Bioengineered vascular grafts provide a promising alternative to autografts for replacing diseased or damaged arteries, but necessitate scaffold designs capable of supporting a confluent endothelium that resists endothelial cell (EC) detachment under fluid flow. To this end, we investigated whether tuning electrospun topography (i.e. fiber diameter and orientation) could impact EC morphology, alignment, and structural protein organization with the goal of forming a confluent and well-adhered endothelium under fluid flow. To test this, a composite polymer blend of Poly(ε-caprolactone) (PCL) and type I collagen was electrospun to form scaffolds with controlled fiber diameters ranging from approximately 100 nm to 1200 nm and with varying degrees of fiber alignment. ECs were seeded onto scaffolds, and cell morphology and degree of alignment were quantified using image analysis of fluorescently stained cells. Our results show that ECs form confluent monolayers on electrospun scaffolds, with cell alignment systematically increasing with a larger degree of fiber orientation. Additionally, cells on aligned electrospun scaffolds display thick F-actin bundles parallel to the direction of fiber alignment and strong VE-cadherin expression at cell-cell junctions. Under fluid flow, ECs on highly aligned scaffolds had greater resistance to detachment compared to cells cultured on randomly oriented and semi-aligned scaffolds. These results indicate that scaffolds with aligned topographies may be useful in forming a confluent endothelium with enhanced EC adhesion for vascular tissue engineering applications.

No abstract

(Abstract)Biodegradable polyesters, such as poly(DL-lactic-co-glycolic acid) (PLGA), have been used to fabricate porous bone scaffolds to support bone tissue development. These scaffolds allow for cell seeding, attachment, growth and extracellular matrix production in vitro and are replaced by new bone tissue when implanted into bone sites in vivo.Hydroxyapatite (HAP) and β-tricalcium phosphate (β-TCP) ceramics have been incorporated into PLGA bone scaffolds and have been shown to increase their osteoconductivity (support cell attachment). Although HAP, β-TCP, and biodegradable polyesters are osteoconductive, there is no evidence that these scaffold materials are osteoinductive (support cell differentiation). Calcium and phosphate ions, in contrast, have been postulated to be osteogenic factors that enhance osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized which permits controlled release of calcium and phosphate ions and thus is hypothesized to be osteoinductive. Incorporation of Zr-ACP into a highly porous poly(DL lactic-co-glycolic acid) (PLGA) scaffold could potentially increase the osteoinductivity of the scaffold and therefore promote osteogenesis when implanted in vivo.To determine the osteoinductivity of Zr-ACP, a MC3T3-E1 mouse calvarialderived osteoprogenitor cell line was used to measure cell response to Zr-ACP. To accomplish this objective, Zr-ACP was added to cell culture at different stages in cell maturation (days 0, 4 and 11). DNA synthesis, alkaline phosphatase (ALP) activity, iii osteopontin synthesis and collagen synthesis were determined. Results indicate that culture in the presence of Zr-ACP significantly increased cell proliferation, ALP activity and osteopontin synthesis but not collagen synthesis. To determine the feasibility of incorporating Zr-ACP into a PLGA scaffold, PLGA/Zr-ACP composite foams (5% or 10% (w/v) polymer:solvent with 25 wt% or 50 wt% Zr-ACP) were fabricated using a thermal phase inversion technique. Scanning electron microscopy revealed a highly porous structure with pores ranging in size from a few microns to about 100 µm. The amorphous structure of the Zr-ACP was maintained during composite fabrication as confirmed by X-ray diffraction measurements. Composite scaffolds also showed significantly greater compressive yield strengths and moduli as compared to pure polymer scaffolds.The results of this study indicate that Zr-ACP enhances the osteoblastic phenotype of MC3T3-E1 cells in vitro and can be incorporated into a porous PLGA scaffold. Porous PLGA/Zr-ACP composites are promising for use as bone scaffolds to heal bone defects.iv

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 mm thick scaffold with celllevel 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 mm, 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.

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.