Microfluidics and biomaterials to study angiogenesis
Introduction
Angiogenesis is the growth of new capillaries from extant blood vessel [1]. This vessel formation is a key part of developmental morphogenesis, response to injury, and pathogenesis [1]. In angiogenesis, endothelial cells (ECs) follow a stepwise migration by first degrading the basement membranes, following directional proangiogenic cues such notch signaling and vascular endothelial growth factor (VEGF) [2] that lead EC's to migrate, proliferate, form tubes, and finally create mature vessels. This processes is induced by neighboring cells types, which can vary from tumor cells to even fibroblasts or macrophages [3]. Angiogenesis is typically up regulated when the tissue becomes hypoxic or inflamed, which commonly occurs in wound and tumor micro-environments [4, 5, 6]. Low oxygen, hypoxia, sensing is a critical effector of angiogenesis which leads to the up regulation of many critical molecular mediators such as hypoxia-inducible factor (HIFs), up regulating factors like VEGF and PDGF-B, and Ang-1 [7, 8, 9]. Angiogenesis is commonly seen during cancer development. Tumor vessels however grow in an uncontrolled mode without the regulation of anti-angiogenic factors, leading to a disorganized tortuous structures [10]. As a result, tumor vasculature varies in diameter and have an increased permeability due to poorly invested perivascular cells and discontinuous ECs [11, 12]. From a functionally perspective, these vessels deliver small amounts of blood, which consequentially results in limited oxygen and nutrients delivered to the tumor. Another form of vessel development that is studied using these devices is vasculogenesis. Vasculargenesis is the formation of new vasculature from non-existing ones. Vasculogenesis, prevalent during embryonic development, gives rise to the heart and first primitive vascular plexus [13].
When vessel formation is not controlled it can lead to a wide variety of diseases such as atherosclerosis, or chronic wounds [14, 15, 16]. The average cost to develop a new therapeutic was reported by Mullard to cost $2.6 billion [17]. In order minimize these costs, researchers have been developing ‘lab on a chip’ devices that allow for better screening of potential therapeutics. Lab-on-chip (LoC) technologies are microscale devices developed originally by the semiconductor industry and expanded to include micro-eletromechanical devices and microfluidics [18]. The benefit of LoC devices is that they are commonly used to streamline complex protocols, have an increased sensitivity, reduce the amount of materials necessary to perform an assay, as well as provide finite control over the microenvironment [19]. The ability to control the surface characteristics, the flow profile, as well as concentration gradients have been used to create in vivo mimicking LoC systems [18]. These systems are utilized as a physiologically relevant cell culture platform, acting as a bridge between in vitro and in vivo systems as well as reducing the use of animals for research.
In order to study in vivo angiogenesis, three animal models have been developed to study the microcirculatory environments including chick, zebrafish, or rodent embryos. [20]. Folkman and colleagues created one of the first in vitro assays for angiogenesis by culturing endothelial cells (ECs) on a Petri dish in tumor conditioned media, so that they formed capillary tubes [21]. However, since then researchers have developed a number of two-dimensional (2D) and three-dimensional (3D) culture systems to explore angiogenesis in regards to wound healing and cancer. In order to increase the physiological relevance of these assays, there is a need to incorporate chemical gradients, shear stress, and a relevant extracellular matrix (ECM) that regulate angiogenesis. In order to accomplish this, recent studies combine hydrogels and microfluidics in order to create physiologically relevant systems.
Section snippets
Microfluidics
Microfluidics are miniaturized devices made of a variety of substances from polydimethylsiloxane (PDMS) to hydrogels. These systems use minimal reagents volumes, can create highly specific assays, and allow for the precise control liquids [22]. These devices have cross-sectional geometries ranging from 10 to 100 μm [23], and exhibit low Reynolds number flow. Through advancements in microfabrication techniques, microfluidics has emerged as a prominent platform to explore cellular responses,
Hydrogel biomaterials
Hydrogels are a new class biomaterial that use a hydrophilic polymer network. These networks may absorb 10–20% up to thousands times their weight in water. These gels can be properly functionalized so that they can degrade over time or are chemically stable [39]. One of the great advantages is that hydrogels can be synthesized from natural or synthetic materials. These materials each have their own advantages and disadvantages. Natural materials used for hydrogels are commonly made from
Combining microfluidics and hydrogels to model angiogenesis
Traditional in vitro angiogenesis assays exhibit some of the key physiological properties, however, they don’t fully explore the complex microenvironments necessary to study angiogenesis. The ability to combine microfluidics and hydrogels may lead to the advance control of chemical gradients, through physiologically relevant biomaterials and finite control over shear stress profiles. Figure 2 details several approaches that are being applied to generate such devices.
In these systems we consider
Conclusion and future directions
The studies described here demonstrate the opportunities of creating LoC that mimic the complex 3D angiogenic microenvironment and thus enabling the study of the regulatory mechanisms governing angiogenesis. These devices have been used to study regenerative angiogenesis as well as the pathology as it relates to tumor development and migration. It should be noted that the end goal of all of the fabrication techniques described here is to create a tubular structure inside a hydrogel. Each
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
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• of special interest
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•• of outstanding interest
Acknowledgements
This work was support by fellowship from the National Cancer Institute, T-32, Nanotechnology Cancer Research training grant (to DM.L.), American Heart Association Established Investigator Award and the President's Frontier Award from Johns Hopkins University (both to S.G.).
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