Description:
Inventors: Matt Kipper, Ketul Popat and Melissa Reynolds, Victoria Leszczak, Raimundo Romero
Patent status: US and PCT patents pending
This invention is a method for modifying the surfaces of vascular stents and orthopedic devices to introduce multiple biological functions to the surface. Its use can be extended to other blood contacting devices (e.g. heart valves, biosensors and extracorporeal devices), and other orthopedic materials (internal fixation devices and intraosseous transcutaneous amputation prostheses).
The invention is a surface treatment for materials that includes the formation of metal oxide (e.g. titania) nanotubes, and their modification with nanostructured polyelectrolyte complexes (nanoparticles, nanofibers, and porous foams) that introduce multiple biological functions to the surface. The polyelectrolyte complexes contain chitosan and/or chitosan modified with a nitrous oxide donating group (NO-chitosan) as the polycation, and glycosaminoglycans, such as heparin, heparan sulfate, and chondroitin sulfate, as the polyanion. These surfaces are further modified with potent growth factors, such as vascular endothelial growth factor (VEGF), members of the fibroblast growth factor family (e.g. FGF-2), or the transforming growth factor beta superfamily (e.g. BMP-2) bound and stabilized by the glycosaminoglycans. The titania nanotubes provide tunable nanotopographical features, and the polyelectrolyte complexes introduce a number of biochemical functions to the surface.
The beneficial effects of the surface nanotopography introduced by the nanotubes have been demonstrated on a number of cell types. Surface nanotopographical features promote surface endothelialization and osteoblast attachment and differentiation. These effects would promote healing in both orthopedic and vascular applications. Furthermore, the nanotopography has been shown to prevent smooth muscle cell proliferation and increase their differentiation, and to reduce both platelet adhesion and macrophage activation. These effects reduce the potential for blood clotting, inflammation, and foreign body reaction to the surface.
With respect to growth factor delivery. Heparin and other glycosaminoglycans bind many growth factors and stabilize them in the extracellular matrix. Glycosaminoglycans also bind growth factor receptors and thereby regulate growth factor signaling. By modifying the surfaces with these glycosaminoglycans, growth factors are stabilized and presented to cells in a context that mimics their biochemical presentation in the native extracellular matrix. This enhances growth factor stability and signaling. As an example orthopedic application, we have delivered FGF-2 from nanostructured glycosaminoglycan-based surfaces, using physiologically relevant growth factor doses (ng/cm2) rather than costly and potentially dangerous superphysiologic doses. Mitogenic activity of FGF-2 with respect to mesenchymal stem cells has been maintained for more than 30 days.Chitosan also promotes the induction of cytokine profiles that lead to the waning of inflammation and healthy integration with the host tissue.
As an example vascular application, heparin and other glycosaminoglycans bind VEGF and its to primary receptors, VEGFR1and VEGFR2. This VEGF and VEGFR binding helps promote endothelialization, by stimulating endothelial cell proliferation, without inducing smooth muscle cell proliferation. Heparin also potentiates the activity of antithrombin III and inhibits blood coagulation. Nitrous oxide (NO) released from the surfaces improving resistance to thrombosis by preventing platelet activation and adhesion, promoting vasorelaxation, and inhibiting neointimal hyperplasia.
Nanotexturing provides unique geometries, including nanofibers, porous foam, nanotubes, nanopillars or nanowires, which each can be tuned using polysaccharide chemistries, nitrous oxide-releasing polymers, and growth factors. These include:
• Nanotopography promotes endothelial cell attachment and inhibits thrombus formation.
• Chitosan has antimicrobial activity.
• Heparin interacts favorably with enzymes in the coagulation cascade to prevent coagulation.
• Nitrous oxide prevents platelet activation and acts as a vasodilator.
• Growth factors stabilized by binding to sulfated polysaccharides promote cell proliferation, differentiation, and extracellular matrix production at the surface.
• Combinations of multiple growth factors can be used, e.g. TGF? growth factors to promote bone healing and VEGF to promote angiogenesis.
Key features of this work:
• It is scalable and conformal over surfaces with large (cm-scale) to very small (10-100 nm) features.
• surfaces can be applied to many surface types, including flat polymers, medical-grade titanium and stainless steel, and glass, nano-structured surfaces, and even cortical bone.
• The surface modification can be automated and modified to precisely tune the composition, amount, and location of growth factors.
• The polysaccharides used have specific binding sequences that stabilize many proteins of interest in the native extracellular matrix. These include vascular endothelial growth factor, parathyroid hormone, members of the fibroblast growth factor family, the transforming growth factor beta superfamily (including some bone morphogenetic proteins), and many more. These stabilizing sequences reduce the amount of bound protein required to achieve the desired biological response.3
• Even in the absence of specific binding sequences, saccharide-based chemistries are well-known stabilizers of protein secondary, tertiary, and quaternary structure, and are used extensively in the pharmaceutical industry as excipients.
• Coatings made from these polysaccharides have broad spectrum antimicrobial activity.
• In general, tissue healing around implants based on the polysaccharide chitosan is characterized by waning of the inflammation response, good tissue integration, and lack of chronic inflammation
Related Publications:
• Kipper, et al. Chitosan-Heparin Polyelectrolyte Multilayers on Cortical Bone: Periostuem-Mimetic, Cytophilic, Antibacterial Coatings. Biotechnology and Bioengineering, 2013 Feb; 110(2): 609-18
• V. Leszczak, L.W. Place, N. Franz, and M.J. Kipper, “Nanostructured Biomaterials from Demineralized Bone Matrix: A Survey of Processing and Crosslinking Strategies” ACS Applied Materials and Interfaces, (In press, 2014).
• L.W. Place, M. Sekyi, and M.J. Kipper, “Aggrecan-Mimetic, Glycosaminoglycan-Containing Nanoparticles for Growth Factor Delivery” Biomacromolecules, 15, 680-689, 2014. (DOI: 10.1021/bm401736c).
• F. Zomer Volpato, J. Almodóvar, K. Erickson, K.C. Popat, C. Migliaresi, and M.J. Kipper, “Controlled Release of Bioactive FGF-2 from Electrospun Chitosan Fibers Using Heparin-Based Nanoparticles”, Acta Biomaterialia 8, 1551-1559, 2012.
• J. Almodóvar and M.J. Kipper, “Coating electrospun chitosan nanofibers with polyelectrolyte multilayers using the polysaccharides heparin and N,N,N-trimethyl chitosan” Macromolecular Bioscience, 11, 72-76, 2011.
• J. Almodóvar, S. Bacon, J. Gogolski, J.D. Kisiday, M.J. Kipper, “Polysaccharide-based polyelectrolyte multilayer surface coatings can enhance mesenchymal stem cell response to adsorbed growth factors” Biomacromolecules, 11, 2629-2639, 2010.
