Biocompatibility and tissue regenerating capacity of crosslinked dermal sheep collagen

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 cytoxicity. After subcutaneous implantation in rats, hexamethylenediisocyanatecrcrosslinked 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

Calcification of subcutaneously implanted collagens in relation to cytotoxicity, cellular interactions and crosslinking

In general, calcification of biomaterials occurs through an interaction of host and implanted material factors, but up to now the real origin of pathologic calcification is unknown. In this study we aimed to investigate incidence of calcification of (crosslinked) dermal sheep collagens (DSCs) with respect to their specific properties, during subcutaneous implantation in rats. Three types of DSCs were commercially obtained: non-crosslinked DSC (NDSC), and DSC crosslinked with glutaraldehyde (GDSC) and hexamethylenediisocyanate (HDSC). NDSC, HDSC and GDSC were (enzymatically) tissue culture pretreated to eliminate their cytotoxic products. Beside this, crosslinking methods were modified to optimize mechanical properties and to decrease cytotoxicity, which resulted in HDSC* and GDSC*. Furthermore, DSC was crosslinked by activation of the carboxylic groups, i.e. by means of acyl azide and carbodiimide, resulting in AaDSC and CDSC, respectively. After implantation of HDSCs and GDSCs a relation between cytotoxicity and calcification of crosslinked DSC could be made. No relation was found between cellular infiltration of DSCs and calcification. However, from the use of different types and modification of crosslinking methods it might be concluded that calcification is mainly related to stable crosslinks, i.e. to the chemical properties of the obtained material

Influence of ethylene oxide gas treatment on the in vitro degradation behavior of dermal sheep collagen

The influence of ethylene oxide gas treatment on the in vitro degradation behavior of noncrosslinked, glutaraldehyde crosslinked or hexamethylene diisocyanate crosslinked dermal sheep collagen (DSC) using bacterial collagenase is described. The results obtained were compared with the degradation behavior of either nonsterilized or γ-sterilized DSC. Upon ethylene oxide sterilization, reaction of ethylene oxide with the free amine groups of DSC occurred, which resulted in a decreased helix stability, as indicated by a lowering of the shrinkage temperature of all three types of DSC. Except for the low strain modulus the mechanical properties of the ethylene oxide sterilized materials were not significantly altered. γ-Sterilization induced chain scission in all three types of DSC, resulting in a decrease of both the tensile strength and the high strain modulus of noncrosslinked and crosslinked DSC. When exposed to a solution of bacterial collagenase, ethylene oxide sterilized materials had a lower rate of degradation compared with nonsterilized DSC. This has been explained by a reduced adsorption of the collagenase onto the collagen matrix as a result of the introduction of pendant N-2-hydroxy ethyl groups

Changes in the mechanical properties of dermal sheep collagen during in vitro degradation

The changes in tensile strength, elongation at break, and high strain modulus of dermal sheep collagen (DSC) during in vitro degradation using bacterial collagenase were studied. The changes in mechanical properties were compared with the change in weight of the samples as a function of degradation time. DSC was crosslinked with either glutaraldehyde (GA) or hexamethylene diisocyanate (HMDIC). During degradation, the changes in mechanical properties of the N-DSC, H-DSC or G-DSC samples were more pronounced than the changes in the weight of the samples. Of the mechanical properties studied, the tensile strength was most susceptible to degradation of the DSC samples. After 2.5 h, N-DSC samples had lost only 55% of their initial weight, but the samples had no tensile strength left. Similar results were obtained for H-DSC, which retained no tensile strength after 24 h degradation, whereas only 45% of the initial weight was lost. G-DSC lost 3.5% of its weight after 24 h degradation, but only 25% of the initial tensile strength remained

Secondary cytotoxicity of (crosslinked) dermal sheep collagen during repeated exposure to human fibroblasts

We investigated commercially available dermal sheep collagen either cross-linked with hexamethylenediisocyanate, or cross-linked with glutaraldehyde. In previous in vitro studies we could discriminate primary, i.e. extractable, and secondary cytotoxicity, due to cell-biomaterial interactions, i.e. enzymatic actions. To develop dermal sheep collagen for clinical applications, we focused in this study on the release, e.g. elimination, of secondary cytotoxicity over time. We used the universal 7 d methylcellulose cell culture with human skin fibroblasts as a test system. Hexamethylenediisocyanate-cross-linked dermal sheep collagen and glutaraldehyde-cross-linked dermal sheep collagen were tested, with intervals of 6 d, over a culture period of 42 d. With hexamethylenediisocyanate-cross-linked dermal sheep collagen, cytotoxicity, i.e. cell growth inhibition and deviant cell morphology, was eliminated after 18 d of exposure. When testing glutaraldehyde-cross-linked dermal sheep collagen, the bulk of cytotoxic products was released after 6 d, but a continuous low secondary cytotoxicity was measured up to 42 d. As a control, non-cross-linked dermal-sheep collagen was tested over a period of 36 d, but no secondary cytotoxic effects were observed. The differences in release of secondary cytotoxicity between hexamethylenediisocyanate-cross-linked dermal sheep collagen, glutaraldehyde-cross-linked dermal sheep collagen and non-cross-linked dermal sheep collagen are explained from differences in cross-linking agents and cross-links obtained. We hypothesize that secondary cytotoxicity results from enzymatic release of pendant molecules from hexamethylene-diisocyanate-cross-linked dermal sheep collagen, e.g. formed after reaction of hydrolysis products of hexamethylenediisocyanate with dermal sheep collagen. Glutaraldehyde-cross-linked dermal sheep collagen contains residual cross-linking agents, which induce the bulk cytotoxicity. Apart from being sensitive to enzymatic degradation, glutaraldehyde-cross-linked dermal sheep collagen was also found to be sensitive to aqueous hydrolysis. Hydrolysis of cross-links may release cytotoxic products and introduce new pendant molecules within glutaraldehyde-cross-linked dermal sheep collagen, which in turn induce cytotoxicity after enzymatic attack

Crosslinking of dermal sheep collagen using hexamethylene diisocyanate

The use of hexamethylene diisocyanate (HMDIC) as a crosslinking agent for dermal sheep collagen (DSC) was studied. Because HMDIC is only slightly water soluble, a surfactant was used to obtain a clear and micellar crosslinking solution and to promote the penetration of HMDIC in the DSC matrix. Using optimized conditions treatment of non-crosslinked DSC (N-DSC) with HMDIC (H-DSC) increased the shrinkage temperature (Ts) of N-DSC from 56°C to 74°C for H-DSC. A linear relation between the decrease in free amine group content and the increase in Ts was observed. Crosslinking with HMDIC did not influence the tensile strength of the N-DSC samples but increased the elongation at break from 141% to 163% and decreased the high-strain modulus from 26 MPa to 16 MPa for the H-DSC samples, respectively

Cross-linking of dermal sheep collagen using a water-soluble carbodiimide

A cross-linking method for collagen-based biomaterials was developed using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC). Cross-linking using EDC involves the activation of carboxylic acid groups to give O-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 (Ts) 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 Ts of non-cross-linked DSC samples from 56 to 73 °C for E-DSC and to 86 °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 15MPa and from 26 to 16MPa, respectively. The elongation at break of E/N-DSC increased upon cross-linking from 142 to 180%

Does proteolysis explain glutamine release from the septic brain?

Berg and colleagues report on amino acid exchange across the human brain during endotoxin infusion. Lipopolysaccharide infusion induced a decrease in the ratio between branched chain amino acids and aromatic amino acids, increased unidirectional phenylalanine uptake, and increased net brain glutamine release. Cerebral proteolysis is suggested to play a role, but the question is whether this is the case and why this would happen