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

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

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%

Adhesion and spreading of cultured endothelial cells on modified and unmodified poly(ethylene terephthalate): a morphological study

The in vitro adhesion and spreading of human endothelial cells (HEC) on hydrophobic poly(ethylene terephthalate) (PETP) and moderately wettable tissue culture polyethylene terephthalate) (TCPETP) were studied with light microscopy and electron microscopy. Numbers of HEC adhering on TCPETP were always higher than those found on PETP. When cells were seeded in the presence of serum, extensive cell spreading on both PETP and TCPETP was observed after the first 30 min. Thereafter, spread cells appeared to withdraw from the PETP surface, resulting in irregularly shaped cells. Complete cell spreading occurred on TCPETP. Complete cell spreading also occurred on PETP and TCPETP when HEC had first been seeded from phosphate buffer solution and serum was supplied after 30 min. Furthermore, HEC spread on both PETP and TCPETP when the surfaces were precoated with protein(s), which promotes cell adhesion. However, when plasma was used for the coating, spread cells did not proliferate in a monolayer pattern. This study shows that TCPETP is, in general, a better surface for adhesion and proliferation of HEC than is PETP, suggesting that vascular prostheses with a TCPETP-like surface will perform better in vivo than prostheses made of PETP

Evaluation of toxicity and neural uptake in vitro and in vivo of superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIO-NPs) have great potential to be used in different pharmaceutical applications, due to their unique and versatile physical and chemical properties. The aim of this study was to quantify in vitro cytotoxicity of dextran 70,000-coated SPIO-NPs labelled/unlabelled with rhodamine 123, in C6 glioma cells and primary hippocampal neural cells. In addition, we analyzed the in vitro and in vivo cellular uptake of labelled SPIO-NPs. The nanoparticles, with average size of 10⁻50 nm and polydispersity index of 0.37, were synthesized using Massart's co-precipitation method. The concentration-dependent cytotoxicity was quantified by using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Intracellular localization of SPIO-NPs was detected by confocal laser microscopy. In vivo confocal neuroimaging (ICON) was performed on male Wistar rats after intravitreal injection followed by ex vivo retina whole mount analysis. When used for in vitro testing concentrations in the range of diagnostic and therapeutic dosages, SPIO-NPs proved to be non-cytotoxic on C6 glioma cells for up to 24 h incubation time. The hippocampal cell culture also did not show impaired viability at low doses after 24 h incubation. Our results indicate that our dextran-coated SPIO-NPs have the potential for in vivo drug delivery applications

Amine functionalization of cholecyst-derived extracellular matrix with generation 1 PAMAM dendrimer

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Biomacromolecules, copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/pdf/10.1021/bm701055k.A method to functionalize cholecyst-derived extracellular matrix (CEM) with free amine groups was established in an attempt to improve its potential for tethering of bioactive molecules. CEM was incorporated with Generation-1 polyamidoamine (G1 PAMAM) dendrimer by using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide cross-linking system. The nature of incorporation of PAMAM dendrimer was evaluated using shrink temperature measurements, Fourier transform infrared (FTIR) assessment, ninhydrin assay, and swellability. The effects of PAMAM incorporation on mechanical and degradation properties of CEM were evaluated using a uniaxial mechanical test and collagenase degradation assay, respectively. Ninhydrin assay and FTIR assessment confirmed the presence of increasing free amine groups with increasing quantity of PAMAM in dendrimer-incorporated CEM (DENCEM) scaffolds. The amount of dendrimer used was found to be critical in controlling scaffold degradation, shrink temperature, and free amine content. Cell culture studies showed that fibroblasts seeded on DENCEM maintained their metabolic activity and ability to proliferate in vitro. In addition, fluorescence cell staining and scanning electron microscopy analysis of cell-seeded DENCEM showed preservation of normal fibroblast morphology and phenotype

Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells

July 3, 2012Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure–function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin α[subscript v]β[subscript 3] and α[subscript v]β[subscript 5] engagement with adsorbed vitronectin to promote colony formation. The structure–function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)National Institutes of Health (U.S.) (Grant DE016516)Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract W911NF-07-D-0004