Elsevier

Biomaterials

Volume 30, Issue 11, April 2009, Pages 2122-2131
Biomaterials

The influence of the sequential delivery of angiogenic factors from affinity-binding alginate scaffolds on vascularization

https://doi.org/10.1016/j.biomaterials.2008.12.057Get rights and content

Abstract

This study describes the features of tissue-engineering scaffold capable of sequentially delivering three angiogenic factors. The scaffold consists of alginate-sulfate/alginate, wherein vascular endothelial growth factor (VEGF) platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) are bound to alginate-sulfate with an affinity similar to that realized upon their binding to heparin. Factor release rate from the scaffold was correlated with the equilibrium binding constants of the factors to the matrix, thus enabling the sequential delivery of VEGF, PDGF-BB and TGF-β1. In alginate scaffolds lacking alginate-sulfate, release of the adsorbed proteins was instantaneous. After subcutaneous implantation for 1 and 3 months in rats, the blood vessel density and percentage of mature vessels were 3-fold greater in the triple factor-bound scaffolds than in the factor-adsorbed or untreated scaffolds. Moreover, vascularization within the triple factor-bound scaffolds was superior to that found in scaffolds delivering only basic fibroblast growth factor. Application of this novel scaffold may be extended to the combined delivery of additional heparin-binding angiogenic factors or combinations of growth factors active in different tissue regeneration processes.

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.

References (30)

  • E.M. Conway et al.

    Molecular mechanisms of blood vessel growth

    Cardiovasc Res

    (2001)
  • D.C. Darland et al.

    Blood vessel maturation: vascular development comes of age

    J Clin Invest

    (1999)
  • G.D. Yancopoulos et al.

    Vascular-specific growth factors and blood vessel formation

    Nature

    (2000)
  • R.R. Chen et al.

    Integrated approach to designing growth factor delivery systems

    FASEB J

    (2007)
  • R.R. Chen et al.

    Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation

    Pharm Res

    (2007)
  • Cited by (220)

    View all citing articles on Scopus
    View full text