Preseeding with autologous fibroblasts improves endothelialization of glutaraldehyde-fixed porcine aortic valves.

OBJECTIVE: This study represents the development of a treatment and seeding procedure to improve endothelial cellular adhesion on glutaraldehyde-fixed valves. METHODS: Porcine aortic valves were fixed with 0.2% glutaraldehyde. Wall pieces of these valves had either no additional treatment (n = 4), incubation in M199 Earle (1x), with sodium carbonate at 2.2 g/L without l-glutamine for 24 hours (n = 4), or additional pretreatment with 5%, 10%, or 15% citric acid (three groups, n = 4 each). Thereafter the pieces were washed and buffered to a physiologic pH. This was followed by seeding of human endothelial cells (5 x 10(6) cells). On the basis of the results of these pilot tests, complete glutaraldehyde-fixed aortic roots treated with 10% citric acid were subjected to cell seeding. The valves were seeded with endothelial cells (4.3 x 10(6) cells) either alone (n = 4) or in combination with preseeding of autologous fibroblasts (2.4 x 10(7) cells, n = 4). After each seeding procedure specimens of the free wall of the grafts were taken. In addition, one leaflet was taken for histologic examination after endothelial cell seeding, after 7 days, and after 21 days. Finally, two commercially available stentless aortic valve prostheses (Freestyle; Medtronic, Inc, Minneapolis, Minn) were treated with 10% citric acid and seeded with human fibroblasts and endothelial cells. Specimen were taken according to the glutaraldehyde-fixed aortic roots. Specimen of all experiments were examined with scanning electron microscopy. Frozen sections were stained immunohistochemically for collagen IV, factor VIII, and CD31. RESULTS: On untreated glutaraldehyde-fixed aortic wall pieces, only poor adhesion (24%) was seen. No viable cells were found after 1 week. Cellular adhesion was best on aortic wall pieces pretreated with 10% citric acid. After 7 days, the cells formed a confluent layer. Endothelial cell seeding on citric acid-treated complete aortic valves showed 45% adhesion, but no confluent layer was found after 1 week. Preseeding of these valves with autologous fibroblasts resulted in an endothelial cellular adhesion of 76% and a confluent endothelial cell layer after 7 days. The layer remained stable for at least 21 days. Results of staining for collagen IV, factor VIII, and CD31 were positive on the luminal side of these valves, indicating the synthesis of matrix proteins and viability of the cells. Pretreatment of commercially available porcine valves with 10% citric acid and preseeding with autologous fibroblasts followed by endothelial cell seeding resulted in an adhesion of 78%. The cells formed a confluent cell layer after 7 days. CONCLUSIONS: Pretreatment of glutaraldehyde-fixed porcine aortic valves with citric acid established a surface more suitable for cellular attachment. Preseeding these valves with autologous fibroblasts resulted in a confluent endothelial cell layer on the luminal surface. Flow tests and animal experiments are necessary for further assessment of durability and shear stress resistance.

Is there an advantage in using homografts in patients with acute infective endocarditis of the aortic valve?

BACKGROUND AND AIM OF THE STUDY: Acute infective endocarditis is a surgical challenge, particularly when paravalvular abscesses and annular destruction are present. The choice of a homograft or mechanical valve prosthesis is an important issue in these patients. The study aim was to compare the outcome with homografts and mechanical valves in patients with acute infective endocarditis. METHODS: A total of 77 patients (mean age 49+/-9 years) operated on for acute endocarditis of the aortic valve was included in the study and analyzed retrospectively. The causative bacterium was isolated from blood cultures in 71 cases. Preoperatively, 21 patients required artificial ventilation and 24 had inotropic support due to hemodynamic instability. Aortic homografts were implanted in 43 patients, and mechanical valve prostheses in 34. The two patient groups were similar in terms of gender, age and preoperative inotropic support. In total, 31 patients (44%) had paravalvular abscesses, and a homograft was used significantly more often (77%, p <0.05) in these cases. Follow up examinations (clinical examination, ECG and transthoracic echocardiography) were performed six months postoperatively and continued on an annual basis. Endocarditis relapse was defined as persisting infection, whereas re-endocarditis indicated a new infection after an interval of at least six months. RESULTS: Perioperative mortality was 11.5% (5/43) in homograft patients. In the 38 survivors, follow up was complete and averaged 5.0+/-1.2 years. One patient had an endocarditis relapse three months after surgery. Re-endocarditis occurred in three patients after two or three years. One other patient had pseudoaneurysm formation without a need for intervention, and one had repeat aortic valve replacement due to dysfunction of the graft after four years. The other 33 patients had an uneventful follow up. Echocardiography revealed aortic insufficiency grade 1 in 12 cases (36%), with no progression during follow up. Perioperative mortality in mechanicat valve patients was 20.5% (n = 7) (p <0.05 versus homograft), and in those with paravalvular abscess, perioperative mortality was even higher than in homograft patients (4/7, 57.1% versus 3/24, 12.5%; p <0.05). When considering only patients without paravalvular abscess, there was no significant difference between groups (10.5% versus 12.5%). Three relapses occurred in mechanical valve patients (10.3%), but no endocarditis recurred during follow up. One late death (3.7%) occurred due to bleeding complicating long-term anticoagulation. CONCLUSION: The study results do not permit a general recommendation to be made for homograft use in patients with acute endocarditis. In cases with paravalvular abscesses, however, there was a trend towards improved outcome in the homograft group.

Ten years' experience in aortic valve replacement with homografts in 389 cases.

BACKGROUND AND AIM OF THE STUDY: Aortic valve replacement using homografts is an accepted alternative to the use of other replacement devices, and has been established at the authors' institution for more than 10 years. METHODS: Since 1992, a total of 389 homografts was implanted, and 332 patients (mean age 54 years, 72% males) were followed up. The initial patients (n = 75) had subcoronary implantation, all subsequent patients had root replacement. Both aortic grafts (AG) and pulmonary grafts (PG) were used. Follow up was conducted with regard to the factors 'graft origin', 'implantation technique' and 'gender', and included clinical examination, ECG and transthoracic echocardiography on an annual basis. RESULTS: Overall 30-day mortality was 5.4% (AG patients 3.9%, PG patients 13.5%; p = 0.09). Among late deaths (n = 22), six were valve-related (all prosthetic infection). Four minor thrombembolic events were recorded due to amaurosis fugax and transient ischemic attacks (TIA). Freedom from reoperation was 86.5%. Indication for graft replacement was greater after subcoronary implantation than after root implantation (p = 0.04). Reoperation was necessary in 24 patients due to restenosis (n = 4), regurgitation grade >II (n = 5), paravalvular leak (n = 2) and prosthetic infection (n = 13). At the latest echocardiographic follow up, mean peak pressure gradient was 15.60 +/- 11.76 mmHg, homograft regurgitation grade was 0.82 +/- 0.66, left ventricular end-diastolic diameter (EDD) was 49.1 +/- 7.54 mm, and mean aortic root diameter was 30.54 +/- 5.48 mm. When comparing parameters at a mean of five years postoperatively, the pressure gradient increased from 10.26 to 15.02 mmHg, regurgitation grade increased from 0.53 to 0.81, and EDD decreased from 52.3 to 50.4 mm. Other variables showed no significant differences. CONCLUSION: The present results confirmed good midterm-results for aortic valve replacement with homografts. These prostheses are vulnerable to infection, and root replacement was superior to the subcoronary implantation technique.

Seeding of human endothelial cells on valve containing aortic mini-roots: development of a seeding device and procedure.

PURPOSE: Complete covering of an artificial valvular scaffold with endothelial cells may prevent thromboembolic complications and lead to an excellent biocompatibility. For this purpose, we developed a seeding device for reproducible cell seeding on valve containing aortic roots. DESCRIPTION: Human endothelial cells and fibroblasts were obtained from saphenous vein pieces. Cryopreserved aortic roots (n = 25) were put into an especially developed tube, set on a rotator, and incubated with the cell suspension. The device rotated in two axes (sagittal and axial), ensuring slight movements of the leaflets. The rotation alternated with resting periods, allowing cell attachment to the surface. Different resting periods were tested (groups 1, 2, and 3 were 30, 45, and 60 min, respectively; n = 5 each). Total incubation time was 24 hours followed by further culturing for 6 days. In two further groups (groups 4 and 5; n = 5 each), a modified inlay was used to allow the cell suspension to flow around the entire graft. In group 4 the grafts were again incubated with human endothelial cells; however, in group 5 pre-seeding with autologous fibroblasts was done in addition. Immunohistochemical staining with antibodies against factor VIII, CD31, laminin, collagen IV, and CD90 were done, and scanning electron microscopy was done after initial seeding and after 6 days in culture. EVALUATION: Seeding resulted in homogenous cell layers on the luminal surface of the free walls in all groups. With resting periods of 45 minutes, these results were also obtained on the leaflets, whereas the other resting times resulted in defects of the endothelial cell layer on the cusps. After 6 days under culture conditions, the endothelial cell layers were confluent and viable, with the exception of the leaflets in group 1. With the modified inlay (groups 4 and 5), confluent cell layers were also achieved on the outer surface. In group 5 pre-seeding with autologous fibroblasts resulted in enhanced synthesis of extracellular matrix proteins, as was demonstrated with immunohistochemical staining for collagen IV and laminin. CONCLUSIONS: With this newly developed seeding device, confluent cell layers on valve containing aortic roots were reproducibly achieved. The technique enables further experimental research and even clinical application.

Myoblasts survive intracardiac transfer and divide further after transplantation.

OBJECTIVE: Skeletal myoblasts have been proven to survive transplantation into myocardial scar tissue. The objective of this study was to evaluate whether these cells can also be transferred into vital myocardium and maintain their ability for cell division after transplantation. In addition, an intravital fluorescence dye for marking these cells was evaluated. MATERIAL AND METHODS: Skeletal myoblasts were harvested from male Lewis rats (n = 6) and then expanded in culture. Before implantation, these cells were trypsinized and labeled using an intravital fluorescence dye (PKH-26). Syngenic myoblast transfer to recipient female Lewis rats (n = 36) was used to simulate autologous transplantation. Under general anesthesia, the rats received injections of 106 myoblasts via a subxyphoidal approach into the apex of the heart. The animals were then divided into 3 groups (n = 10 each). The animals were sacrificed at several time points, and the hearts were harvested for histologic examination: group A, 7 days postoperatively; group B, 14 days postoperatively; and group C, 28 days postoperatively. An additional group, group D (n = 6), served as a control group; these animals were injected with only cell medium. Corresponding to the study groups, 2 animals of this control group were sacrificed at each time point, and the hearts were explanted. At histological examination, 8- m sections were investigated to identify surviving stained cells. For further evaluation, the sections were stained using monoclonal antibodies against n-cam, desmin, and a- actin. RESULTS: No fluorescing cells were found in any hearts of rats in the control group. Surviving fluorescing myoblasts were found in 9 of 10 hearts of groups A and C and 8 of 10 hearts of group B. Labeled myoblasts were located in the intercellular spaces between the myocardial fibers. Fibrotic or inflammatory reactions could not be identified around the injection site in any hearts of the study groups. Immunohistochemical staining results showed that the labeled cells expressed n-cam, desmin, and a-actin. The myoblasts had regained their physiologic structures and had started to form myofibers. In groups B and C, more n-cam-positive cells than labeled cells were found, indicating further cell division. CONCLUSIONS: Intravital fluorescence staining with PKH- 26 dye proved to be an easy and reliable method for identifying cells after cellular transplantation. Myoblasts survived an intracardiac transfer, regaining their physiologic structures and maintaining their ability for further cell division.