Elsevier

Journal of Controlled Release

Volume 184, 28 June 2014, Pages 58-66
Journal of Controlled Release

Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture

https://doi.org/10.1016/j.jconrel.2014.04.007Get rights and content

Abstract

Timely vascularization is essential for optimal performance of bone regenerative constructs. Vascularization is efficiently stimulated by vascular endothelial growth factor (VEGF), a substance with a short half-life time. This study investigates the controlled release of VEGF from gelatin microparticles (GMPs) as a means to prolong VEGF activity at the preferred location within 3D bioprinted scaffolds, and the effects on subsequent vascularization. The release of VEGF from GMPs was continuous for 3 weeks during in vitro studies, and bioactivity was confirmed using human endothelial progenitor cells (EPCs) in migration assays. Traditional and real-time migration assays showed immediate and efficient EPC migration in the presence of GMP-released VEGF, indistinguishable from VEGF-solution that was added to the medium. Matrigel scaffolds containing EPCs and VEGF, which was released either in a fast or sustained fashion by application of GMPs, were investigated for their in vivo vasculogenic capacity. Implantation in subcutaneous pockets in nude mice for one week demonstrated that vessel formation was significantly higher in the VEGF sustained-release group compared to the fast release group. In addition, regional differences with respect to VEGF release were introduced in 3D bioprinted EPC-laden scaffolds and their influence on vasculogenesis was investigated in vivo. The different regions were retained and vessel formation occurred analogous with the results seen in the Matrigel plugs.

We conclude that GMPs are suitable to generate sustained release profiles of bioactive VEGF, and that they can be used to create defined differentiation regions in 3D bioprinted heterogeneous constructs, allowing a new generation of smart scaffold design. The prolonged presence of VEGF led to a significant increase in scaffold vascularization when applied in vivo.

Introduction

Vascularization of tissue-engineered constructs is an ongoing challenge in regenerative medicine. Without sufficient blood supply, oxygen and metabolic needs are not met, which can lead to central necrosis of constructs. This limits scaffold size to the diffusion distance nutrients can travel in the used material [1], [2]. A possible solution to this problem could be to introduce a prevascularization step to stimulate the process of efficient vascularization once the construct is implanted. Grafts with new blood vessels that can connect to the host vasculature could enhance the quantity and quality of newly formed tissues. Endothelial progenitor cells (EPCs) that are present in the circulation play an important role in this process as they can differentiate into endothelial cells that line blood vessels [3]. Late outgrowth EPCs (sometimes referred to as endothelial colony forming cells or ECFCs in literature [4], [5]) have a high capacity for proliferation and vessel formation in vitro and in vivo [6], [7]. Vessel formation properties of the EPCs have been studied extensively in established 2D tubulogenesis and 3D vasculogenesis assays, and indicate that they are suitable to prevascularize tissue engineered constructs. A potent angiogenic and vasculogenic growth factor that is often applied in regenerative medicine is vascular endothelial growth factor (VEGF), a heparin-binding, homodimeric glycoprotein of 45 kDa of which VEGF165 is the predominant isoform [8]. It is a key regulator of physiological vessel formation during embryogenesis, mainly by preventing apoptosis of endothelial cells [9]. VEGF is degraded rapidly in the bloodstream with a half-life time of less than 1 h following injection [10], [11]. Controlled release of VEGF to accomplish longer growth factor presence at target locations leads to increased vessel formation in scaffolds [12], [13]. As a suitable system for growth factor release gelatin or gelatin microparticles (GMPs) are often applied. Gelatin is a natural product that is used in many FDA-approved devices. Growth factor encapsulation is based on electrostatic interactions with the gelatin as well as the gelatin degradation rate [14], [15], [16], [17]. The main advantages of the GMPs are the diffusional loading of growth factors and the non-covalent nature of the interaction between gelatin and growth factor, thus avoiding chemical reactions that could damage the protein. Furthermore, GMPs are non-cytotoxic, biodegradable and they have previously been used to deliver other growth factors such as BMP-2, TGFβ1 and FGF [18], [19]. Intracardial injection of GMPs loaded with VEGF has led to increased neoangiogenesis in a rat myocardial infarct model [20], [21]. GMPs can be incorporated into hydrogel plugs or 3D bioprinted constructs [22]. This technique enables production of a new generation of scaffolds with defined architecture and the opportunity to include predefined regions of prevascularization in the scaffold. Furthermore, with 3D bioprinting, scaffold porosity can be introduced, which appears to improve the in vivo performance by lowering the diffusion distance for oxygen and nutrients [23].

Based on these considerations the present study aimed to combine controlled VEGF release with 3D bioprinting technology to enable production of novel hydrogel scaffolds with properties that can be tuned both in time and space to optimize vessel formation. Suitability of GMPs for VEGF delivery was first assessed with in vitro release studies and real-time EPC migration studies. Subsequently, in vivo studies were carried out to investigate whether prolonged VEGF presence in scaffolds would improve the degree of vascularization. The suitability of GMP mediated controlled VEGF release in these novel architectural constructs was studied using the 3D bioprinting technology.

Section snippets

EPC isolation and culture

Human umbilical cord blood was collected from full term pregnancies, using a protocol approved by the local ethics committee (01/230 K, Medisch Ethische Toetsings Commissie (METC), University Medical Center Utrecht). Mononuclear cells (MNCs) were isolated by density-gradient centrifugation using Ficoll-paque (density 1.077 g/ml). MNCs were subsequently resuspended in Endothelial Growth Medium-2 (EGM-2) containing 10% fetal calf serum (FCS) and EGM-2 SingleQuots (Lonza, Walkersville, MD, USA) and

Characterization of EPCs from umbilical cord blood (cb-EPCs)

EPCs were isolated from human umbilical cord blood, expanded and characterized as shown in Fig. 1. Flow cytometric analysis shows that progenitor cell markers CD34, CD105, CD133, and endothelial cell markers CD31, CD144 (VE-cadherin), CD90 and KDR (VEGF-R2) were present and leukocyte marker CD45 and macrophage/monocyte marker CD14 were absent in the cell population. Cells were positive for von Willebrand Factor (vWF), and showed characteristic membrane-bound staining for CD31 and VE-cadherin on

Discussion

Slow release of VEGF from GMPs led to a significant increase in the number of perfused vessels in a human EPC containing Matrigel plug compared to fast VEGF release or control group in a vascular ingrowth model. When the VEGF-laden GMPs were regionally applied in 3D bioprinted scaffolds with defined architecture this effect was seen locally, more vessels were present in GMP-containing regions.

In this study, umbilical cord EPCs were used to assess the effect of prolonged VEGF presence. From

Conclusions

GMPs are suitable to generate sustained release profiles of bioactive VEGF, and are effectively used to generate defined differentiation regions in 3D bioprinted heterogeneous constructs. The prolonged presence of VEGF led to a significant increase in scaffold vascularization when applied in vivo.

Acknowledgments

This research forms part of the Project P2.04 BONE-IP of the research program of the BioMedical Materials Institute, co-founded by the Dutch Ministry of Economic Affairs.

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