No abstract

While attempting to find a suitable crosslinking reagent for biopolymers, a naturally occurring proanthocyanidin (PA) obtained from grape seeds was selected to fix biological tissues. The cytotoxicity and crosslinking rate, reflected by the in vitro and in vivo degradation of fixed matrices has been studied. The shrinkage temperature of the fixed bovine pericardium increased from 66 to 86 degrees C. A cytotoxicity assay using fibroblast cultures revealed that PA is approximately 120 times less toxic than glutaraldehyde (GA), a currently used tissue stabilizer. In vitro degradation studies showed that fixed tissue was resistant to digestion by bacterial collagenase. Crosslinks between PA and tissues can be stabilized by decreasing the dielectric constant of the solution during storage. After subcutaneous implantation for periods ranging between 3 and 6 weeks, we found no apparent degradation of the GA- or PA-fixed tissues, whereas fresh tissue controls rapidly disintegrated. Beyond 6 weeks PA crosslinks began to degrade. More fibroblasts migrated and proliferated inside the PA-fixed implants compared with GA counterparts. Tissues crosslinked with PA manifested an enhanced collagen expression and deposition and did not calcify after implantation. GA, on the other hand, even after thorough rinsing continued to be cytotoxic, inhibited collagen synthesis and encouraged dystrophic calcification. Collagen matrices crosslinked with PA are expected to be of value in the design of matrices that will encourage cell ingrowth and proliferation, which are temporary in nature, and that are intended to regenerate or replace missing tissues, which can delay the biogradation of collagen. As such they should be of significant value in the emerging field of tissue engineering.

Glutaraldehyde crosslinking of native or reconstituted collagen fibrils and tissues rich in collagen significantly reduces biodegradation. Other aldehydes are less efficient than glutaraldehyde in generating chemically, biologically, and thermally stable crosslinks. Tissues crosslinked with glutaraldehyde retain many of the viscoelastic properties of the native collagen fibrillar network which render them suitable for bioprostheses. Implants of collagenous materials crosslinked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing crosslinking through bridging of activated carboxyl groups with diamines and using glutaraldehyde to crosslink the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic diamines. This crosslinking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypropane-1, 1-diphosphonic acid (3-APD), and chondroitin sulfate to collagen or to the crosslink-enhanced collagen network reduces its potential for calcification. Platelet aggregation is also reduced by glutaraldehyde crosslinking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde--leaching through the interstices of the collagen fibrils or the tissue matrix--and of reactive aldehydes associated with the bound polymeric glutaraldehyde can be minimized by neutralization and thorough rinsing after crosslinking and storage in a nontoxic bacteriostatic solution.

The reversibility of glutaraldehyde crosslinks has been suggested as a reason for failure of long-term bioprosthetic implants. The stability of such crosslinks was investigated in tendons and model compounds. Small but cytotoxic levels of glutaraldehyde were still released from crosslinked tendons even after these tendons were extensively rinsed for up to 6 months. The toxic effect was evidenced by the death of fibroblasts surrounding a midsection piece of rinsed crosslinked tendon, while the end section pieces did not show toxic effects. The formation and stability of glutaraldehyde modified [14C]-L-lysine derivatives were investigated. The polymerization of glutaraldehyde with amino compounds was initially fast but continued to proceed slowly for months. Degradation of high-molecular-weight soluble polymers was detected by gel filtration chromatography. Low-molecular-weight soluble materials were also released from insoluble products which were formed when high concentrations of glutaraldehyde and radioactive lysine were reacted. These chemical and biological studies suggest that local cytotoxicity of glutaraldehyde crosslinked bioprostheses may be due to unstable glutaraldehyde polymers that persist in the interstices of crosslinked tissues.