Detoxified glutaraldehyde cross-linked pericardium: tissue preservation and mineralization mitigation in a subcutaneous rat model.
OBJECTIVE
Glutaraldehyde is considered a promoter of calcification by the action of toxic aldehyde group residuals from cross-linking. Post-fixation treatment with homocysteic acid (HA), besides bonding aldehyde groups and neutralizing toxicity, should enhance biocompatibility due to the strongly electronegative sulfonic group. The aim of this investigation was to evaluate HA efficacy on tissue preservation and dystrophic calcification mitigation in glutaraldehyde cross-linked bovine pericardium (BP) using a subcutaneous rat model.


METHODS
Four samples of BP, two with glutaraldehyde-HA and two with glutaraldehyde treatment, were implanted in each of 24 male Sprague-Dawley rats. Three rats were killed at 14 days, eight at 28 days, eight at 56 days and five at 84 days. Unimplanted glutaraldehyde-HA- and glutaraldehyde-treated samples served as controls. All samples were studied by gross examination, mammography, light transmission and scanning electron microscopy, and atomic absorption spectroscopy. The nature of mineralization was investigated by coupling techniques of scanning electron microscopy, electron microprobe analysis and X-ray powder diffraction.


RESULTS
No histological and ultrastructural differences were found between glutaraldehyde-HA- and glutaraldehyde-treated BP, whether implanted or unimplanted. In both groups, calcification progressed with time, but significantly less after glutaraldehyde-HA treatment than after glutaraldehyde alone and at all time intervals (14.63 +/- 21.34 versus 43.17 +/- 15.99 at 28 days, p = 0.003; 56.42 +/- 40.20 versus 90.59 +/- 32.90 at 56 days, p = 0.008; 91.68 +/- 67.68 versus 156.23 +/- 17.85 at 84 days, p = 0.01). Differences were evident by mammography and histology (von Kossa stain). Electron microprobe analysis in both groups showed the composition of calcified nuclei to be calcium phosphate, stoichiometrically close to apatite (Ca5(PO4)3(OH)). The occurrence of crystallized apatite was supported by X-ray powder diffraction findings, the amount of crystallized apatite being higher in glutaraldehyde-treated samples.


CONCLUSIONS
Post-fixation treatment with HA preserves BP structural properties and significantly mitigates mineralization of long-term subcutaneous implants.
Valente
M
M
University of Padua and Modena, Sorin Biomedica-Cardio, Saluggia, Italy.
Pettenazzo
E
E
Thiene
G
G
Molin
G M
GM
Martignago
F
F
De Giorgi
G
G
Gatti
A M
AM
Giaretta
A
A
Pasquino
E
E
Talenti
E
E
Rinaldi
S
S
D000818:Animals; D001672:Biocompatible Materials; D001705:Bioprosthesis; D002114:Calcinosis / Q000517:prevention & control*; D002417:Cattle; D004577:Electron Probe Microanalysis; D005976:Glutaral / Q000494:pharmacology; D006350:Heart Valve Prosthesis; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010496:Pericardium / Q000737:chemistry / Q000637:transplantation* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014021:Tissue Preservation / Q000379:methods; D014961:X-Ray Diffraction
D001672:Biocompatible Materials; D006710:Homocysteine; C007956:homocysteic acid; D005976:Glutaral