Objective-We have previously demonstrated the ability to construct 3-dimensional microvascular beds in vitro via angiogenesis from isolated, intact, microvessel fragments that retain endothelial cells and perivascular cells. Our objective was to develop and characterize an experimental model of tissue vascularization, based on the implantation of this microvascular construct, which recapitulated angiogenesis, vessel differentiation, and network maturation. Methods and Results-On implantation in a severe combined-immunodeficient mouse model, vessels in the microvascular constructs rapidly inosculated with the recipient host circulation. Ink perfusion of implants via the left ventricle of the host demonstrated that vessel inosculation begins within the first day after implantation. Evaluation of explanted constructs over the course of 28 days revealed the presence of a mature functional microvascular bed. Using a probe specific for the original microvessel source, 91.7%Ϯ11% and 88.6%Ϯ19% of the vessels by day 5 and day 28 after implantation, respectively, were derived from the original microvessel isolate. Similar results were obtained when human-derived microvessels were used to build the microvascular construct. Key Words: vascularization Ⅲ microcirculation Ⅲ angiogenesis Ⅲ human Ⅲ vascular remodeling V ascularization is the process by which perfusion pathway length and vessel segment number are increased and organized into a functional vascular bed. In normal situations, this effective increase in vessel density delivers more blood to the tissue, facilitating tissue growth and/or increased tissue activity. 1,2 Consequently, vascularization is a primary component of tissue growth and repair, such as occurs during development, 3 after an upstream occlusive event leading to tissue ischemia, 4 or during proliferative events, as seen in tissue healing 5,6 and tumor growth. 7 Although we know of many factors and signals that initiate or terminate the vascularization process, little is known about the rules that govern vascularization as an integrated process that includes angiogenesis, 3 arteriogenesis, 8 vascular remodeling, 9 vessel adaptation, 10 and arterio-venous polarization. 11 We have previously shown that isolated intact microvessel fragments retain angiogenic potential and are capable of forming a simple microvascular bed when cultured in a 3-dimensional collagen I gel. 12 In this microvascular construct, the vessel fragments undergo stereotypical angiogenesis, forming neovessels that maintain patent lumen and perivascular cell associations. Furthermore, the vessel fragments within this culture system are responsive to proangiogenic conditions. 12,13 All of this occurs in the absence of blood flow and relatively few nonvascular cells. Conclusions-WithHere we report the development and characterization of an experimental model of tissue vascularization based on the implantation of this microvascular construct. Precultured or freshly formed microvascular constructs implanted subcutaneously inosculate with the...
The primary emphasis of tissue engineering is the design and fabrication of constructs for the replacement of nonfunctional tissue. Because tissue represents a highly organized interplay of cells and extracellular matrix, the fabrication of replacement tissue should mimic this spatial organization. This report details studies evaluating the use of a three-dimensional, direct-write cell deposition system to construct spatially organized viable structures. A direct-write bioassembly system was designed and fabricated to permit layer-by-layer placement of cells and extracellular matrix on a variety of material substrates. Human fibroblasts suspended in polyoxyethylene/polyoxypropylene were coextruded through a positive displacement pen delivery onto a polystyrene slide. After deposition, approximately 60% of the fibroblasts remained viable. Bovine aortic endothelial cells (BAECs) suspended in soluble collagen type I were coextruded via microdispense pen delivery onto the hydrophilic side of flat sheets of polyethylene terephthalate. After deposition with a 25-gauge tip, approximately 86% of the BAECs were viable. When maintained in culture for up to 35 days, the constructs remained viable and maintained their original spatial organization. These results indicate the potential for utilizing a direct-write, three-dimensional bioassembly tool to create viable, patterned tissue-engineered constructs.
Tissue engineering combines the fields of medicine and engineering to build replacement tissue capable of restoring, maintaining, or improving damaged tissue. Researchers have recently developed techniques to fabricate tissue in which both the cells and matrix have a carefully defined architecture. This report details studies evaluating the use of a direct-write, 3-dimensional (3D) bioassembly tool (BAT) capable of extruding cells and matrix into spatially organized, 3D constructs. This system has been characterized by its ability to fabricate viable 2-dimensional and 3D constructs containing up to 2 separate cellular solutions suspended in type I collagen. The effects of various environmental factors, such as extrusion pressure, humidity, and stage heating, were examined with respect to the viability of the extruded cells. The data indicate that the system parameters required to extrude cells suspended in collagen do not adversely affect the viability of those cells. Maintaining a high humidity, especially when stage heat was applied, is critical in maintaining the viability of the printed cells. These results demonstrate that the BAT is capable of spatially organizing separate cellular solutions into a defined architecture; however, when cells were extruded in a supporting matrix of 3.0 mg/mL type I collagen, it was not possible to consistently generate adjacent, touching, but nonoverlapping lines of separate solutions. Thus, when a fabrication system such as BAT is used to generate complex, 3D viable constructs, the supporting matrix for the cells should be carefully chosen on the basis of such characteristics as its rate of polymerization and stiffness.
Optical coherence tomography (OCT) is an imaging modality capable of acquiring cross-sectional images of tissue using back-reflected light. Conventional OCT images have a resolution of 10-15 microm, and are thus best suited for visualizing tissue layers and structures. OCT images of collagen (with and without endothelial cells) have no resolvable features and may appear to simply show an exponential decrease in intensity with depth. However, examination of these images reveals that they display a characteristic repetitive structure due to speckle. The purpose of this study is to evaluate the application of statistical and spectral texture analysis techniques for differentiating living and non-living tissue phantoms containing various sizes and distributions of scatterers based on speckle content in OCT images. Statistically significant differences between texture parameters and excellent classification rates were obtained when comparing various endothelial cell concentrations ranging from 0 cells/ml to 25 million cells/ml. Statistically significant results and excellent classification rates were also obtained using various sizes of microspheres with concentrations ranging from 0 microspheres/ml to 500 million microspheres/ml. This study has shown that texture analysis of OCT images may be capable of differentiating tissue phantoms containing various sizes and distributions of scatterers.
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