Richard E. Phillips

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.

Blood and tissue compatibility of modified polyester: Thrombosis, inflammation, and healing

Poly(ethylene terephthalate) (PET) has been reported in literature to be moderately inflammatory and thrombogenic. To moderate the inflammatory response, PET fabric was surface modified by either Fluoropassiv fluoropolymer (FC), or an RGD-containing peptide (RGD). Samples were subsequently autoclave sterilized and implanted subcutaneously in Sprague Dawley rats for 2 to 4 weeks. Retrieved samples were evaluated histopathologically for indications of material toxicity and healing. Minimal acute or chronic inflammation was associated with the fabrics after 2 and 4 week implant duration. However, fibroblast proliferation into FC modified fabric (PET/FC) was less than that into unmodified (PET) and RGD modified fabric (PET/RGD) after 4 weeks, suggesting that FC modification of PET may inhibit excessive tissue growth. Additional samples of modified and unmodified fabrics were placed in stainless steel mesh cages, which were then implanted subcutaneously for 4 weeks. Cellular exudate was extracted weekly and cell concentrations within the exudate measured. Total leukocyte count (TLC) (reflective of local inflammation) at 1 week for PET/RGD was greater than that for PET/FC and PET. TLCs after 4 week implant decreased for all sample groups. In a separate experiment, PET vascular grafts surface modified by either FC or RGD were contacted 1 h with blood using the baboon arteriovenous (AV) shunt model of thrombosis in both the presence and absence of heparin. Accumulation of 111In labeled platelets (reflective of thrombus accumulation) upon grafts was less in the presence of heparin (effect significant at p = 1.2 x 10(-6), two-way ANOVA). Accumulation (in the presence of heparin) upon PET/RGD was less (p = 0.19), and upon PET/FC significantly less (p = 0.016) than that upon the unmodified PET control, suggesting that FC modification of PET may inhibit thrombus accumulation.

Calcification of polyurethanes implanted subdermally in rats is enhanced by calciphylaxis

Calcification complicates the use of the polymer polyurethane in cardiovascular implants. To date only costly experimental circulatory animal models have been useful for investigating this disease process. In this paper we report that polyurethane calcification in rat subdermal implants is enhanced by overdosing with a vitamin-D analog. The calcification-prone state, known as calciphylaxis, was induced in 4-week old rats by oral administration of a vitamin-D analog, dihydrotachysterol. We studied two commercially available polyurethanes (Biomer and Mitrathane) and two proprietary polyurethanes (PEU-2000 and PEU-100). PEU-100 is unique because it is derivatized with ethanehydroxy-bisphosphonate (EHBP) for calcification resistance. Polyurethane calcium and phosphate levels and morphological changes due to calciphylaxis were compared with those of control rat subdermal explants in 60-day studies. Increased polyurethane mineralization was observed due to calciphylaxis with 60-day rat subdermal explants of Biomer, Mitrathane, and PEU-2000 (calcium levels, respectively, 4.13 +/- 0.56, 18.61 +/- 2.73, and 3.37 +/- 0.22 microgram/mg, mean +/- standard error) as compared to control explants (calcium levels, respectively, 1.22 +/- 0.1, 12.57 +/- 0.86, and 0.20 +/- 0.86 microgram/mg). The study also demonstrated that with 60-day implants calciphylaxis had no side effects on somatic growth and serum calcium levels. Explant surface morphology of these polyurethane explants examined by scanning electron microscopy, back scattering electron imaging coupled with energy dispersive X-ray spectroscopy, and light microscopy demonstrated the presence of predominantly surface-oriented calcification. PEU-100, derivatized with 100 n.moles/ mg of EHBP, resisted calcification with explant calcium levels 0.51 +/- 0.01 (calciphylaxis) and 0.38 +/- 0.01 (control) microgram/mg. It is concluded that calciphylaxis enhances superficial polyurethane calcification in rat subdermal implants and that an EHBP-modified polyurethane resists calcification despite calciphylaxis. Rat subdermal implants using calciphylaxis may be generally useful for evaluating the calcification potential of various biomedical polymers.

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.

Degradation of polyurethanes in vitro and in vitro: comparison of different models

This study compares and contrasts mechanisms of polyetherurethane (PEU) degradation m vitro and in vivo. Models comprising incubation with hydrogen peroxide in vitro (H202). m vtvo subcutaneous rat implant (SUBQ). and subcutaneous rat cage implant (CAGE) are described and compared with in vivo degradation of the pacemaker lead device retrieved after human implant (PACE). Experimental results support the hypothesis that stress accelerates PEU degradation. Scanning electron microscopy (SEMI, gel permeation chromatography (GPC), and Fourier transform IR spectroscopy/attenuated total reflectance (FT-IR/ATR) evaluation of tested PEU samples suggests, for all models. decreased soft segment and increased ester functtonality at the polymer surface These observations are consistent with a single, metal ton catalyzed. polyester intermediate, oxidative degradation mechanism common to all models, and with device performance m VIVO. Model comparison suggests that m vitro H,Oz and in vivo SUBQ and CAGE models accurately mimic m vtvo degradation of the pacemaker lead device (PACE).