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

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

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%

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

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

CD34+cells augment endothelial cell differentiation of CD14+endothelial progenitor cells in vitro

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34+ cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34+ cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14+ cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14+ cell-derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34+ and CD14+ cells augments endothelial differentiation of the CD14+ cells. In vitro, the influence of CD34+ cells on the endothelial differentiation capacity of CD14+ cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14+-derived cells adopted an endothelial cell phenotype, whereas in CD34+/CD14+ co-cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34+/CD14+ co-cultures compared to both monocultures. CD34-conditioned medium also increased endothelial differentiation of CD14+ cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin-8 and monocyte chemoattractant protein-1 neutralizing antibodies. We show that co-culturing of CD34+ and CD14+ cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction

Material dependent differences in inflammatory gene expression by giant cells during the foreign body reaction

Multinucleated giant cells (GCs) are often observed in the foreign body reaction against implanted materials. The in vivo function of GCs in this inflammatory process remains to be elucidated. GCs degrade collagen implants in rats and may also orchestrate the inflammatory process via the expression and secretion of modulators, such as cytokines and chemokines. In this study, we show that the gene expression of PMN chemoattractants, CXCL1/KC and CXCL2/MIP-2, is high in GCs micro-dissected from explanted Dacron, cross-linked collagen (HDSC), and bioactive ureido-pyrimidinone functionalized oligocaprolactone (bioactive PCLdiUPy). Conversely, the gene expression levels of TGF beta and pro-angiogenic mediators VEGF and FGF were found to be low in these GCs as compared with the expression levels in total explants. GCs in bioactive PCLdiUPy displayed high cytokine and angiogenic mediator expression compared with GCs isolated from the two other studied materials, whereas chemokine gene expression in GCs isolated form HDSC was low. Thus, GCs adopt their expression profile in response to the material that is encountered. (c) 2007 Wiley Periodicals, In

In vivo testing of crosslinked polyethers. I: Tissue reactions and biodegradation

The in vivo biocompatibility and biodegradation of crosslinked (co)polyethers with and without tertiary hydrogen atoms in the main chain and differing in hydrophilicity were studied by means of subcutaneous implantation in rats. After 4 days, 1 month, and 3 months postimplantation, the tissue reactions and interactions were evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Poly(tetrahydrofuran) (poly(THF)), poly(propylene oxide) (poly(POx)), and poly(tetrahydrofuran-co-oxetane) (poly-(THF-co-OX)) were tested as relatively hydrophobic polyethers, and poly(ethylene oxide) (PEO) and a poly(THF)/PEO blend were used as more hydrophilic materials. In general, all polyethers showed good biocompatibility with respect to tissue reactions and interactions, with low neutrophil and macrophage infiltration, a quiet giant cell reaction, and formation of a thin fibrous capsule. For the relatively hydrophobic polyethers studied, the biostability increased in the order poly(POx) < poly(THF-co-OX) < poly(THF), probably indicating that the absence of tertiary hydrogen atoms has a positive effect on the biostability. Concerning the more hydrophilic materials, crosslinked PEO showed the highest rate of degradation, probably due to the mechanical weakness of the hydrogel in combination with the highest presence of giant cells as a result of the high porosity. A frayed surface morphology was observed after implantation of the crosslinked poly(THF)/PEO blend, which might be due to preferential degradation of PEO domains