The DIP chamber was developed to provide a means to circulate medium around and through a prevascularized tissue construct. While designing the DIP chamber, we incorporated features to support four objectives: (1) provide flexibility in bioreactor utility, (2) support the shape and integrity of soft tissue constructs, (3) support microvessel viability and neovascular growth, and (4) provide a potential avenue for intravascular perfusion of the neovascular network. The modular character of the DIP chamber provided flexibility in its use by combining any number of stacked modules. In the described configuration, we selected a three-module bioreactor with each module accommodating direct circulation. A module either circulated medium (i.e., the top and bottom modules) or supported an MVC (the central module). Since we use soft, collagen-based constructs, we developed biocompatible internal supports to maintain the size and position of the MVC within the module. The supports were also designed to permit collagen polymerization around a removable mandrel. This mandrel, once removed, formed an artificial lumen within the construct that was contiguous with the inflow and outflow ports of the surrounding module, thereby providing a central conduit for potential intravascular perfusion.
In the DIP chamber, medium circulation through the central lumen substantially impacted the development of a neovascular network via angiogenesis; microvessel density, microvessel distribution throughout the construct, and angiogenic activity were all influenced by the method of chamber circulation. Surprisingly, the E-circulation scheme, which presumably provides submaximal medium delivery, was clearly the most effective at promoting angiogenesis and construct prevascularization. Constructs supported by E-circulated conditions exhibited significantly greater microvessel density and neovessel sprouting. Microvessels in the E+I-circulated DIP chambers maintained lumen structure and viability but exhibited no angiogenic sprouting. The differences between the two circulation protocols suggest that the potential shortcomings in culture conditions, assumed to be present in the E-circulated protocol, are important in inducing angiogenesis from intact vessel elements (or a preexisting microvessel network) in vitro. Conversely, conditions that are perhaps more ideal in supporting tissue health, such as those presumed to be present in the E+I-circulated scheme, may act to stabilize and mature microvessels. In addition to the implications on the relationship between microvessel stability and angiogenesis, these particular findings suggest that it may be possible to control angiogenesis in tissue constructs simply by modulating the level of stress to the system. When angiogenesis is desired, the system would be stressed (by reducing medium flow to the bioreactor in our case), while reestablishing high levels of chamber circulation may act to return the system to a stable situation (i.e., mature the forming neovessels).
The reduced medium delivery in the E-circulated condition could result in limited delivery of a nutrient (i.e., oxygen) or factor important in maintaining microvessel stability and/or accumulation of a destabilizing factor (i.e., angiogenic factor). Even though we did not measure discrete oxygen concentrations in DIP chamber constructs, the increased expression of HIF1α, a protein synthesized in response to hypoxia,19
in microvessels (both parent microvessels and neovessels) in E-circulated chambers strongly argues for a condition of reduced oxygen levels in these cultures. Given the known role of hypoxia in causing microvessel instability and promoting angiogenesis,20,21
it seems very likely that hypoxia is contributing to angiogenesis in the E-circulated DIP chambers. However, we cannot rule out the possibility that an important factor for angiogenesis accumulates in the E-circulated condition but is washed out in the E+I-circulated condition. If oxygen delivery is important, it is not yet clear as to the range of oxygen tensions necessary for promoting angiogenesis in the constructs; clearly, extreme hypoxia (i.e., 1% O2
), as was present in the static MVC cultures, negatively affects microvessel sprouting.
Increased production of HIF1α has also been correlated to increased production of VEGF in endothelial and other cell types.15
Therefore, it seemed likely that the increased expression of HIF1α in the constructs would lead to increased autocrine VEGF production, which would then lead to angiogenic sprouting. However, blockage of VEGF signaling via two different inhibitors did not cause a profound decrease in angiogenesis in MVCs. Therefore, it is unlikely that VEGF is responsible for the angiogenic sprouting observed in the E-circulated cultures. Whether or not VEGF is influencing neovessel phenotypes in a way other than neovessel growth (e.g., changing diameters) is unclear. HIF1α is known to regulate the expression of a number of factors relevant to angiogenesis,14
which could very possibly be an important stimulus for neovessel sprouting and growth in our microvessel system.
We have not yet systematically examined if any of the neovessels in the constructs cultured in the DIP chamber, particularly in the E-circulated condition, are perfusion competent. Ultimately, we intend to assemble a new microcirculation within the DIP chambers capable of transporting, intravascularly, a circulating medium. From our data, it would appear that the parent MVFs maintained a patent lumen (in both the E- and E+I-circulated conditions), whereas the neovessels that sprouted from the MVFs in the E-circulated conditions did not. However, the absence of a clear lumen in the new sprouts and neovessels may reflect the absence of hemodynamic conditioning thought important in postangiogenesis maturation and neovessel stability.7,8
Therefore, it may be necessary to initiate intravascular perfusion, perhaps via a strategy involving the central circulation port and a potential connection to the neovascular network, to cause the neovascular network that does form in the DIP chamber to progress toward a mature microvessel network complete with contiguous lumen. However, such a strategy would have to take into account the antiangiogenic conditions created by perfusing the chamber through the central port (i.e., E+I-circulated protocol). However, if sprouting angiogenesis in the DIP chamber constructs reflects a specific oxygen tension, then perhaps it is possible, by culturing under defined partial pressures of oxygen in the incubator, to manipulate angiogenesis in constructs, not by altering medium delivery but by changing starting oxygen levels. With such modifications, it may be possible to form, in vitro
, functional microvascular networks.