Influence of different chemical cross-linking treatments on the properties of bovine pericardium and collagen

Abstract

The use of biological materials in the construction of bioprostheses requires the application of different chemical or physical procedures to improve the mechanical performance of the material without producing any undesirable effects. A number of cross-linking methods have been tested in biological tissues composed mainly of collagen. The basis for most of them is the use of glutaraldehyde (GA), which acts on the Lys or Hyl residues. We have studied the effects of alternative chemical treatments: diphenylphosphorylazide (DPPA) and ethyldimethylaminopropyl carbodiimide (EDAC). Their mechanism of action is based on the activation of the carboxyl groups, which then permits their cross-linking to amino groups. As a control, we employed conventional treatment with GA, applying it to bovine pericardium and collagen membranes removed from bovine pericardium. The analysis of the Lys and Hyl residues showed that DPPA and EDAC produced 50% of the chemical change provoked by GA. This value was even lower in the trials with collagen. In terms of the resistance to collagenase degradation, chemical cross-linking with GA provided much greater protection in both materials (3.81±3.47 nmol of amino acid/mg dry tissue for pericardium and 4.41±1.13 nmol of amino acid/mg dry tissue for collagen). Treatment with DPPA also protected pericardium (13.11±6.57 nmol amino acid/mg dry tissue) although the values for collagen was lower (50.0±32.4 nmol amino acid/mg dry tissue). Treatment with EDAC was much less protective than the other two chemical reagents (43.28±17.4 and 55.85±14.57 nmol amino acid/mg dry tissue for pericardium and collagen, respectively). The degree of tissue calcification after implantation of the chemically treated materials into young rats was considerably greater for GA and DPPA (32.9±18.8 and 36.3±13.3 mg g -1 dry tissue, respectively) than with EDAC (18.0±7.2 mg g -1 dry tissue; P<0.001). After 60 days of implantation, the values for GA and EDAC were higher (124.1±31.3 and 124.6±21.0 mg g -1 dry tissue, respectively) versus 34.6±19.2 mg g -1 dry tissue for DPPA. There were no significant differences in collagen levels in samples treated with GA or EDAC after 30 days of implantation, although both groups showed significant differences when compared with DPPA-treated samples ( P<0.001). After 60 days of implantation, there were no significant differences among these three treatments in terms of the calcium accumulated on samples.