The effect of different treatment modalities on the calcification potential and cross-linking stability of bovine pericardium

Abstract

English: In the quest for suitable substitution materials to be used in surgical procedures, a large variety of different kinds of materials have been investigated. In cardiothoracic surgery, biological tissues such as porcine heart valves and bovine pericardium exhibited the most suitable properties for use as substitute material, while a variety of synthetic materials are also being used. Biological materials must meet a lengthy list of requirements, before it can be successfully and safely employed as substitution material. Amongst others, it needs to be stable against biological breakdown, easily sterilizable, express minimal immunogenicity, maintain mechanical strength and tissue stability, resist calcification, be non-carcinogenic and non-toxic and permit easy handling. Numerous chemicals and methodologies have been investigated in order to produce the most suitable materials attaining these properties. Glutaraldehyde has emerged as the chemical agent rendering most of these requirements to tissues following fixation and cross-linking with it. Despite the continued use of GA-fixed bovine pericardium worldwide, calcification and tissue degradation after 10-12 years post-implant remains a big problem. The main objective of this study was to try and identify additional biochemical treatment/s which can be employed in the fixation and storage of bovine pericardium, that will minimize the calcification potential of the tissue significantly without compromising the physical properties or the quality of the tissue. GA-fixed pericardial tissue was used as the control, and the outcomes of all the parameters for the other tissue treatments were compared against it. Numerical and categorical data were collected. In the first phase of study, four different methods of tissue treatment were compared for extractable calcium and water contents following 8 weeks implantation of treated samples in the subcutaneous rat model. Aluminium as treatment model was discarded due to the severe calcification of the implants. Results of tissue treated with GAGs were promising and compared favorably with commercial Glycar-treated tissue, and this prompted more detailed investigation. In the next phase of the study, mechanical properties (tensile strength) and crosslinking stability (thermal denaturation temperatures) of tissues treated with different concentrations of GAGs were compared with GA and Glycar-treated tissue. Treatment with a GAG concentration of 0.01M yielded tissue with comparable tensile strength and thermal denaturation temperatures above the minimum benchmark. This concentration was identified as the optimal GAG concentration to be investigated in subcutaneous rat implant studies. In the final phase, treated pericardial samples were implanted into weanling rats for 8 weeks and evaluated on the calcification potential, water content, antigenicity and extent of cross-linking of the collagen in the tissues. Tissue treated with 0.01M GAG compared favorably with the commercial Glycar patches regarding all of these parameters, outperforming GA-fixed control tissue significantly. Significant evidence was however found that added GAGs were still not effectively stabilized despite adding metaperiodate as fixative. GAGs leached out of tissue following an extended storage period. Only a limited amount of GAGs was visible on the outer surface of the explants compared to the layer of GAGs superficially bound to the tissue before implantation. Despite decreasing the tissue calcification substantially while maintaining good mechanical strength and low antigenicity, stabilization of the GAGs in treated tissues will have to be adequately addressed before clinical application of such tissues can be approved.

Afrikaans: In die soeke na geskikte produkte om as plaasvervangende materiale tydens chirurgiese prosedures te kan gebruik, is ‘n groot verskeidenheid van verskillende tipes produkte reeds ondersoek. Vir gebruik tydens kardiotorakale prosedures het biologiese weefsels soos varkkleppe en beesperikardium die beste aan die vereistes vir so ‘n produk voldoen. ‘n Verskeidenheid sintetiese materiale word egter ook vir hierdie doel gebruik. Biologiese produkte moet aan ‘n lang lys van vereistes voldoen, voordat dit suksesvol en veilig as plaasvervangende materiaal gebruik kan word. Die produk moet onder andere stabiel teen biologiese (ensimatiese) afbraak wees, maklik steriliseerbaar wees, minimale immuunreaksie van die ontvanger ontlok en genoegsame meganiese sterkte en weefselstabiliteit na prosessering behou. Verder moet die produk ook nie maklik verkalk nie, nie toksies of kankerverwekkend wees nie en maklike hantering toelaat. Verskeie chemikalieë en metodes is reeds ondersoek om die mees geskikte materiaal wat aan al hierdie vereistes voldoen, te lewer. Die chemiese reagens wat die meeste van hierdie vereistes aan biologiese weefsels kon toevoeg nadat hulle daarmee gefikseer is, is gluteraldehied. Ten spyte van die voortgesette wêreldwye gebruik van gluteraldehied-gefikseerde beesperikardium, bly die kalsifikasie en weefseldegenerasie na 10-12 jaar na inplantering steeds ‘n groot problem. Die hoofdoel van hierdie studie was om addisionele biochemiese behandelingsmetodes wat vir die fiksering en berging van die weefsel gebruik kan word, te identifiseer. Hierdie metodes moet die kalsifikasiepotensiaal van die perikardiale weefsel aansienlik verlaag, maar terselfdertyd nie die fisiese eienskappe en kwaliteit daarvan nadelig beїnvloed nie. Numeriese en kategoriese data is tydens die studie versamel. Gluteraldehiedgefikseerde perikardiale weefsel is deurgaans as kontrole gebruik om die uitkomste van al die parameters wat vir die ander behandelingsmetodes bepaal is, mee te vergelyk. Tydens die eerste fase van die studie is weefsel op vier verskillende metodes (gluteraldehied, aluminium, glikosaminoglikane en Glycar) behandel en gefikseer. Die weefselmonsters is hierna vir agt weke subkutaan in rotte ingeplant en die effektiwiteit van die behandelings is vergelyk ten opsigte van ekstraeërbare vogen kalsiuminhoud. Kontrole- en aluminiumbehandelde weefsel het uitermatig verkalk, en daar is op grond hiervan besluit om aluminium as behandelingsmetode te staak. Weefsel wat met glikosaminoglikane (GAG) gefikseer is het belowende resultate getoon en baie goed met kommersiële Glycar-weefsel vergelyk, en daar is besluit om verdere ondersoek hierna in te stel. In die volgende fase van die studie is weefsel wat met vyf verskillende konsentrasies GAG behandel is, vergelyk met GA-gefikseerde en Glycarweefsel ten opsigte van meganiese eienskappe (tensiele sterkte) en stabiliteit van die kruisbindings (protein denaturasie temperatuur). Die tensiele sterkte van weefsel wat met 0.01M GAG behandel is, was vergelykbaar met die ander twee metodes, terwyl die stabiliteit van die kruisbindings ook bo die aanvaarbare minimum standaard van 80oC was. Op grond van hierdie resultate is 0.01M GAG geїdentifiseer as die optimale GAG-konsentrasie om vir behandeling van weefsel vir verdere inplantings in rotte te gebruik. In die finale fase van die studie is weefsel wat met 0.0025M, 0.01M en 0.2M GAG behandel is, sowel as GA-gefikseerde en Glycar-behandelde weefsel vir agt weke in jong rotte ingeplant. Na herwinning is die weefsel vergelyk ten opsigte van water- en kalsiuminhoud en antigenisiteit, terwyl die omvang van die kruisbindings in die weefsels voor inplantering deur middel van die weerstand teen ensimatiese vertering bepaal is. Weefsel wat met 0.01M GAG behandel is het baie goed vergelyk met Glycarweefsel ten opsigte van al die parameters, en die GAgefikseerde kontroleweefsel beduidend oortref. Ten spyte daarvan dat metaperiodaat addisioneel gebruik is om die bygevoegde GAG in die perikardium te fikseer, is beduidende bewys gevind dat die GAG steeds nie effektief gestabiliseer was nie. GAG het gedurende ‘n lang stoorperiode uit die weefsel geloog, en baie min GAG was na inplantering in die rotte steeds op die oppervlak van die weefsel sigbaar vergeleke met voor inplantasie. Alhoewel behandeling van perikardium met GAG die kalsifikasiepotensiaal in rotte beduidend verlaag het terwyl goeie tensiele sterkte en lae antigenisiteit behou is, sal die doeltreffende stabilisering van GAG eers voldoende aangespreek moet word alvorens hierdie weefsel met vertroue vir kliniese gebruik aangewend kan word.