ABSTRACT
Collagen-based scaffolds are extensively utilized as an analog for the extracellular matrix in cultured skin substitutes (CSS). To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed. In this study, freeze-dried collagen-GAG sponges were crosslinked with increasing concentrations of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC; 0, 1, 5, 10, 50mm). Cross-linking with EDC at concentrations >1mm was shown to greatly decrease degradation by collagenase up to 21 days. Ultimate tensile strength (UTS) of acellular collagen sponges scaled positively with EDC concentration up to 10mm. At 50mm EDC, the UTS decreased dramatically likely due to the brittle nature of the highly crosslinked material. Co-culture of human fibroblasts (HF) and keratinocytes (HK) on these substrates reveals an apparent cytotoxicty of the EDC at high concentrations with reduced cell viability and poor cellular organization in CSS fabricated with scaffolds crosslinked with 10 or 50mm EDC. From the data gathered in this study, intermediate concentrations of EDC, specifically 5mm, increase collagen sponge stability and strength while providing an environment in which HF and HK can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration.
Subject(s)
Collagen/metabolism , Collagen/ultrastructure , Cross-Linking Reagents/chemistry , Ethyldimethylaminopropyl Carbodiimide/chemistry , Glycosaminoglycans/metabolism , Glycosaminoglycans/ultrastructure , Skin, Artificial , Coculture Techniques , Collagen/chemistry , Ethyldimethylaminopropyl Carbodiimide/toxicity , Fibroblasts/drug effects , Freeze Drying , Glycosaminoglycans/chemistry , Humans , Keratinocytes/drug effects , Materials Testing , Microscopy, Electron, Scanning , Stress, MechanicalABSTRACT
The haemocompatibility of vascular, Dacron prostheses was improved by coating with albumin and/or collagen crosslinked with glutaraldehyde (GTA) or carbodiimide (CDI). F.p.l.c. (fast protein liquid chromatography) analysis of the products desorbed from polymeric matrices incubated in physiological conditions for periods extended up to 10 d did not detect the monomers or polymers of collagen and albumin but small amounts of degradation products, the molecular weights of which were less than 45,000, thus minimizing an eventual immunogenic response after implantation. However GTA and CDI matrices required extensive washing to neutralize the cytotoxic effect of GTA and achieve the release of CDI from protein complexes.
Subject(s)
Biocompatible Materials , Blood Vessel Prosthesis , Proteins , Animals , Blood Vessel Prosthesis/adverse effects , Cell Survival/drug effects , Cells, Cultured , Chromatography, High Pressure Liquid , Collagen , Cross-Linking Reagents/toxicity , Ethyldimethylaminopropyl Carbodiimide/toxicity , Glutaral/toxicity , In Vitro Techniques , Permeability , Serum Albumin, BovineABSTRACT
For the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate the reactive reagent N-ethyl-N'-(dimethylaminopropyl) carbodiimide is used. The application of this reagent results in a number of stable linkages (viz. "peptide" linkages between the polysaccharide and tetanus toxoid, intrachain ester linkages in the polysaccharide component and binding of the N-acylurea derivative of the reagent) and less stable ones (viz. anhydride linkages). As a consequence of the reaction, the reagent is converted to a non-reactive urea derivative. The toxic properties of the reagent and of the converted reagent were studied. These properties do not contraindicate the use of the coupling reagent for the preparation of vaccines for human use. In addition analytical methods have been developed for the quantitative evaluation of the coupling reagent, the reaction products and for the N-acylurea derivative of the reagent and of the residual reactivity of conjugates for primary aminogroups. Although no test was performed for the assay of ester linkages in the polysaccharide component of the conjugate, evidence is presented that such linkages may be present. The results of the test for residual reactivity indicated a spontaneous rearrangement of linkages after the preparation of the conjugate. In addition the influence of the ratio of coupling reagent-to-polysaccharide and tetanus toxoid on antigenic and immunogenic activities of the conjugate was studied. An increase of the ratio resulted in a decrease of the antigenic activity of the polysaccharide component but in an increase of its immunogenic activity as to the induction of IgG antibodies to the polysaccharide. The immunogenic activity of the polysaccharide component correlated rather well with the antigenic activity measured in heterologous enzyme-linked immunosorbent assay using antibodies to both components.
