluorescent staining, Fluorescence Microscopy, Cell Culture, Biochemical Assays, Western Blotting
Daily Supervisor: João Crispim
Supervisor: Prof.dr. Daniel Saris and Prof. dr. Pascal Jonkheijm
If you are interested in this assignment, please contact: Dr. Janneke Alers, contact person for all DBE assignments
Fig 1. Antibody staining against growth factors on biomaterial. Flourescence only present when the biomaterial was fucntionalized with GF binding peptide, showing that is possible to achieve immobilization of GFs on biomaterials towards the affinity with synthetic peptides
Fig. 2. Immobilized hTGF-β1 induces translocation of Smad2/3 into the nucleus in human-derived hamstring cells showing the activation of the TGF signaling Pathway (Scale bar: 50 µm).