The influence of electrospun scaffold topography on endothelial cell morphology, alignment, and adhesion in response to fluid flow.

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 100-1,200 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.

3D viability imaging of tumor phantoms treated with single-walled carbon nanohorns and photothermal therapy.

A new image analysis method called the spatial phantom evaluation of cellular thermal response in layers (SPECTRL) is presented for assessing spatial viability response to nanoparticle enhanced photothermal therapy in tissue representative phantoms. Sodium alginate phantoms seeded with MDA-MB-231 breast cancer cells and single-walled nanohorns were laser irradiated with an ytterbium fiber laser at a wavelength of 1064 nm and irradiance of 3.8 W cm(-2) for 10-80 s. SPECTRL quantitatively assessed and correlated 3D viability with spatiotemporal temperature. Based on this analysis, kill and transition zones increased from 3.7 mm(3) and 13 mm(3) respectively to 44.5 mm(3) and 44.3 mm(3) as duration was increased from 10 to 80 s. SPECTRL provides a quantitative tool for measuring precise spatial treatment regions, providing information necessary to tailor therapy protocols.

Imaging and characterization of bioengineered blood vessels within a bioreactor using free-space and catheter-based OCT.

BACKGROUND AND OBJECTIVE: Regenerative medicine involves the bioengineering of a functional tissue or organ by seeding living cells on a biodegradable scaffold cultured in a bioreactor. A major barrier to creating functional tissues, however, has been the inability to monitor the dynamic and complex process of scaffold maturation in real time, making control and optimization extremely difficult. Current methods to assess maturation of bioengineered constructs, such as histology or organ bath physiology, are sample-destructive. Optical coherence tomography (OCT) has recently emerged as a key modality for structural assessment of native blood vessels as well as engineered vessel mimics. The objective of this study was to monitor and assess in real time the development of a bioengineered blood vessel using a novel approach of combining both free-space and catheter-based OCT imaging in a new quartz-walled bioreactor. Development of the blood vessel was characterized by changes in thickness and scattering coefficient over a 30-day period. MATERIALS AND METHODS: We constructed a novel blood vessel bioreactor utilizing a rotating cylindrical quartz cuvette permitting free-space OCT imaging of an installed vessel's outer surface. A vascular endoscopic OCT catheter was used to image the lumen of the vessels. The quartz cuvette permits 360 degree, free-space OCT imaging of the blood vessel. Bioengineered blood vessels were fabricated using biodegradable polymers (15% PCL/collagen, ∼300 µm thick) and seeded with CH3 10t1/2 mesenchymal stem cells. A swept-source OCT imaging system comprised of a 20 kHz tunable laser (Santec HSL2000) with 1,300 nm central wavelength and 110 nm FWHM bandwidth was used to assess the vessels. OCT images were obtained at days 1, 4, 7, 14, 21, and 30. Free-space (exterior surface) OCT images were co-registered with endoscopic OCT images to determine the vessel wall thickness. DAPI-stained histological sections, acquired at same time point, were evaluated to quantify wall thickness and cellular infiltration. Non-linear curve fitting of free-space OCT data to the extended Huygen-Fresnel model was performed to determine optical scattering properties. RESULTS: Vessel wall thickness increased from 435 ± 15 µm to 610 ± 27 µm and Vessel scattering coefficient increased from 3.73 ± 0.32 cm⁻¹ to 5.74 ± 0.06 cm⁻¹ over 30 days. Histological studies showed cell migration from the scaffold surface toward the lumen and cell proliferation over the same time course. The imaging procedure did not have any significant impact on scaffold dimensions, cell migration, or cell proliferation. CONCLUSIONS: This study suggests that combination of free-space and catheter-based OCT for blood vessel imaging provides accurate structural information of the developing blood vessel. We determined that free-space OCT images could be co-registered with catheter-based OCT images to monitor structural features such as wall thickness or delamination of the developing tissue-engineered blood vessel within a bioreactor. Structural parameters and optical properties obtained from OCT imaging correlate with histological sections of the blood vessel and could potentially be used as markers to non-invasively and non-destructively assess regeneration of engineered tissues in real time.

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.

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.

Fabrication and characterization of poly(DL-lactic-co-glycolic acid)/zirconia-hybridized amorphous calcium phosphate composites.

Several minerals, such as hydroxyapatite and beta-tricalcium phosphate, have been incorporated into bioresorbable polyester bone scaffolds to increase the osteoconductivity both in vitro and in vivo. More soluble forms of calcium phosphate that release calcium and phosphate ions have been postulated as factors that increase osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized allowing controlled release of calcium and phosphate ions. When incorporated into bioresorbable scaffolds, Zr-ACP has the potential to induce osteoconductivity. In this study, 80-90% (w/v) porous poly(DL-lactic-co-glycolic acid) (PLGA) scaffolds were formed by thermal phase separation from dioxane while incorporating Zr-ACP. Scanning electron microscopy revealed a highly porous structure with a pore size ranging from a few microm to about 100 microm, smaller than we had hoped for. Zr-ACP particles were evenly dispersed in the composite structure and incorporated into the pore walls. The amorphous structure of the Zr-ACP was maintained during composite fabrication, as found by X-ray diffraction. Composite scaffolds had larger compressive yield strengths and moduli compared to pure polymer scaffolds. These initial efforts demonstrate that PLGA/Zr-ACP composites can be formed in ways that ultimately serve as promising bone scaffolds in tissue engineering.

Osteoblast response to zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate.

Calcium phosphate bioceramics, such as hydroxyapatite, have long been used as bone substitutes because of their proven biocompatibility and bone binding properties in vivo. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized, which is more soluble than hydroxyapatite and allows for controlled release of calcium and phosphate ions. These ions have been postulated to increase osteoblast differentiation and mineralization in vitro. The focus of this work is to elucidate the physicochemical properties of Zr-ACP and to measure cell response to Zr-ACP in vitro using a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line. Cells were cultured in osteogenic medium and mineral was added to culture at different stages in cell maturation. Culture in the presence of Zr-ACP showed significant increases in cell proliferation, alkaline phosphatase activity (ALP), and osteopontin (OPN) synthesis, whereas collagen synthesis was unaffected. In addition, calcium and phosphate ion concentrations and medium pH were found to transiently increase with the addition of Zr-ACP, and are hypothesized to be responsible for the osteogenic effect of Zr-ACP.