Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their ␣-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and Nterminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

The network structure of native and carbodiimide cross-linked gelatin A and B gels was studied based on their rheological behavior. Gelatin A and B contain different numbers of carboxylic acid groups caused by different preparation conditions and had previously shown different characteristics in controlled release applications. It was evaluated to which extent chemical cross-linking densified the network structure of physical gelatin gels. After normalization of the equilibrium shear modulus (G e) with respect to swelling (Q), it was observed that the normalized G e values largely depend on the way gelatin is prepared from collagen. At an equal number of chemical junctions, chemically cross-linked gelatin B gels had a lower elasticity modulus than chemically cross-linked gelatin A gels. This seemed contradictory as gelatin B contains more carboxylic acid groups, available for cross-linking, but is related to a higher probability for intramolecular cross-linking, as was validated quantitatively by chemical and rheological analysis of the number of cross-links. Assuming an ideal network, the average molecular weight of the elastic network chains (M c) was calculated for physical and chemical gelatin A and B networks, and on the basis of M c the mesh sizes of the gels were estimated. The calculated mesh sizes were experimentally confirmed by lysozyme and albumin diffusion. Chemical cross-linking increased the resistance of the gels toward thermal degradation, resulting in a more gradual disintegration of physical cross-links upon heating. Moreover, chemical cross-linking prevented recombination of these cross-links upon cooling.

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOHgelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOHgelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40 degrees C.

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Gelatin gels were applied to porous Dacron meshes with the aim of using these gels for local drug delivery. In this article, the biocompatibility and degradation of gelatin gels with different crosslink densities applied in Dacron were studied in vivo by subcutaneous implantation in rats. Dacron discs were treated with carbon dioxide gas plasma to improve hydrophilicity, and subsequently impregnated with gelatin type B. The gelatin samples were crosslinked to different extents using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). After 6 h, 2, 5, and 10 days, and 3, 6, and 10 weeks of postimplantation, the tissue reactions and biodegradation were studied by light microscopy. The early reaction of macrophages and polymorphonuclear cells to crosslinked gelatin was similar to or milder than Dacron. Giant cell formation was predominantly aimed at Dacron fibers and was markedly reduced in the presence of a crosslinked gelatin coating. At week 10 of implantation, the crosslinked gelatin gels were still present in the Dacron matrix. The gelatin degradation was less for samples with the highest crosslink density. The gelatin gel with the lowest crosslink density showed clear cellular ingrowth, starting after 6 weeks of implantation. The intermediate and high crosslinked gelatin gels showed little or no ingrowth. In these gels, giant cells were involved in the phagocytosis of gelatin parts at week 10. Application of carbodiimide crosslinked gelatin gels in Dacron is suitable for medical applications because of the good biocompatibility of the gels and the possibility of adapting the degradation rate of gelatin to a specific application.

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Chemically cross-linked gelatin−chondroitin sulfate (ChS) hydrogels were prepared for the controlled release of small cationic proteins. The amount of chondroitin sulfate in the gelatin gels varied between 0 and 20 wt %. The chemical cross-link density, the degree of swelling, and the rheological behavior were determined to characterize the cross-linked hydrogels. Chemically cross-linked gelatin−ChS hydrogels were loaded with lysozyme, and the release was measured using phosphate-buffered saline. The lysozyme loading capacity of the hydrogels significantly increased with increasing chondroitin sulfate content of the gels. Compared to plain gelatin gels, the release rate of lysozyme slowed for the hydrogels containing 5 and 10 wt % of chondroitin sulfate, while the release was faster for hydrogels containing 20 wt % of chondroitin sulfate. The permeation of lysozyme through gelatin−ChS gels was measured using a two-compartment diffusion cell, and the effective diffusion coefficient was calculated. The effective diffusion of lysozyme in the gels was also qualitatively studied using fluorescence recovery after photobleaching. The Langmuir isotherms of lysozyme adsorption to gelatin−ChS gels and the lysozyme diffusion in the gels in the absence of electrostatic interactions were determined to evaluate the contributions of unspecific interaction between lysozyme and chondroitin sulfate and diffusion to the release. Both the interaction and the diffusion increase with increasing chondroitin sulfate content of the hydrogels, which resulted in a minimum value of the effective release rate for gels containing 5 wt % chondroitin sulfate.

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