Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (∼200 mg ml⁻¹). Young's modulus (E) measurements from acellular collagen gels (range 0.5-12 kPa) exhibited a power-law concentration dependence (range 3-9 mg ml⁻¹) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5-27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ∼5-200 mg ml⁻¹). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R²=0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R²=0.83, exponents of -0.7 and -1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.

This paper reports a method for generating an intact and perfusable microvascular network that connects to microfluidic channels without appreciable leakage. This platform incorporates different stages of vascular development including vasculogenesis, endothelial cell (EC) lining, sprouting angiogenesis, and anastomosis in sequential order. After formation of a capillary network inside the tissue chamber via vasculogenesis, the adjacent microfluidic channels are lined with a monolayer of ECs, which then serve as the high-pressure input ("artery") and low pressure output ("vein") conduits. To promote a tight interconnection between the artery/vein and the capillary network, sprouting angiogenesis is induced, which promotes anastomosis of the vasculature inside the tissue chamber with the EC lining along the microfluidic channels. Flow of fluorescent microparticles confirms the perfusability of the lumenized microvascular network, and minimal leakage of 70 kDa FITC-dextran confirms physiologic tightness of the EC junctions and completeness of the interconnections between artery/vein and the capillary network. This versatile device design and its robust construction methodology establish a physiological transport model of interconnected perfused vessels from artery to vascularized tissue to vein. The system has utility in a wide range of organ-on-a-chip applications as it enables the physiological vascular interconnection of multiple on-chip tissue constructs that can serve as disease models for drug screening.

The functionality of vascular networks within implanted prevascularized tissues is difficult to assess using traditional analysis techniques, such as histology. This is largely due to the inability to visualize hemodynamics in vivo longitudinally. Therefore, we have developed dynamic imaging methods to measure blood flow and hemoglobin oxygen saturation in implanted prevascularized tissues noninvasively and longitudinally. Using laser speckle imaging, multispectral imaging, and intravital microscopy, we demonstrate that fibrin-based tissue implants anastomose with the host (severe combined immunodeficient mice) in as short as 20h. Anastomosis results in initial perfusion with highly oxygenated blood, and an increase in average hemoglobin oxygenation of 53%. However, shear rates in the preformed vessels were low (20.8±12.8s-1), and flow did not persist in the vast majority of preformed vessels due to thrombus formation. These findings suggest that designing an appropriate vascular network structure in prevascularized tissues to maintain shear rates above the threshold for thrombosis may be necessary to maintain flow following implantation. We conclude that wide-field and microscopic functional imaging can dynamically assess blood flow and oxygenation in vivo in prevascularized tissues, and can be used to rapidly evaluate and improve prevascularization strategies. © Copyright 2012, Mary Ann Liebert, Inc.