Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium.

Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.

Scanning-fiber-based imaging method for tissue engineering.

A scanning-fiber-based method developed for imaging bioengineered tissue constructs such as synthetic carotid arteries is reported. Our approach is based on directly embedding one or more hollow-core silica fibers within the tissue scaffold to function as micro-imaging channels (MIC). The imaging process is carried out by translating and rotating an angle-polished fiber micro-mirror within the MIC to scan excitation light across the tissue scaffold. The locally emitted fluorescent signals are captured using an electron multiplying CCD camera and then mapped into fluorophore distributions according to fiber micro-mirror positions. Using an optical phantom composed of fluorescent microspheres, tissue scaffolds, and porcine skin, we demonstrated single-cell-level imaging resolution (20 to 30 µm) at an imaging depth that exceeds the photon transport mean free path by one order of magnitude. This result suggests that the imaging depth is no longer constrained by photon scattering, but rather by the requirement that the fluorophore signal overcomes the background "noise" generated by processes such as scaffold autofluorescence. Finally, we demonstrated the compatibility of our imaging method with tissue engineering by visualizing endothelial cells labeled with green fluorescent protein through a ≈ 500 µm thick and highly scattering electrospun scaffold.

A fiber-optic-based imaging system for nondestructive assessment of cell-seeded tissue-engineered scaffolds.

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ~500 µm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30 µm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.

Monte Carlo fluorescence microtomography.

Fluorescence microscopy allows real-time monitoring of optical molecular probes for disease characterization, drug development, and tissue regeneration. However, when a biological sample is thicker than 1 mm, intense scattering of light would significantly degrade the spatial resolution of fluorescence microscopy. In this paper, we develop a fluorescence microtomography technique that utilizes the Monte Carlo method to image fluorescence reporters in thick biological samples. This approach is based on an l(0)-regularized tomography model and provides an excellent solution. Our studies on biomimetic tissue scaffolds have demonstrated that the proposed approach is capable of localizing and quantifying the distribution of optical molecular probe accurately and reliably.

Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds.

Electrospun polymer/apatite composite scaffolds are promising candidates as functional bone substitutes because of their ability to allow pre-osteoblast attachment, proliferation, and differentiation. However these structures usually lack an adequate pore size to permit sufficient cell migration and colonization of the scaffold. To overcome this limitation, we developed an apatite-coated electrospun PLLA scaffold with varying pore size and porosity by utilizing a three-step water-soluble PEO fiber inclusion, dissolution, and mineralization process. The temporal and spatial dynamics of cell migration into the scaffolds were quantified to determine the effects of enhanced pore size and porosity on cell infiltration. MC3T3-E1 pre-osteoblast migration into the scaffolds was found to be a function of both initial PEO content and time. Scaffolds with greater initial PEO content (50% and 75% PEO) had drastically accelerated cell infiltration in addition to enhanced cell distribution throughout the scaffold when compared to scaffolds with lower PEO content (0% and 25% PEO). Furthermore, scaffolds with an apatite substrate significantly upregulated MC3T3-E1 alkaline phosphatase activity, osteocalcin content, and cell-mediated mineralization as compared to PLLA alone. These findings suggest that such a scaffold enhances pre-osteoblast infiltration, colonization, and maturation in vitro and may lead to overall improved bone formation when implanted in vivo.