A cross-linking method for collagen-based biomaterials was developed using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EN). Cross-linking using EDC involves the activation of carboxylic acid groups to give 0-acylisourea groups, which form cross-links after reaction with free amine groups. Treatment of dermal sheep collagen (DSC) with EDC (E-DSC) resulted in materials with an increased shrinkage temperature (T5) and a decreased free amine group content, showing that cross-linking occurred. Addition of N-hydroxysuccinimide to the EDC-containing cross-linking solution (E/N-DSC) increased the rate of cross-linking. Cross-linking increased the 7, of non-cross-linked DSC samples from 56 to 73°C for E-DSC and to 66°C for E/N-DSC samples, respectively. For both cross-linking methods a linear relation between the decrease in free amine group content and the increase in Ts was observed. The tensile strength and the high strain modulus of E/N-DSC samples decreased upon cross-linking from 18 to 15 MPa and from 26 to 16 MPa, respectively. The elongation at break of E/N-DSC increased upon cross-linking from 142 to 180%. Keywords:Collagen, cross-linking, carbodiimide Received 16 March 1995; accepted 15 June 1995The high enzymatic turnover rate of collagen in the body makes stabilization of collagen-based biomaterials by chemical cross-linking methods necessary to give materials that maintain the desired mechanical properties and stability during the desired implantation period. Several cross-linking methods have been reported and in principle they can be divided into two groups. First, bifunctional reagents can be used to bridge amine groups of lysine or hydroxylysine residues of different polypeptide chains by monomeric or oligomeric cross-links. Second, amide-type cross-links can be formed by activation of the carboxylic acid groups of glutamic and aspartic acid residues followed by reaction of these activated carboxylic acid groups with amine groups of another polypeptide chain.Based on the use a4 bifunctional reagents for crosslinking, glutaraldehyde (GA) has generally been applied for the stabilization of collagen-based materials'.The use of hexamethylene diisocyanate (HMDIC) as a cross-linking agent was introduced by Chvapil et al. '. A promising class of cross-linking agents for collagen :more recently described is the polyepoxy compounds3,4.GA cross-linking involves the formation of short (branched) aliphatic chains' and pyridinium compounds5V6, while -in HMDIC cross-linking aliphatic Correspondence to Professor Dr J. Feijen. chains containing urea bonds are introduced between two adjacent amine groups7. Both GA and HMDIC cross-linking may lead to the presence of unreacted functional groups (probably aldehyde or amine groups after hydrolysis of isocyanate groups) in the collagen matrix, which can result in a cytotoxic reaction upon degradation of the collagens9'. Furthermore, it has been reported that GA cross-linked collagen-based biomaterials releases toxic GA (related...

The formation of Schiff bases during crosslinking of dermal sheep collagen (DSC) with glutaraldehyde (GA), their stability and their reactivity towards GA was studied. All available free amine groups had reacted with GA to form a Schiff base within 5 rain after the start of the reaction under the conditions studied (0.5% (w/w) GA). Before crosslinks are formed the hydrolysable Schiff bases initially present were stabilized by further reaction with GA molecules. An increase in shrinkage temperature (Ts) from 56 °C for non-crosslinked DSC (N-DSC) to 78°C for GA crosslinked DSC (G-DSC) was achieved after crosslinking for 1 h. From the relationship between the free amine group content and the Ts during crosslinking it was concluded that higher GA concentrations and longer reaction times will result in the introduction of pendant-GA-related molecules rather than crosslinks. After 24 h crosslinking an average uptake of 3 GA molecules per reacted amine group was found. No increase in the tensile strength of the materials was observed after crosslinking, which may be a result of formation of crosslinks within the fibres rather than in between fibres. Aligning of the fibres by applying a pre-strain to the samples and subsequent crosslinking yielded materials with an increased tensile strength.

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Bacterial collagenase was used to study the susceptibility of dermal sheep collagen (DSC) cross-linked with a mixture of the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide (E/N-DSC) towards enzymatic degradation. Contrary to non-cross-linked DSC (N-DSC), which had a rate of weight-loss of 18.1% per hour upon degradation, no weight loss was observed for E/N-DSC during a 24 h degradation period. The tensile strength of the E/N-DSC samples decreased during this time period, resulting in partially degraded samples having 80% of the initial tensile strength remaining. The susceptibility of E/N-DSC samples towards enzymatic degradation could be controlled by varying the degree of cross-linking of the samples. Ethylene oxide sterilization of E/N-DSC samples made the material more resistant against degradation compared with non-sterilized E/N-DSC samples. This may be explained by a decrease of the adsorption of bacterial collagenase onto the collagen owing to reaction of ethylene oxide with remaining free amine groups in the collagen matrix.

The biocompatibility and tissue regenerating capacity of four crosslinked dermal sheep collagens (DSC) was studied. In vitro, the four DSC versions were found to be noncytotoxic or very low in cytotoxicity. After subcutaneous implantation in rats, hexamethylenediisocyanate-crosslinked DSC (HDSC) seldom induced an increased infiltration of neutrophils or macrophages, as compared with normal wound healing; whereas new formation of collagen was observed. DSC crosslinked with glutaraldehyde (GDSC) followed by reaction with NaBH4 shortly after implantation showed an increased infiltration of neutrophils with a deviant morphology. Furthermore, a high incidence of calcification was observed, which may explain the minor ingrowth of giant cells and fibroblasts, and the poor formation of new rat collagen. Acyl azide-crosslinked DSC (AaDSC) first induced an increased infiltration of macrophages, and then of giant cells, both with high lipid formation. AaDSC degraded at least twice as slowly as HDSC and GDSC, finally leaving a matrix of newly formed rat collagen. Samples crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (ENDSC) induced the same mild cellular reaction as HDSC; whereas, similar to AaDSC, the degradation rate was slow and an optimal rat collagen matrix was formed. Of the crosslinked DSC samples, ENDSC seems most promising for tissue regeneration.

This year we celebrate the first centennial of the discovery of germinal centers by Flemming in 1884. The present paper reviews and adds new data to the functional anatomy of a germinal center. Emphasizing its reactive nature, we first describe a germinal center reaction and then deal with its infrastructural aspects and constituent cell populations, both lymphoid and nonlymphoid. Elements involved in the de novo formation of a germinal center, like antigen, T cells, and the mysterious germinal-center-precursor cell, are discussed. Next, attention is paid to the requirements for lymphoid cells to migrate into germinal centers, and novel features of germinal-center-seeking cells are presented. Subsequently, we discuss kinetic aspects of the high proliferative activity in a germinal center; and finally a description of the functional capacities of germinal-center-derived cells, such as B memory cells and IgM-antibody-forming cell precursors, completes this picture of present-day knowledge of the germinal center, a structure which has yet to reveal its last secrets.

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