The influence of the sequential delivery of angiogenic factors from affinity-binding alginate scaffolds on vascularization
Introduction
The assembly and maintenance of organized tissue patterns are tightly regulated by the spatial and temporal presentation of multiple growth factors and cytokines. In vasculogenesis and angiogenesis, vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) serve as key biosignalling molecules for inducing stable and functional networks of blood vessels [1], [2], [3], [4]. These factors are attached to the extracellular matrix via specific interactions with heparan sulfate and act in spatio-temporal gradients to regulate vessel density, size and distribution [5].
The complex mechanism of angiogenesis implies that both strategies designed to induce new vasculature in tissues affected by ischemic diseases as well as tissue engineering applications, may benefit from controlled delivery of combinations of growth factors. Current approaches for growth factor delivery employ different variations of controlled release systems [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Most systems are designed for the delivery of one [6], [7], [9], [12], [14], [15], [18] or two factors from a single system [8], [10], [11], [13], [16], [17]. Such factors are either physically entrapped in polymeric matrices [7], [8], [9], [14], [17] or bound to matrices integrated with heparin [16], [18] and heparan sulfate [15].
Here, we present a simple and controllable strategy for attaining sequential delivery of three common angiogenic factors, namely VEGF, PDGF-BB and TGF-β1, from a single tissue-engineering scaffold. We hypothesized that the incorporation of alginate-sulfate, which is capable of binding multiple heparin-binding proteins [19] into such alginate scaffolds would result in a system able to sequentially release the proteins at a rate reflected by their equilibrium binding constants. The concept of this system was tested in vitro, and after implantation into rat, the extent of scaffold vascularization and vessel maturation was determined and compared to results obtained in systems wherein the triple factors were delivered following adsorption to the alginate matrix. In addition, we tested the efficacy of our triple factor system as relative to the delivery of basic fibroblast growth factor (bFGF), either bound or adsorbed to the scaffold.
Section snippets
Materials and animals
Sodium alginate (LVG, >65% guluronic acid content) came from NovaMatrix, FMC Biopolymers (Drammen, Norway). Alginate-sulfate (7.9 ± 0.3 wt%. sulfur by Sheniger method) was synthesized as previously described [19]. Recombinant human bFGF, PDGF-BB, VEGF and TGF-β1, as well as ELISA Development Kits for bFGF (900-K08), VEGF (900-K10) and PDGF-BB (900-K04) were purchased from CytoLab (Rehovot, Israel). A human TGF-β1 DuoSet ELISA Development System came from R&D (Minneapolis, MN). A streptavidin (SA)
Factor binding to alginate-sulfate
We have recently shown that heparin-binding proteins can specifically interact with alginate-sulfate, with equilibrium binding constants closely mimicking those observed upon interaction with heparin [19]. Among these proteins were the angiogenic factors, bFGF, VEGF and PDGF-BB (Table 2). Here, we estimated the equilibrium binding of TGF-β1 to alginate-sulfate via SPR analysis, and studying this interaction over a range of protein concentrations (80–300 nm) (Fig. 1). TGF-β1 was found to be
Discussion
The formation of a stable and functional network of blood vessels requires the participation of multiple angiogenic factors, presented in the proper spatio-temporal manner. In the present study, we provide evidence that the sequential delivery of three angiogenic factors from an alginate-sulfate/alginate scaffold resulted in the formation of higher density vasculature with more mature blood vessels, as compared to when the three factors were adsorbed to the matrix and released instantaneously.
Conclusions
This paper provides evidence that sequential delivery of three angiogenic factors (VEGF, PDGF-BB and TGF-β1) from macroporous scaffold could be achieved via their differential affinity binding to alginate-sulfate, leading to the formation of stable and mature blood vessels within the scaffold after implantation in rat. Application of this system may be extended to include additional heparin-binding factors that play important roles in angiogenesis and/or that act in different tissue
Acknowledgements
We thank Ms. Parvin Zerin for histological studies and Dr. Ervin Elias for his assistance with animal surgery. The research was supported by a grant from the Israel Science Foundation (793/04). Smadar Cohen holds the Claire & Harold Oshry Professor Chair in Biotechnology.
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