Mark A. Moore

Shrinkage temperature versus protein extraction as a measure of stabilization of photooxidized tissue.

A rise in thermal denaturation temperature has been utilized as an indication of stabilization of collagen-containing materials such as pericardial tissue and porcine heart-valve leaflets following treatment with glutaraldehyde, Denacol, or other chemical agents. In contrast, stabilization of bovine pericardial tissue by dye-mediated photooxidation does not result in a significant rise in shrinkage temperature comparable with these treated materials. It was therefore hypothesized that a rise in shrinkage temperature is not a necessary indication for tissue stabilization. A sensitive protein extraction assay has been developed which can be used to monitor the stabilization of pericardial tissue by a variety of treatment methods, including photooxidation. A reduction in extractable protein, as analyzed by polyacrylamide gel electrophoresis, is noted for pericardial tissue treated with photooxidation, glutaraldehyde, or Denacol. Loss of extractable protein, as a function of treatment time, correlates well with a significant rise in shrinkage temperature for pericardium treated with glutaraldehyde or Denacol but not with photooxidation. This difference is attributed to the stabilization processes of glutaraldehyde and Denacol, which involve extensive crosslinking and polymer formation within and in addition to the native pericardial matrix, leading to a rise in matrix complexity and thermal stability. In contrast, photooxidation is a catalytic process involving modification and crosslink formation within existing matrix components, resulting in a material with little added matrix complexity.

Evaluation of porcine valves prepared by dye-mediated photooxidation

BACKGROUND Previous studies demonstrated that dye-mediated photooxidation can stabilize bovine pericardium. Here, photooxidized porcine valve cusp and root tissue were assessed in comparison to fresh and glutaraldehyde-treated samples. METHODS AND RESULTS In an in vitro tissue solubility test, both photooxidized and glutaraldehyde-treated tissues were resistant to protein extraction compared to fresh tissue. A rat subcutaneous model was used to test in vivo stability and calcification potential. In this study, four of the six fresh leaflets were not visible because of resorption while both photooxidized and glutaraldehyde-treated tissues were biostable. Mineral contents of the rat explants were much lower for both fresh and photooxidized leaflets when compared with glutaraldehyde-treated leaflets. Also, the aortic root calcified whether treated or not with the most mineral being associated with glutaraldehyde-treated root. Analysis of photooxidized porcine valves explanted from the mitral position in sheep indicated a material that was biostable and contained only minor calcification, perhaps due to deformed stents. CONCLUSIONS Porcine valve tissue treated by dye-mediated photooxidation is biostable and resistant to calcification, and has potential for use in heart valve bioprostheses.

Crosslink formation in porcine valves stabilized by dye-mediated photooxidation.

Bovine pericardial and porcine valve materials stabilized by dye-mediated photooxidation have shown potential for bioprosthetic valve use. Previously, in vitro and in vivo stability of these materials was demonstrated through enzymatic, chemical, extraction, rat subcutaneous, and functional challenges. Here, we examine the stability of photooxidized porcine aortic valves through amino acid, crosslink, and hydrothermal isometric tension analysis. Photooxidation reduced intact histidine residues from 17.0 to 0 residues per 1000, indicating the photooxidative alteration of this amino acid. Diphenyl borinic acid-derivitized hydrolyzates of proteins were separated by high-performance liquid chromatography, which identified several amino acid crosslinks that appeared with photooxidation that were absent in untreated controls. Thermal relaxation analysis indicated a significantly higher (p < 0.0002) thermal stability for photooxidized porcine cusps than that of untreated controls, with mean relaxation times for untreated cusps of 14,000 +/- 4650 versus 22,900 +/- 2480 s for photooxidized cusps. In summary, porcine aortic valve tissue treated by dye-mediated photooxidation contains new chemical species and exhibits properties consistent with intermolecular crosslink formation, which explain the increased biostability of this material and its potential for use in bioprosthetic devices.

Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm(-1)) and a decrease in reducible cross-links (1690 cm(-1)). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.

Nonaldehyde sterilization of biologic tissue for use in implantable medical devices

Biologic tissue stabilized by dye-mediated photooxidation has found application in implantable devices. The desire to avoid aldehydes in the processing of photooxidized tissues led to the development of a nonaldehyde, iodine based sterilant. The interaction of tissue with iodine was indicated by a change in tissue shrinkage temperature, dependent upon solution and incubation parameters. The amino acid tyrosine also was altered, presumably because of aromatic ring iodination. Transmission electron microscopic study indicated no change in the quarter staggered array structure of collagen under controlled iodine treatment conditions. The D10 values for iodine kill of several organisms, in the absence of tissue, were determined in 0.1% iodine (pH 6.5) at 37°C for Bacillus subtilis (12 min), Aspergillus niger, Escherichia coli, Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa (all <1 min). In a separate experiment, samples of 0.1 % iodine (pH 6.5) containing photooxidized pericardial tissue were inoculated with 1.6 × 107 Bacillus subtilis, 4.6 × 106 Pseudomonas aeruginosa, or 7.2 × 106 Staphylococcus aureus and incubated at 37°C. No survivors were detected on the tissue samples after exposure of 48 hr. Photooxidized pericardial tissue samples inoculated with either 3.2 × 105 porcine parvovirus or 1 × 109 infectious bovine rhinotracheitis were exposed to 0.1 % iodine (pH 6.5) at 36°C for 12hr. No viral particles were detected after exposure, yielding minimum viral log reduction factors of 3.0 and 6.5, respectively. The results presented indicate the potential for a nonaldehyde, iodine based solution to sterilize implantable devices containing biologic tissue.