Preseeding with autologous fibroblasts improves endothelialization of glutaraldehyde- fixed porcine aortic valves Helmut Gulbins, MD, Angelika Goldemund,

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Preseeding with autologous fibroblasts improves endothelialization of glutaraldehyde- fixed porcine aortic valves Helmut Gulbins, MD, Angelika Goldemund, Ingrid Anderson, Ulrike Haas, Antje Uhlig, Bruno Meiser, MD, Bruno Reichart, MD The Journal of Thoracic and Cardiovascular Surgery Volume 125, Issue 3, Pages 592-601 (March 2003) DOI: 10.1067/mtc.2003.48 Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 1 Cell seeding device. Valve is fixed in cylinder so that all parts are in contact with cell suspension. During seeding procedure, cylinder is rotated every 30 minutes to expose different parts of valve to cell seeding. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 2 Aortic wall piece, glutaraldehyde fixed and incubated with M-199 for 24 hours, 7 days after EC seeding. No viable cells were observed under scanning electron microscopy (original magnification 500×). The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 3 EC seeding on glutaraldehyde-fixed aortic wall piece after pretreatment with 5% citric acid after 7 days under culture conditions. Confluent EC layer is seen under scanning electron microscopy (original magnification 500×). The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 4 Toxicity testing. A, Arrows indicate margin of glutaraldehyde-fixed aortic wall piece that was only washed in physiologic sodium chloride solution for 10 minutes. Surrounding wall piece, no viable ECs are seen. B, Aortic wall piece pretreated with 10% citric acid. Wall piece lies directly on still confluent cell layer. There is no evidence of cell death. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 5 Islands of viable ECs on group C valves 7 days after seeding procedure. Arrows indicate margin of EC layer (scanning electron microscopy, original magnification 100×). The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 6 Fibroblasts seeded on group C valves (scanning electron microscopy, original magnification 1000×). The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 7 Confluent EC layer on group C valves preseeded with autologous fibroblasts (scanning electron microscopy, original magnification 1000×). The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 8 Immunohistochemical staining for collagen IV of group C valves after seeding with fibroblasts and ECs. A, Specimen clearly are stained positively for collagen IV at luminal surface, as indicated by red peroxidase reaction. B, Negative control. Small cell layer (stained blue) is seen, but no positive peroxidase reaction is found. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 9 A, Immunohistochemical staining with factor VIII antibody and counterstaining with fluorescein of leaflets of commercially available porcine heart valve 21 days after cell seeding. Cells on surface are stained positively for factor VIII (fluorescing green), proving them to be viable ECs. B, Immunohistochemical staining with CD31-antibody and peroxidase reaction of leaflets of commercially available porcine heart valve 21 days after cell seeding. Cells on surface are stained positively for CD31 (stained red). The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 10 Aldol condensation is physiologic process in the cross-linking of collagen fibers.21 Glutaraldehyde forms Schiff base with free amino groups of collagen fibers. Cross-linking can take place either as formation of another Schiff base with amino group of second fiber or by aldol condensation. Result, however, is rather hydrophobic. Citric acid (strong organic acid with four hydroxy groups) salifies amino groups, thereby inducing hydrophilia on surface of collagen fiber. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 592-601DOI: (10.1067/mtc.2003.48) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions