Elsevier

IRBM

Volume 29, Issue 4, September 2008, Pages 223-230
IRBM

Original article
Thrombogenicity studies of three different variants of processed bovine pericardiumÉtudes de thrombogénécité de trois différents variants des péricardes de bovins traités

https://doi.org/10.1016/j.rbmret.2007.07.003Get rights and content

Abstract

Versatile use of bovine pericardium in clinical cardiovascular surgery requires processing, especially cross-linking, to make the tissue non antigenic and mechanically strong. Forty-nine bovine pericardia were made acellular and then cross-linked by two different methods, group A (20) with formalin and group B (29) with heparin and subsequently, as a final measure, five of group B pericardia were gamma-ray sterilized with stipulated dose of 25 kGy and classified as group C. The surface property of thrombogenesis and haemolysis were compared in these three groups to identify a suitable method of processing so that it could be used in the vascular system without any thromboembolic complication. Extensive microscopical examination, mechanical testing and other physical property for biocompatibility studies were conducted on these three different groups with key focus on in vitro thrombogenicity studies. Heparin treated group B bovine pericardium appeared to be the best method of processing among these three by the above studies and was confirmed by laser confocal microscopy. Heparin cross-linked and heparin sodium treated processing had gained the higher tensile strength, and appeared to be nonthrombogenic, noncalcifiable (by animal experiments), biocompatible biomaterial, which can be used clinically.

Résumé

L’utilisation du péricarde bovin en chirurgie cardiovasculaire nécessite en particulier, un traitement par réticulation, afin de rendre le tissu non antigénique et de le renforcer mécaniquement. Quarante péricardes bovins ont été rendus acellulaires et ont été réticulés par deux méthodes différentes : le groupe A avec du formol (20), et le group B (29) avec de l’héparine. Cinq péricardes du groupe B ont été stérilisés par rayonnement gamma (25 kGy) et classés dans le groupe C. Les propriétés de la surface des péricardes traités vis-à-vis de la thrombogénèse et de l’hémolyse ont été comparées pour les trois groupes afin d’identifier la méthode de traitement appropriée en vue d’une utilisation clinique pour le système vasculaire sans risque de complications thromboemboliques.

Introduction

Bovine pericardium (BP) processing towards a clinical biomaterial is a common practice. Bovine pericardium tissue patches have been used for pericardial sac closure after open-heart surgery [2], vascular graft repair [1], [18] and in making a heart valve [12], [24].

It basically comprises of collagen type I, which has got low antigenicity, but high thrombogenicity [14]. It is prepared acellular and cross-linked by chemical or physical methods to reduce antigenicity [8], thereby preventing calcification this enhances the mechanical strength and minimizes xenogenic tissue solubility [3], [17]. Standard practice of glutaraldehyde cross-linking carries a high-risk of tissue fatigue and calcific degeneration [7], partly due to cytotoxicity and inflammatory changes [11], and fragmentation of collagen because of continuous wear and tear. To address the issue of calcification, Lee et al. developed a novel methodology for the chemical modification of biological tissues by directly coupling heparin to bovine pericardium (BP). Their heparinization involved pretreatment of BP using glutaraldehyde (GA) and was followed by grafting heparin to BP by the reaction of residual aldehyde with amine group of heparin. They evaluated the effect of heparin coupling on calcification by in vitro as well as in vivo studies. Their results revealed that heparinized BP exhibit greater resistance to collagenase digestion than control tissue [25].

The aim of this study was to find a better processing technique with effective cross-linking to produce a durable biomaterial, which remains flexible, biocompatible and nonthrombogenic. While analyzing the various methods for processing of the biomaterial, our focus was on thrombogenic studies. In this study, we made the tissue acellular, followed by cross-linking with heparin and subsequent treatment via anticalcification and then analysed for major thrombogenic parameters.

Section snippets

Materials and methods

Bovine pericardium was harvested and procured from an inspected abattoir in sterile environment. The specimens were stored in Hanks balanced salt solution (HBSS) having an antibiotic cocktail of cifran, gentamycin, streptomycin, cephalosporin and amphotericin B, and later brought into a current Good Manufacturing Practice (cGMP) laboratory for further processing.

Forty-nine bovine pericardial pieces were taken for study, where 20 numbers identified as group A processing were treated with

Results

All the groups had complete decellularisation by the standard method followed in the laboratory. High-resolution optical microscopy had shown better tissue homogeneity and integrity in group B which was supported by confocal microscopy (CM), and transmission electron microscopy (TEM). Higher tissue density was noticed by ESEM, in case of group B. Gamma irradiated tissue showed granular appearance by CM and TEM, fragmentation by ESEM, whereas group A ones showed nonintegrated separated matrix,

Discussion

Though bovine pericardium has potential to emerge as an excellent graft material, it has been plagued by problems inherent or due to inadequate processing techniques [7] (Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8). Decellularized bovine pericardium has propensity for calcification, unless the collagen is effectively cross-linked. Further calcification can induce thrombosis or vice versa [4] Cross-linking with heparin prevents early tissue deterioration by forming an artificial matrix made up of

Conclusion

Use of heparin as a cross-linking agent results in a superior biomaterial reflected by its favorable thrombogenic profile, mechanical and surface properties (smooth without pitting). Our study also indicates that gamma sterilization results in deleterious effect on the surface and mechanical properties of the tissue. It is to be noted that heparin cross-linking without gamma irradiation produces the best biomaterial and has been proved to be sterile. The validity of these results and

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