Browsing by Author "Whited, Bryce Matthew"

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    Design and nondestructive imaging of a bioengineered vascular graft endothelium
    Whited, Bryce Matthew (Virginia Tech, 2013-02-01)
    Cardiovascular disease is currently the leading cause of death in the U.S. that frequently requires bypass surgery using vascular grafts for treatment. Current limitations with fully synthetic grafts have led researchers to bioengineered alternatives that consist of a combination of vascular scaffolds and cells. A major challenge in creating a functional bioengineered vascular graft is development of a confluent endothelium on the lumen that is able to resist detachment under physiologic fluid flow. In addition, methodologies used to assess the growth and maturation of the endothelium in a noninvasive and dynamic manner are severely lacking. Therefore, the overall goal of this research is to advance the field of vascular tissue engineering by 1) creating methodologies to enhance EC adherence to a vascular graft and 2) development of a noninvasive and real-time imaging system capable of assessing the graft endothelium.  To achieve these objectives, three separate studies were performed. In the first study, electrospun scaffold fiber diameter and alignment were systematically varied to determine their effect on endothelial cell (EC) morphology and adherence under fluid flow. ECs on uniaxially aligned nanofibers displayed elongated and aligned morphologies leading to higher adherence to the scaffolds under physiologic levels of fluid flow as compared to those on randomly oriented scaffolds. In the second study, a fiber optic based (FOB) imaging system was developed to image fluorescent ECs through a thick electrospun scaffold.  Results demonstrated that the FOB imaging system was able to accurately visualize fluorescent ECs in a noninvasive manner through the thick and highly opaque scaffold. In the final study, the FOB imaging system was used to noninvasively quantify vascular graft endothelialization, EC detachment, and apoptosis through the vessel wall with greater imaging penetration depth than two-photon microscopy. Additionally, the FOB method was capable of continuously tracking EC migration and endothelialization of a bioengineered graft in a bioreactor. Overall, these results demonstrate that aligned scaffold topographies enhance EC adherence under fluid flow and the FOB imaging system is a promising tool to monitor endothelium development and response to fluid flow in a manner that has not previously been afforded using conventional imaging methods.
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    Osteoblast Response to Zirconia-Hybridized Pyrophosphate Stabilized Amorphous Calcium Phosphate
    Whited, Bryce Matthew (Virginia Tech, 2005-05-04)
    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 μ-tricalcium phosphate (μ-TCP) ceramics have been incorporated into PLGA bone scaffolds and have been shown to increase their osteoconductivity (support cell attachment). Although HAP, μ-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 calvarial-derived 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, 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.