Bioengineered vascular graft grown in the mouse peritoneal cavity

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Bioengineered vascular graft grown in the mouse peritoneal cavity Lei Song, MD, Lai Wang, MD, Prediman K. Shah, MD, Aurelio Chaux, MD, Behrooz G. Sharifi, PhD Journal of Vascular Surgery Volume 52, Issue 4, Pages 994-1002.e2 (October 2010) DOI: 10.1016/j.jvs.2010.05.015 Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 1 Generation of tissue capsule in the peritoneal cavity of a mouse. Siastic tubing (A) was inserted into peritoneal cavity of C57BL/6 mice and removed 14 days after implantation. B shows that the two ends of the tube are covered with tissues, whereas the midsection of the tube remained uncovered (arrows). C shows the implanted tube that was harvested after 8 weeks, which is completely covered with tissue. D illustrates the removal of the tissue capsule from the tube by cutting one end of the capsule. E shows the harvested plastic tube and the tissue capsule that was used for autografting into the abdominal aorta of a mouse. Similar results were obtained with implanted polyethylene tubing. For details on the number of tubes and methodology, please see the Method section in the Appendix (online only). Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 2 Representative immunohistochemical analysis of tissue capsule. The frozen sections of tissue capsule harvested at 8 weeks from the peritoneum of MRL mice were stained with 1:50 dilution of anti-smooth muscle α-actin antibody (A), 1:50 dilution of anti-CD31 antibody (B), and 1:50 dilution of anti-MOMA-2 antibody (C). The vast majority of the sections from the three strains of mice were negative for MOMA-2 expression, and only one section from one mouse was positive for moma-2 expression (arrow). D shows collagen staining. We stained five tissue capsules that were harvested from each strain of mice. See method section in the Appendix (online only) for details. Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 3 Expression profiling of freshly-harvested tissue capsules isolated from the three strains of mice. Total RNA was isolated from freshly-harvested tissue capsule from the three strains of mice (three mice/strain), and after pooling and quality control evaluation, they were analyzed by microarray using mouse Affymetrix GeneChip MOE-430 (Affymetrix, Santa Clara, CA) that contains 22,000 cDNA and ESTs. A, Venn diagram showing the number of genes enriched in each strain-specific cell population and their overlaps. Note the high overlap between the three strains of mice. In a small percentage of the cases, the same gene is recognized by more than one probe set in the Affymetrix array; hence, the 669 probe sets enriched in all peritoneal cells actually correspond to 628 unique genes. B shows the distribution within the shared transcriptome for gene products with known or putative functions. Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 4 Grafting of tissue capsule into abdominal aorta of the same MRL mice. A, Freshly harvested tissue capsule was everted and grafted into abdominal aorta of the same mice (ie, the capsule grafts are autografts). Please note the smoothness of the surface of the graft (arrow). B shows the capsule autograft 4 months after transplantation. C and D show venous isografts (arrow) and arterial isografts (arrow) 4 months after grafting, respectively. The number of grafts, patency rate, and animal survival rate are shown in the Table. Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 5 Representative micro-ultrasound analysis of the recipient of the capsule graft. The micro-ultrasound was used to evaluate patency of the capsule graft 4 months after grafting in MRL mouse. A and B show that the capsule autografts remained distended with average length of 7.38 mm and diameter of 1.23 mm. C shows blood flow as measured by pulse wave Doppler. Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 6 Representative laser Doppler images from the bilateral hind limbs of the recipients of capsule, venous, and arterial grafts. A, The laser Doppler images were recorded at three different times: 1 day prior to grafting (baseline), 1 day, and 1 month after grafting in MRL mice. Images taken of hind limbs with the capsule graft (II and III) were compared with those of baseline (I). Similarly, venous graft (V and VI) as well as arterial graft images (VIII and IX) were compared with their respective baseline images (IV and VII), respectively. Representative color-coded images reflect red blood cell velocity (red is highest velocity, green intermediate, and blue, lowest velocity). B, Cumulative results of laser Doppler image-derived blood flow of the bilateral hind limbs are shown over the experimental time course. Each data point represents the mean value ± SD. Venous graft (VG) is compared with arterial graft (AG; P < .05), and the capsule graft (CG) is compared with arterial graft (AG; P < .01) at 1 day following transplantation. For a detailed number of grafts and the patency rates, please see the Table. Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 7 Representative immunohistochemical analysis of the capsule graft harvested 4 months after transplantation in MRL mice. Frozen sections harvested from five MRL mice tissue capsule autografts were analyzed by histochemical staining and immunostaining. A shows Movat staining. B and C show smooth muscle α-actin staining of autografts and venous isografts, respectively. D shows staining of capsule autograft for the expression of CD31 endothelial cell marker. The presence of macrophages was evaluated by anti-MOMA-2 staining (E). We stained five tissue capsule autografts, five venous isografts, and five aortic isografts. Journal of Vascular Surgery 2010 52, 994-1002.e2DOI: (10.1016/j.jvs.2010.05.015) Copyright © 2010 Society for Vascular Surgery Terms and Conditions