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.

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.

Chemical modification of bovine tissues by dye-mediated photooxidation.

BACKGROUND AND AIMS OF THE STUDY: Photooxidation of pericardium has been shown chemically to alter and stabilize tissue. The characterization of photooxidatively induced, chemical modifications of bovine pericardial and arterial tissue is reported here. METHODS: Tissues were prepared by various methods of photooxidation and analyzed for thermal denaturation temperature, protein extraction, amino acid content and crosslink content. RESULTS: Photooxidation of tissue resulted in no significant time-dependent changes in thermal denaturation temperature, but did result in a time-dependent alteration and reduction in extracted proteins. This reduction is consistent with chemical alteration and stabilization of the tissue. Photooxidation also resulted in a time-dependent reduction of histidine content in treated tissues by histidine being converted to a non-detectable form. No other amino acid alteration was detected by amino acid analysis. Crosslink analysis of tissue hydrolyzates showed a time-dependent alteration in crosslink content of photooxidized tissue and an apparent addition of several types of new crosslinks. CONCLUSIONS: These chemical modifications are consistent with oxidative modification of amino acids in the tissues, resulting in an alteration of existing crosslinks and possible addition of new crosslinks in the tissues. This treatment process leads to in vivo and in vitro stability of pericardial and arterial tissues with potential use as bioprosthetic materials.

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 degrees 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 X 10(7) Bacillus subtilis, 4.6 X 10(6) Pseudomonas aeruginosa, or 7.2 X 10(6) Staphylococcus aureus and incubated at 37 degrees C. No survivors were detected on the tissue samples after exposure of 48 hr. Photooxidized pericardial tissue samples inoculated with either 3.2 X 10(5) porcine parvovirus or 1 X 10(9) infectious bovine rhinotracheitis were exposed to 0.1% iodine (pH 6.5) at 36 degrees C for 12 h. 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 the nonaldehyde, iodine based solution to sterilize implantable devices containing biologic tissue.

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.

Stabilization of pericardial tissue by dye-mediated photooxidation.

Bovine pericardial tissue was stabilized through a dye-mediated photooxidation reaction. Shrink temperature analysis of the stabilized tissue indicated a material with similar properties to untreated pericardial tissue and unlike identical tissue treated with glutaraldehyde. Photooxidized tissue was resistant to extraction when compared with untreated tissue or control tissues treated in the absence of light or dye. Photooxidized tissue was also resistant to enzymatic digestion by pepsin and to chemical digestion by cyanogen bromide (CNBr). In contrast, untreated or control treated tissues were readily digested by these reagents. Reduction of photooxidized tissue with beta-mercaptoethanol prior to CNBr digestion partially restored susceptibility of the tissue to CNBr digestion, indicating the photooxidation of methionine residues. Soluble collagen derived from bovine pericardium was used as a model compound for the photooxidation reaction. Polyacrylamide gel electrophoresis analysis indicated the photooxidative conversion of collagen into higher molecular weight aggregates consistent with intermolecular crosslink formation. Photooxidized tissue was stable to in vivo degradation when compared with control tissue. Results presented here indicate a crosslinked pericardial tissue produced by dye-mediated photooxidation possessing properties of chemical stability, enzymatic stability, in vivo stability, and biomechanical integrity suitable for use as a biomaterial.

A continuous fluorescence assay for protein kinase C.

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.

Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin.

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.