Genipin cross-linked engineered tissues are 10000 times less toxic than glutaraldehyde cross-linked tissues. Hence, genipin is a better fixative to support the recellularization of tissue-engineered constructs such as vascular grafts. Here, we demonstrate the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of genipin cross-linked vascular grafts with high speed and specificity.
Current results indicate that the fluorescence lifetime of AR-BP shortens upon GE cross-linking. Raman spectroscopy reveals secondary structural changes occurring in the extracellular matrix of pericardia that correspond to Amide I, Amide III and C-C stretch vibrations.
We conclude that FLIm guided Raman imaging can detect cross-linking signatures with biochemical specificity and that this imaging modality provides a non-destructive and label-free method to assess the quality of vascular grafts
Tissue engineered vascular graft (TEVG) are used when native vessels are not available to repair vascular damage. At the time of implantation in human body, these constructs present poor cellularity. To understand the cellularization kinetics under physiological conditions in a setting suitable for experimentation, bioreactors are often used in laboratory setting because of its controllable culture parameters including seeding conditions, flow type, pressure and temperature. Therefore, a non-destructive, label-free imaging modality that is capable of evaluating cell migration on luminal surfaces of TEVGs inside bioreactors is valuable for studying cellularization kinetics and providing a potential quality control method for manufacturing mature TEVGs. A multispectral Fluorescence Lifetime Imaging (ms-FLIm) using 355 nm excitation was configured to accommodate a rotating side-firing scanning probe for intraluminal imaging of tubular-shaped bovine pericardium (BP) scaffolds. The scanning was realized by reciprocal rotation and pullback of the fiber probe. Mesenchymal stem cells were seeded on BP-based TEVGs and cultured in the prototype bioreactor for up to one week. Distinct experimental conditions including the seeding side (i.e. BP serious and fibrous side) and media flow (i.e. static and dynamic pulsatile flow) were evaluated. Using ms-FLIm, the migration of cells on antigen removed BP TEVGs was periodically examined over a week; and the migration rates under different conditions were analyzed. Current results suggest helical ms-FLIm has potential to monitor in situ tissue recellularization process in bioreactors.
A fiber-based, label-free multispectral fluorescence lifetime imaging and intravascular ultrasound (FLIm/IVUS) system was evaluated as a new tool for monitoring variations in biochemical and structural composition of vascular biomaterials, including native arteries and engineered vascular grafts both in vitro and in vivo. Fiber-based FLIm was adapted to assess the hollow geometry of vasculature, allowing for imaging of the luminal surface of vessels. The capacity of FLIm to resolve tissue cellular location (i.e. scaffold reendothelialization) and collagen to elastin ratio on the vessel wall was investigated. Quantitative imaging parameters derived from spectrally- and temporally-resolved autofluorescence (i.e. intensity ratios and fluorescence lifetime) provide benchmark indicators to identify areas of recellularized tissue, and to distinguish wall matrix compositions within and across biomaterials. In addition, fiber-based FLIm was complemented with intravascular ultrasound (IVUS) for simultaneous in vivo evaluation of biochemical and structural tissue properties. Here, we performed an in vitro evaluation of pig carotid arteries and show correlations between FLIm parameters and biochemical composition in different anatomical locations. We discuss the spectral and lifetime differences between native pig carotid artery, acellular antigen removed bovine pericardium grafts, and reendotheliarized grafts. Finally, we translate the findings to an in vivo clinical FLIm/IVUS imaging study with antigen removed bovine pericardium grafted on healthy pig native carotid artery. Upon implantation, the graft is expected to repopulate with cells, and change composition as cells remodel it. These experiments demonstrate the feasibility of fiber-based FLIm/IVUS to examine vascular engineered tissue in research and clinical settings.
Techniques that dynamically assess the maturation of tissue engineered constructs allow more efficient longitudinal control of developmental parameters than traditional destructive analyses, enhancing the likelihood of successful outcomes. We present a non-destructive and minimally invasive imaging method to monitor the growth of engineered vascular tissue based on label-free fluorescence lifetime imaging (FLIm) using a single fiber optic interface. We demonstrate the potential of the fiber-based FLIm system on vascular grafts composed of antigen removed bovine pericardium extracellular scaffolds seeded with human endothelial or mesenchymal stem cells. Tissue constructs are illuminated with 355 nm pulsed laser light that excites tissue autofluorescence, stemming from scaffold proteins (e.g., collagen), and cellular metabolic co-factors (i.e., NADH and FAD). Fluorescence lifetime images are acquired by scanning the distal tip of a multimode fiber across the sample surface, to deliver fluorescence excitation and collect fluorescence emission. A wavelength selection module is used to spectrally separate autofluorescence into four spectral bands that were selected to match the emission peaks of the main tissue fluorophores. By examining the relative intensity and mean fluorescence lifetime in each spectral band we identify the composition of engineered tissues, and evaluate the progression of recellularization. The fiber-based apparatus is compatible with imaging a range of sample geometries including planar and tubular constructs, and imaging in regions of restricted space such as inside tissue bioreactors, or in vivo. Future applications for the system include longitudinal monitoring of the luminal surface of engineered vascular tissues, or intravascular imaging in vivo to monitor viability of vascular implants.
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