Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels

Abstract

The field of tissue engineering is severely limited by a lack of microvascularization in tissue engineered constructs. Biomimetic poly(ethylene glycol) hydrogels containing covalently immobilized platelet-derived growth factor BB (PDGF-BB) were developed to promote angiogenesis. Poly(ethylene glycol) hydrogels resist protein absorption and subsequent non-specific cell adhesion, thus providing a “blank slate”, which can be modified through the incorporation of cell adhesive ligands and growth factors. PDGF-BB is a key angiogenic protein able to support neovessel stabilization by inducing functional anastomoses and recruiting pericytes. Due to the widespread effects of PDGF in the body and a half-life of only 30 min in circulating blood, immobilization of PDGF-BB may be necessary. In this work bioactive, covalently immobilized PDGF-BB was shown to induce tubulogenesis on two-dimensional modified surfaces, migration in three-dimensional (3D) degradable hydrogels and angiogenesis in a mouse cornea micropocket angiogenesis assay. Covalently immobilized PDGF-BB was also used in combination with covalently immobilized fibroblast growth factor-2, which led to significantly increased endothelial cell migration in 3D degradable hydrogels compared with the presentation of each factor alone. When a co-culture of endothelial cells and mouse pericyte precursor 10T1/2 cells was seeded onto modified surfaces tubule formation was independent of surface modifications with covalently immobilized growth factors. Furthermore, the combination of soluble PDGF-BB and immobilized PDGF-BB induced a more robust vascular response compared with soluble PDGF-BB alone when implanted into an in vivo mouse cornea micropocket angiogenesis assay. Based on these results, we believe bioactive hydrogels can be tailored to improve the formation of functional microvasculature for tissue engineering.

Introduction

While organ transplantation saves thousands of lives each year, in the USA over 100,000 people are currently waiting on the organ transplant list and 16 patients die every day awaiting an organ (United Network for Organ Sharing, www.unos.org). The increasing demand for donated organs to replace damaged or diseased tissues cannot be met by the current supply from cadaveric and living donors. The field of tissue engineering aims to meet this demand by replacing injured and diseased tissues with functional engineered counterparts. However, these efforts are severely limited by a lack of microvascularization within engineered constructs, as diffusion-based transport of nutrients within the timeframe and at the concentrations necessary to support cell survival is limited to a few hundred microns [1].

Platelet-derived growth factor (PDGF) is a molecule important in the establishment of the vasculature that also has roles in the development of the kidneys, lungs and central nervous system. Four isoforms of PDGF are known, PDGF-A–PDGF-D, with PDGF-A and PDGF-B forming homo- and heterodimers. The homodimer PDGF-BB, often involved in angiogenesis, is released in high concentrations by endothelial cells at the sprouting tip of forming capillaries in response to hypoxia, growth factors and shear stress, and serves to mediate pericyte recruitment [2], [3]. Although not involved in initial vessel formation, PDGF-BB is involved in neovessel stabilization and functionalization by inducing anastomoses and recruiting pericytes. Vessel stabilization has been shown to be dependent upon expression of PDGF β-receptors, which are expressed by fibroblasts, endothelial cells and smooth muscle cells [4], [5]. PDGF-BB also stimulates pericyte production of extracellular matrix proteins, including fibronectin, collagen and proteoglycans, necessary for the basement membrane of capillaries. In addition, PDGF-BB increases expression levels of vascular endothelial growth factor (VEGF) in mural cells and stimulates fibroblasts to produce and secrete collagenases, key for cell migration in angiogenesis [6].

Growth factors with widespread effects, such as PDGF-BB, have seen relatively few clinical successes despite documented in vitro efficacy [7]. This is possibly due to a short circulating half-life [8] or the potential for unintended action due to the ubiquity of PDGF-BB targets. Covalent immobilization enables controlled, spatial presentation of potent growth factors to stimulate a desired response and mediate potential drawbacks. Immobilization of biomolecules can be accomplished by attaching them to a polymer, such as poly(ethylene glycol) (PEG), which is then incorporated into a larger scaffold network. The conjugation of growth factors to PEG has been shown to improve solubility, decrease immunogenicity and increase stability [9], [10], while at the same time retaining bioactivity of the original molecule. For example, PEG-conjugated epidermal growth factor (EGF), which does not diffuse or become endocytosed, has been shown to be bioactive and capable of inducing DNA synthesis in a manner comparable with soluble EGF [7]. In another study, PEG–VEGF incorporated into a biodegradable gel not only increased endothelial cell tubulogenesis, but also increased endothelial cell motility 14-fold and cell–cell connections 3-fold [11].

Synthetic polymer matrices facilitate the design of scaffold materials with reproducible and modifiable characteristics, such as drug delivery rates, degradation rates and mechanical properties. Poly(ethylene glycol) diacrylate (PEGDA) is a hydrophilic and biocompatible polymer which can be designed to mimic the mechanical properties of soft tissue [12]. A PEGDA hydrogel acts as a “blank slate” by resisting protein absorption and cell adhesion, enabling precise modification with bioactive ligands and growth factors to induce desired responses, such as cell adhesion, migration and proliferation [13]. In addition, matrix metalloproteinase (MMP)-sensitive sequences can be incorporated into the monomer backbone to enable biodegradation of the synthetic matrix [14]. PEGDA can be photocrosslinked by introducing a photosensitive chemical catalyst into the prepolymer solution, and mild crosslinking conditions permit cellular encapsulation [11]. Upon stimulation with growth factors that induce migration, encapsulated cells will secrete MMPs, which enable degradation of the specifically tailored matrix.

The current studies used bioactive, PEG-based hydrogels modified with covalently immobilized PDGF-BB to promote in vitro tubule formation and stabilization, as well as in vivo angiogenesis. Bioactive, immobilized PDGF-BB was shown to enhance the angiogenic activities of tubule formation on modified surfaces in two-dimensional (2D) and promote cell migration into three-dimensional (3D) degradable hydrogels. Since angiogenesis and vessel stabilization in vivo require precise coordination between multiple growth factors, the combination of PDGF-BB with fibroblast growth factor-2 (FGF-2) was also investigated. PDGF-BB and FGF-2 have previously been found to induce a synergistic vascular response in both the mouse cornea and ischemic hindlimb models [15]. In the current work the combination of covalently immobilized PDGF-BB and FGF-2 showed enhanced cell migration in 3D degradable hydrogels.

Previous research has shown that a co-culture of human umbilical vein endothelial cells (HUVECs) and 10T1/2 pericyte precursor cells formed long-term stable vessels in vivo on a fibronectin–type I collagen matrix [16]. Additionally, 10T1/2 cells, when cultured with HUVECS, displayed a smooth muscle cell morphology and began expressing pericyte markers, such as smooth muscle α-actin, smooth muscle myosin and calponin via the transforming growth factor β1 (TGF-β1) pathway [17]. However, FGF-2 has been shown to act as an antagonist of TGF-β1-induced smooth muscle cell gene expression in 10T1/2 cells [18]. In the current studies a co-culture of cells resulted in tubule formation independent of surface modifications with covalently immobilized growth factors, and a FGF-free medium enhanced tubule formation. Finally, bioactive hydrogels containing the combination of both soluble PDGF-BB to initiate angiogenesis and immobilized PEG–PDGF-BB exhibited a significant increase in vessel density when assessed in the mouse cornea micropocket angiogenesis assay. These studies reaffirm that PEG-based hydrogels can be designed with covalently immobilized growth factors to stimulate a desired cellular response.