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Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium.

ABSTRACT: 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.

SUBMITTER: Whited BM

PROVIDER: S-EPMC3621659 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium.

Whited Bryce M BM Hofmann Matthias C MC Lu Peng P Xu Yong Y Rylander Christopher G CG Wang Ge G Sapoznik Etai E Criswell Tracy T Lee Sang Jin SJ Soker Shay S Rylander Marissa Nichole MN

PloS one 20130409 4

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 secti ...[more]

PMID: 23585885

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Project description:We tested the hypothesis that the mouse peritoneum can function like a bioreactor to generate directed bio-engineered tissues such as those used for bypass grafting. Additionally, we reasoned that the mouse animal model would allow us to elucidate the underlying cellular and molecular mechanisms that are responsible for the generation of tissue in peritoneal cavity.Plastic tubes (two tubes/mouse) were implanted into the peritoneal cavity of three strains of mice (C57BL/6, BALB/c, and MRL). The tubes were harvested, tissue capsule surrounding the tubes was removed, and analyzed by immunostaining (five capsules/five mice/strain) and microarray (three capsules/three mice/strain). In addition, the tissue capsules that were harvested from MRL mice (n = 21) were grafted into abdominal aorta of the same mice as autografts. The patency of all grafts was monitored by micro-ultrasound, and their functionality was assessed by laser Doppler imaging of blood flow in femoral arteries. Venous (n = 13) and arterial isografts (n = 11) were used as positive controls. In a negative control group (five mice/strain), the abdominal aorta was occluded by double ligation with 9-0 silk.The implanted plastic tubes required at least 8 weeks of incubation in the peritoneum of the three strains of mice in order to generate useful grafts. No vascular cells were found in the tissue capsules. Microarray analysis of tissue capsules revealed that the capsular cells express a gene expression program that is vastly shared among the three strains of mice, and the cells exhibit a high degree of plasticity. The micro-ultrasound analysis of the grafts showed that 62% of autografts remained patent compared with 77% of venous isografts and 91% of arterial isografts. The laser Doppler imaging analysis showed that blood flow dropped by 40% and 35% in the autografts and vein isografts, respectively, 1 day after surgery. The flow, however, rebounded to the level of arterial isografts 1 month post-surgery and remained unchanged among all grafts for the next 4 months. Immunostaining of the autografts showed a thick vessel wall with endothelial cells that lined the lumen and smooth muscle cells that constituted the graft wall.The mouse peritoneal cavity of mice has the ability to function like a bioreactor to generate bio-engineered tissues. The tissue capsules harvested from peritoneal cavity of a mouse are composed of nonvascular cells that display phenotype of progenitor cells. After grafting, however, the capsule autografts become arterialized and remained patent for at least 4 months after surgery, similar to venous or arterial isografts.

Project description:BackgroundPulmonary hypertension (PH) complicating idiopathic pulmonary fibrosis (IPF) is associated to worse outcome. There is a great need for a non-invasive diagnostic modality to detect and evaluate the severity of pulmonary vascular disease (PVD). 99mTc-PulmoBind is a novel imaging agent that binds to the adrenomedullin (AM) receptor on the pulmonary microvascular endothelium. SPECT imaging employing the endothelial cell tracer 99mTc-PulmoBind was used to assess PVD associated with lung fibrosis.MethodsRats with selective right lung bleomycin-induced fibrosis were compared to control rats. SPECT imaging was performed after three weeks with 99mTc-PulmoBind and 99mTc-macroaggregates of albumin (MAA). PH and right ventricular (RV) function were assessed by echocardiography. Lung perfusion was evaluated by fluorescent microangiography. Lung AM receptor expression was measured by qPCR and by immunohistology. Relevance to human IPF was explored by measuring AM receptor expression in lung biopsies from IPF patients and healthy controls.ResultsThe bleomycin group developed preferential right lung fibrosis with remodeling and reduced perfusion as assessed with fluorescent microangiography. These rats developed PH with RV hypertrophy and dysfunction. 99mTc-PulmoBind uptake was selectively reduced by 50% in the right lung and associated with reduced AM receptor expression, PH and RV hypertrophy. AM receptor was co-expressed with the endothelial cell protein CD31 in alveolar capillaries, and markedly reduced after bleomycin. Quantitative dynamic analysis of 99mTc-PulmoBind uptake in comparison to 99mTc-MAA revealed that the latter distributed only according to flow, with about 60% increased left lung uptake while left lung uptake of 99mTc-PulmoBind was not affected. Lung from human IPF patients showed important reduction in AM receptor expression closely associated with CD31.ConclusionsSPECT imaging with 99mTc-PulmoBind detects PVD and its severity in bleomycin-induced lung fibrosis. Reduced AM receptor expression in human IPF supports further clinical development of this imaging approach.

Dataset Information

Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium.

Publications

Dynamic, nondestructive imaging of a bioengineered vascular graft endothelium.

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