Endothelial cell lining of bioprosthetic heart valve materials.

To investigate the conditions for endothelial cell lining of glutaraldehyde-treated bioprosthetic heart valves, we examined in vitro the growth properties of endothelial cells on clinically used pericardial valve material and on glutaraldehyde-fixed pericardium treated with L-glutamic acid. To improve endothelial cell attachment to the valvular surface, we precoated both materials either with fibronectin or with fibrillar collagen (95% type I, 5% type III). Toxicity of glutaraldehyde, released from clinically used valve material, caused endothelial cell death, independent of the type of precoating. Treatment of the valve material with L-glutamic acid resulted in regular endothelial cell proliferation. We found that collagenous precoating, compared with fibronectin precoating, markedly enhanced endothelial cell proliferation and attachment (p less than 0.05). Maintenance of antithrombogenic potency of the seeded cells on L-glutamic acid-treated valve material was proved by regular release of prostacyclin. We conclude that bioprosthetic heart valve materials can be lined with endothelial cells if toxic glutaraldehyde released from the bioprostheses is eliminated.

New aspects of the degeneration of bioprosthetic heart valves after long-term implantation.

Bioprosthetic heart valves removed 76 to 150 months after implantation were morphologically investigated to correlate structural alterations with clinical failure modes. Traditional morphologic methods of evaluating valvular heterografts, such as microradiography and electron microscopy, were complemented by undecalcified ground sections, a new technique for analyzing the distribution of mineral deposits. Apart from well-investigated mechanisms that accelerate tissue degeneration, our observations point to additional facts: (1) phagocytosis of collagen fibrils and elastic material by macrophages and foreign body giant cells in areas near tears and perforations and (2) initial calcification indicated by delicate crystals in the intercellular space arranged in close relation to the periodicity of the cross-striation pattern of collagen fibrils. The present report not only calls attention to degenerative changes that are enhanced by mechanical stress but also underlines phagocytosis as an important mechanism in the destruction of bioprosthetic heart valves.

Biocompatibility of aldehyde-fixed bovine pericardium. An in vitro and in vivo approach toward improvement of bioprosthetic heart valves.

Aldehyde-induced side effects limit the clinical usefulness of bioprosthetic heart valves. Treatment of aldehyde-fixed pericardium with L-glutamic acid at pH 3.5 and storage in a nontoxic, bacteriostatic solution resulted in a lower degree of calcification in 63-day subcutaneous implants in rats (13.3 +/- 2 mg calcium per gram dry weight of tissue), as compared with commercially available tissue (169 +/- 24 mg/gm, p less than 0.05). Endothelial cells died within 1 day after seeding on the commercial tissue; however, considerable endothelial cell proliferation was measured, even 14 days after seeding on L-glutamic acid-treated pericardium. Improved biocompatibility of this alternative treatment may be due to stable chemical binding of free, reactive aldehyde groups.

Glutaraldehyde affects biocompatibility of bioprosthetic heart valves.

A marked release of glutaraldehyde from commercially available pericardial bioprosthetic heart valve (BHV) material in washing solutions was found by high performance liquid chromatography (up to 1.8 ppm of glutaraldehyde per gram of dry tissue). In vitro endothelial cell proliferation rate was impaired dose-dependently in the presence of increasing glutaraldehyde concentrations of the cultivation medium (r = 0.9; p less than 0.05). Cultivation of endothelial cells was impossible on the surface of commercially available BHV material, but successful and uninhibited when toxic glutaraldehyde ligands of the BHV material were antagonized by treatment with L-glutamic acid.

Improved spontaneous endothelialization by postfixation treatment of bovine pericardium.

The longstanding release of locally cytotoxic glutaraldehyde concentrations from fixed biological materials is one reason for the lack of spontaneous endothelialization in vivo and in vitro of biological grafts (and valves). Preliminary studies have shown that bovine pericardium postfixation-treated with aminoacids may be endothelialized in vitro. To test whether such treatment improves spontaneous endothelialization in vivo 8 cm long segments grafts (6 mm I.D.) were interposed into the carotid arteries of 10 sheep. Ten grafts were sewn from postfixation treated pericardium (PTP), 4 from standard glutaraldehyde fixed pericardium (GAP) and 6 polytetrafluoroethylane (PTFE) grafts were implanted to study the degree of spontaneous endothelialization. In two pregnant animals, all grafts occluded (2 PTP, 1 GAP, 1 PTFE). In the remaining animals 1 additional PTFE graft occluded and 2 PTFE and 1 GA grafts showed significant thrombotic obstruction. The patent grafts were harvested at 3 months and the surface covered with red thrombus was determined by planimetry. PTP grafts had significantly less (12.2% +/- 15%) thrombotic appositions than glutaraldehyde treated grafts (49% +/- 20%; P = 0.01) and PTFE grafts (40.5% +/- 13%; P = 0.01). In the central areas of the PTP grafts, endothelial cells spread directly on the collagenous matrix and produced a basal lamina. In GAP, endothelial cells spread on amorphous material or macrophages and in PTFE grafts, only a neointima composed of myofibroblasts was endothelialized. Neutralization of glutaraldehyde concentrations by postfixation with aminoacids improves spontaneous endothelialization in vivo in biological materials.

Improved endothelialization of postfixation treated biological vascular grafts.

Preliminary in vitro and in vivo studies have shown that endothelialization is improved by a detoxifying postfixation treatment of glutaraldehyde (GA) fixed bovine pericardial patches and grafts. To test whether this is also true for GA tanned human vein (HUV) grafts, patches of commercially available HUV grafts (MHUV), postfixation treated HUV grafts (PTHUV) and GA fixed HUV granfts (GAHUV) were endothelialized in vitro. Eight pairs of MHUV and PTHUV grafts were implanted as femoropopliteal grafts in eight sheep. Endothelial cell adherence was significantly better on PTHUV (11910 +/- 4413 cells/cm2) than on MHUV (6545 +/- 2835 cells/mm2; p = 0.0007) and on GAHUV (3563 +/- 1638; p = 0.0001) one day after cell seeding. After eight days of culture significantly more cells spread on PTHUV material than on MHUV (p = 0.0002), but none of the cultures on GAHUV remained viable. Four PTHUV grafts occluded in the femoropopliteal position, mostly because of kinking, so that only in four pairs of grafts could the thrombus-free surface be compared by planimetry. Again PTHUV material was covered more by endothelial cells than was MHUV material. On PTHUV endothelial cells spread directly on the graft material while on MHUV these cells spread on a layer of fibrin and macrophages. Postfixation treatment of GA-fixed biological graft material by amino-acid solutions improves the biocompatibility of the material and enhances in vitro as well as spontaneous in vivo endothelialization.

Toxic effects of aldehydes released from fixed pericardium on bovine aortic endothelial cells.

Glutaraldehyde (GA) and formaldehyde (FA) were shown to be released from 1 cm2 fixed pericard patches into 2-mL storage solutions after three 2-min washings in concentrations of about 1 mg/L. The cytotoxicity of these aldehyde concentrations on bovine aortic endothelial cells (BAECs) was evaluated in vitro, by proliferation capacity, cellular ATP content, PGI2 release and cyclic AMP synthesis. Continuous incubation of BAECs with GA greater than 0.1 mg/L and FA greater than 0.5 mg/L resulted in a significantly inhibited proliferation and in an increase of the intracellular Ca2+ triggered parameters, PGI2 and cyclic AMP, up to three fold. This strongly suggests an aldehyde-induced inhibition of the plasma-membrane bound Ca2+-ATPase, an enzyme which normally maintains low intracellular Ca2+-level. From these findings there is evidence that aldehydes released from bioprosthetic valve tissue may contribute to the lack of endothelial cell coverage in human implants.

[Glutaraldehyde residues in heart valve prostheses]. / Glutaraldehydrückstände in Herzklappentransplantaten.

Bioprosthetic heart valves prepared from glutaraldehyde-pretreated bovine pericardium are used to replace diseased human cardiac valves. Mineralisation in the course of time and toxic effects are possibly caused by glutaraldehyde residues. Different washing methods carried out before transplantation are compared with regard to the different ability of glutaraldehyde extraction.

Improved biocompatibility of bioprosthetic heart valves by L-glutamic acid treatment.

Treatment of glutaraldehyde-fixed pericardium with L-glutamic acid and storage in bacteriostatic preservatives (paraben) stably antagonizes free, reactive aldehyde groups within the fixed bioprosthetic heart valve tissue. In 63-day subcutaneous implants in rats, the calcification rate of this treatment (13.3 +/- 2 mg calcium/g wt tissue) was markedly reduced as compared to conventionally treated tissue (169 +/- 24 mg/g; p less than 0.05). To test the influence of tissue-released toxic aldehydes on spontaneous endothelial cell ingrowth in vivo, vascular grafts (8-cm long, 6-mm diameter) from fixed pericardium treated with L-glutamic acid were interposed into the carotid arteries in ten sheep. They were compared to grafts from conventionally treated pericardium implanted at the contralateral side. Following 3 months of implantation, planimetry revealed 49% +/- 20% of the surface of conventionally preserved pericardium to be covered with red thrombus, but only 12% +/- 5% in L-glutamic acid treated pericardium (p less than 0.05). The ultrastructural findings of a closed endothelial cell layer on the graft surface reveals the new technique to be a promising approach towards increased biocompatibility of aldehyde-fixed bioprosthetic heart valves.

Endothelial cell lining of bioprosthetic heart valve material.

In this in vitro study, the growth properties of cultured endothelial cells on conventionally treated pericardial valve material were measured. These data were compared to endothelial cell proliferation on an alternatively treated valve material. This alternative preservation procedure was developed in order to bind free, residual glutaraldehyde in the valve tissue by reaction with L-glutamic acid. In order to optimize endothelial cell attachment and proliferation, fibronectin and fibrillar collagen type I were tested as surface precoating substances. Cell viability of the seeded cells was evaluated by means of proliferation kinetics, antithrombotic activity, and morphological appearance. Endothelial cell death occurred within the first 2 days after seeding on conventionally treated valve tissue, independent of the type of precoating. On alternatively treated tissue, regular endothelial cell proliferation was observed. Precoating with fibrillar collagen markedly increased endothelial cell attachment and proliferation as compared to fibronectin. Maintenance of antithrombotic activity of the seeded cells was proven by regular release of prostacyclin.

Decreased tissue reaction to bioprosthetic heart valve material after L-glutamic acid treatment. A morphological study.

Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).

Improved biocompatibility by postfixation treatment of aldehyde fixed bovine pericardium.

Long-standing release of locally cytotoxic aldehyde concentrations is responsible for lack of spontaneous endothelialization and increased calcification of glutaraldehyde fixed bovine pericardium. Postfixation treatment with amino acids made in vitro endothelialization of bioprosthetic heart valves possible. Such treated pericardium calcified significantly less (13 +/- 4 micrograms/mg dry weight) than did conventionally processed pericardium (114 +/- 25 micrograms/mg) after 63 days of subcutaneous implantation in rats. To test the ability for spontaneous in vivo endothelialization, 5 sheep had 6 mm grafts made from postfixation treated pericardium (PTP) implanted into the carotid artery, compared to PTFE grafts on the contralateral side, which spontaneously endothelialize in animal models. In a pregnant animal, both grafts occluded. All remaining pericardial grafts remained patent, but one additional PTFE graft occluded and another one was stenosed. The area covered with red thrombus was significantly smaller in the PTP grafts (3.05 +/- 3.9%) than in the PTFE grafts 42 +/- 14% (p = 0.0036); TEM and SEM showed endothelial cells growing directly on the PTP, but only on myofibroblasts in PTFE grafts. Postfixation treatment of glutaraldehyde fixed pericardium aids spontaneous endothelialization and decreases tissue calcification.