Traditional Crosslinking Research Articles

Bioamine-crosslinked gellan gum hydrogel for neural tissue engineering

Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.

* Collagen Cross-Linking: Biophysical, Biochemical, and Biological Response Analysis.

Extracted forms of collagen are subjected to chemical cross-linking to enhance their stability. However, traditional cross-linking approaches are associated with toxicity and inflammation. This work investigates the stabilization capacity, cytotoxicity and inflammatory response of collagen scaffolds cross-linked with glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 4-arm polyethylene glycol (PEG) succinimidyl glutarate (4SP), genipin (GEN), and oleuropein. Although all cross-linking methods reduced free amine groups, variable data were obtained with respect to denaturation temperature, resistance to collagenase digestion, and mechanical properties. With respect to biological analysis, fibroblast cultures showed no significant difference between the treatments. Although direct cultures with human-derived leukemic monocyte cells (THP-1) clearly demonstrated the cytotoxic effect of GTA, THP-1 cultures supplemented with conditioned medium from the various groups showed no significant difference between the treatments. With respect to cytokine profile, no significant difference in secretion of proinflammatory (e.g., interleukin [IL]-1β, IL-8, tumor necrosis factor-α) and anti-inflammatory (e.g., vascular endothelial growth factor) cytokines was observed between the noncross-linked and the 4SP and GEN cross-linked groups, suggesting the suitability of these agents as collagen cross-linkers.

Probing Protein-Protein Interactions with Genetically Encoded Photoactivatable Cross-Linkers.

Fundamental to all living organisms is the ability of proteins to interact with other biological molecules at the right time and location, with the proper affinity, and to do so reversibly. One well-established technique to study protein interactions is chemical cross-linking, a process in which proteins in close spatial proximity are covalently tethered together. An emerging technology that overcomes many limitations of traditional cross-linking methods is one in which photoactivatable cross-linking noncanonical amino acids are genetically encoded into a protein of interest using the cell's native translational machinery. These proteins can then be used to trap interacting biomolecules upon UV illumination. Here, we describe a method for the site-specific incorporation of photoactivatable cross-linking amino acids into fluorescently tagged proteins of interest in E. coli. Photo-cross-linking and analysis by SDS-PAGE using in-gel fluorescence detection, which provides rapid, highly sensitive, and specific detection of cross-linked adducts even in impure systems, are also described. An example expression and cross-linking experiment involving transmembrane signaling of a bacterial second messenger receptor system that controls biofilm formation is shown. All reagents needed to carry out these experiments are commercially available, and do not require special or unique technology to perform, making this method tractable to a broad community studying protein structure and function.

Improved biodegradation of synthetic azo dye by horseradish peroxidase cross-linked on nano-composite support

A ZnO nanowires/macroporous SiO2 composite was used as support to immobilize horseradish peroxidase (HRP) by in-situ cross-linking method. Using diethylene glycol diglycidyl ether (DDE) as a long-chained cross-linker, it was adsorbed on the surface of ZnO nanowires before reaction with HRPs, the resulted composite was quite different from the traditional cross-linking enzyme aggregates (CLEAs) on both structure and catalytic performance. The immobilized HRP showed high activity in the decolorization of azo dyes. The effect of various conditions such as the loading amount of HRP, solution pH, temperature, contact time and concentration of dye were optimized on the decolorization. The decolorization percentage of Acid Blue 113 and Acid black 10 BX reached as high as 95.4% and 90.3%, respectively. The immobilized HRP gave the highest decolorization rate under dye concentration as 50mg/L and reaction time of 35min. The immobilized HRP exhibited much better resistance to temperature and pH inactivation than free HRP. The storage stability and reusability were greatly improved through the immobilization, from the decolorization of Acid blue 113 it was found that 80.4% of initial efficiency retained after incubation at 4°C for 60 days, and that 79.4% of decolorization efficiency retained after 12 cycles reuse.

In vitro study of strontium doped calcium polyphosphate-modified arteries fixed by dialdehyde carboxymethyl cellulose for vascular scaffolds

Dialdehyde carboxymethyl (DCMC) was select as an optimal crosslinking reagent for its excellent biocompatibility, appropriate degradable property and suitable chemical reactivity in our previous research. However, similar to other traditional crosslinking reagent, the endothelialization of biological vascular scaffolds fixed by DCMC was not sufficient enough. To overcome this limitation presented in DCMC-fixed arteries, a novel strategy was developed by introducing SCPP into arteries to investigate effects of strontium doped calcium polyphosphate (SCPP)-doping on the properties of DCMC-fixed arteries, especially on the endothelialization of fixed arteries. After modifying and crosslinking, their chemical structures, mechanical properties, stability, and cytocompatibility were examined. The result obtained from EDS analysis indicated SCPP was successfully introduced into DCMC-fixed arteries. Compared with purely DCMC-crosslinked ones, DCMC/SCPP modification has no significant effect on mechanical strength of fixed arteries, but a slight tend to improve the stability of fixed samples in D-Hanks solution. Moreover, MTT assay, SEM observation and live/dead assay implied that DCMC/SCPP modification could effectively stimulate HUVECs’ adhesion and proliferation, and thus promote endothelialization process of fixed arteries. In this study, micron-sized SCPP particles were firstly applied to modify DCMC-crosslinked porcine arteries, and the results demonstrated this method could improve the endothelialization of fixed arteries. Therefore, DCMC/SCPP-modified arteries with excellent physicochemical properties and biocompatibility should be a promising materials for fabricating vascular scaffolds.

Biophysical, biochemical and biological properties of nanotextured collagen fibres

Event Abstract Back to Event Biophysical, biochemical and biological properties of nanotextured collagen fibres Anna Sorushanova1, 2, Anne M. Mullen3, Abhay Pandit2 and Dimitrios I. Zeugolis1, 2 1 NUI Galway, REMODEL, Ireland 2 NUI Galway, CURAM, Ireland 3 Food Research Centre, Teagasc, Ireland Introduction: Through self-assembly, macromolecules with chemical complementarity form supramolecular structures,, which are held together by weak bonds. Thus, exogenous cross-links are required to produce stable constructs. Specifically in collagen-based scaffolds, chemical, physical and/or biological cross-linking methods are under investigation to control the biophysical, biochemical and biological properties of the produced devices[1]-[3]. However, biological and physical methods are not suitable for load-bearing tissues, due to the weak-induced stability, whilst chemical stabilisation methodologies are associated with foreign body response. Although the potential of multi-functional poly(ethylene glycol) systems as stabilisation and functionalisation agents for collagen hydrogels / sponges has been shown[4],[5], it is still unclear whether such agents can induce proportional stability to traditional cross-linking methods, without associated cytotoxicity. Herein, we hypothesise that 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate (PEG) can produce cytocompatible collagen fibres for tendon repair and regeneration, with biophysical, biochemical and biological properties, superior to customarily used chemical approaches. Experimental Methods: Collagen fibres were fabricated and cross-linked with PEG of different concentrations; 0.5mM, 1mM, 2.5mM, 5mM and 10mM. Structural characteristics were assessed with Scanning Electron Microscopy (SEM). Thermal stability was assessed with Differential Scanning Calorimetry (DSC). The extent of cross-linking was assessed with ninhydrin and collagenase assays. Mechanical properties were assessed with tensile testing. In vitro cytocompatibility, protein expression and gene analysis was assessed with human skin fibroblasts, human tenocytes and human bone marrow mesenchymal stem cells (BMSCs) after 3, 7, 14, 21 and 28 days in culture. Morphological analysis was assessed using ImageJ. Non-cross-linked and glutaraldehyde (GTA) cross-linked fibres acted as control. Conclusion: PEG-based fibres were characterised by crevices and ridges running parallel to the longitudinal axis of the fibres. This surface topography induced bidirectional cell attachment, elongation and growth. PEG-based fibres had similar mechanical properties to GTA cross-linked fibres. Further, PEG-based fibres did not induce any cell cytotoxicity, whilst GTA fibres were found to be cytotoxic at the effective concentration. PEG fibres maintained tenocyte phenotype and differentiated BMSCs towards tenogenic lineage. Overall, our data clearly indicate the potential of these fibres for tendon repair and regeneration. ReValueProtein Research Project (Exploration of Irish meat processing streams for Recovery of high Value Protein based ingredients for food and non-food uses, Grant Award No. 11/F/043) is supported by the Department of Agriculture, Food and the Marine (DAFM) under the National Development Plan 2007–2013 funded by the Irish Government.

Preparation and self-healing behaviors of poly(acrylic acid)/cerium ions double network hydrogels

This work aims at developing an approach to poly(acrylic acid)/cerium ions (PAA/cerium ions) double network hydrogels and exploring the self-healing properties of the hydrogels, with expectation to provide some clues for constructing new healable gel actuators and enrich the family of self-healable hydrogels. The hydrogel is covalently crosslinked by a traditional crosslinking agent N,N’-methylene bis-acrylamide to form the first network and further physically crosslinked by the ionic interaction between cerium ions and the carboxyl groups in the hydrogel network. The preparation method and self-healing behaviors of the hydrogels are investigated. Open image in new window

A Single Molecular Diels–Alder Crosslinker for Achieving Recyclable Cross‐Linked Polymers

A triol-functional crosslinker combining the thermoreversible properties of Diels-Alder (DA) adducts in one molecule is designed, synthesized, and used as an ideal substitute of a traditional crosslinker to prepare thermal recyclable cross-linked polyurethanes with excellent mechanical properties and recyclability in a very simple and efficient way. The recycle property of these materials achieved by the DA/retro-DA reaction at a suitable temperature is verified by differential scanning calorimetry and in situ variable temperature solid-state NMR experiments during the cyclic heating and cooling processes. The thermal recyclability and remending ability of the bulk polyurethanes is demonstrated by three polymer processing methods, including hot-press molding, injection molding, and solution casting. It is notable that all the recycled cross-linked polymers display nearly invariable elongation/stress at break compared to the as-synthesized samples. Further end-group functionalization of this single molecular DA crosslinker provides the potential in preparing a wide range of recyclable cross-linked polymers.

Synthesis of novel temperature responsive PEG-b-[PCL-g-P(MEO2MA-co-OEGMA)]-b-PEG (tBG) triblock-graft copolymers and preparation of tBG/graphene oxide composite hydrogels via click chemistry

Novel triblock-graft copolymers, poly ethylene glycol-b-[poly(ε-caprolactone)-g-poly(2-(2-methoxyethoxy) ethyl methacrylate-co-oligo (ethylene glycol) methacrylate)]-b-poly ethylene glycol (PEG-b-[PCL-g-P(MEO2MA-co-OEGMA)]-b-PEG) (tBG), were synthesized via ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). In the synthesis process, temperature responsive P(MEO2MA-co-OEGMA) chains were grafted onto the PCL block of triblock copolymer PEG-b-PCL-b-PEG to improve its hydrophilicity. This method succeeded in increasing the solubility of PEG-b-PCL-b-PEG in water, and more importantly, endowing PEG-b-PCL-b-PEG with temperature sensitivity. By adjusting the feed ratio of 2-(2-methoxy ethoxy) ethyl methacrylate (MEO2MA) and oligo (ethylene glycol) methacrylate (OEGMA) monomers, the lower critical solution temperature (LCST) of the tBG can be realized at about 37°C. Taking advantage of the excellent mechanical property of graphene sheets, alkyne-functionalized graphene oxide (alkyne-GO) was introduced to cross-link tBGs and prepare tBG/GO composite hydrogel through click reaction between tBG-N3 and alkyne-GO. Different from traditional cross-linkers, alkyne-GO acts as reinforcing filler in the composite hydrogel. Benefiting from superior properties of PCL, PEG, P(MEO2MA-co-OEGMA) and GO, the as-prepared temperature responsive tBG/GO hydrogel exhibits excellent mechanical strength and toughness, demonstrating future potential applications in tissue engineering and biotechnology fields.

Biocompatible elastin-like click gels: design, synthesis and characterization.

Elastin-like recombinamer click gels (ELR-CGs) for biomedical applications, such as drug delivery or tissue engineering, have been developed by taking advantage of the click reaction (CuAAC) in the absence of traditional crosslinking agents. ELRs are functionalized with alkyne and azide groups using conventional chemical techniques to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. Hydrogels with moduli in the range 1,000-10,000Pa have been synthesized, characterized, and tested in vitro against several cell types. The cells embedded into ELR-CGs possessed high viability and proliferation rate. The mechanical properties, porosity and swelling of the resulting ELR-CGs can easily be tuned by adjusting the ELR concentration. We also show that it is possible to replicate different patterns on the hydrogel surface, thus allowing the use of this type of hydrogel to improve applications that require cell guidance or even differentiation depending on the surface topography.

Feasibility study of the naturally occurring dialdehyde carboxymethyl cellulose for biological tissue fixation

The aim of this study was to evaluate the crosslinking effect of dialdehyde carboxymethyl cellulose (DCMC) on decellularized porcine aortas. Before implanted, biological tissues must be chemically modified to avoid rapid enzymatic degradation and serious immune response. To overcome limitations like high cytotoxicity and susceptibility to calcification caused by glutaraldehyde (GA), a traditional crosslinking reagent, dialdehyde carboxymethyl cellulose (DCMC) was employed to fix biological tissues. The crosslinking characteristics and cytotoxicity of aortas fixed by DCMC were all investigated. The results indicated that DCMC-fixation significantly increased the mechanical strength and the capacity of enzymatic hydrolytic resistance of tissues. The histological examination showed that the microcosmic structures of tissues were all preserved well after DCMC fixation. In addition, the data obtained from MTT assay confirmed that the cytotoxicity of DCMC-fixed tissues was significantly lower than glutaraldehyde-fixed counterparts. In a word, the present study demonstrated DCMC might be an effective crosslinking reagent for biological tissue fixation with low cytotoxicity.

Effective Phosphate Removal from Synthesized Wastewater Using Copper–Chitosan Bead: Batch and Fixed-Bed Column Studies

To remove phosphate from solution, a new class of sorbent based on chitosan bead (CB) was prepared using copper ion (Cu(II)) with/without a traditional crosslinking agent (glutaraldehyde [GLA]); these materials are referred to as CB-G-Cu and CB-Cu, respectively. Copper ions play a key role in the CB synthesis; these species crosslink each polymer chain, and during phosphate removal, they are the active functional group. Overall, 2.5 % (w/w) of chitosan is necessary to maintain the physical properties of the bead. In the FTIR spectra, adding GLA decreased the intensity of the amino group in chitosan, lowering the amount of copper in the CB. The maximum phosphate uptake (Q) for CB-Cu was 53.6 mg g−1 when calculated with the Langmuir isotherm, and the phosphate equilibrium was achieved in 12 h. Although the solution pH was not strongly affected, values below 7 are optimal for phosphate removal. The CB-Cu can be feasibly applied during a fixed column test, revealing that the phosphate breakthrough was 1.5 times higher than with CB-G-Cu.

Crosslinking of Carboxylated Nitrile Rubbers with Resol Resins

This paper discusses at length the metal oxide and sulfur-free crosslinking of rubber compounds for carboxylated nitrile rubber (XNBR) using resols based on p-alkyl phenols (resol crosslinking). It demonstrates that, with the best grade and dosage of resol and the right Krynac X nitrile rubber grade, such a compound can be adjusted to match the overall property profile resulting from traditional crosslinking with zinc oxide/sulfur with even better processing characteristics. Significant improvements can be achieved, for instance in the compression set characteristics. Resol crosslinking is sensitive to the grade of rubber used. The best and most balanced property profile can be achieved with XNBR grades containing 1% carboxylic acid (Krynac X 146, Krynac X 160). When the principles explained are observed, vulcanisates based on XNBR grades containing 7% carboxylic acid (Krynac X 750) also offer advantages. In addition to vulcanisates containing carbon black, compounds containing silica are also discussed and it is shown that, in this case, the additional use of silanes is indispensable for effective vulcanisation.

Synergistic effects in the processes of crosslinking of elastomers

Radiation crosslinking of elastomers is an example of the modification of polymers by ionizing radiation. In practice, often parallel both traditional crosslinking (with peroxide) and radiation treatment is applied (Bik et al., 2003, 2004). Elastomers can be irradiated both before and/or after vulcanization products. The aim of this study was to investigate the system of the mixed radiation/peroxide and peroxide/radiation crosslinking of selected elastomers (Engage 8200, HNBR). In particular, attention was directed to the influence of the protective effects of aromatic additives in elastomers (peroxides, thermal- and light stabilizers) on the phenomenon of crosslinking and postradiation oxidation. Aromatic peroxides may undergo modifications during the preirradiation, which affect the subsequent processes of vulcanization. In this way the method of gas chromatography (GC) was applied for determination of hydrogen and oxidation effects, never described before for Engage 8200. Using that approach, radiation efficiency of hydrogen evolution and oxygen absorption efficiency of the polymers has been identified. To describe the phenomena of postradiation oxidation of elastomers, the method of Diffuse Reflection Spectrophotometry (DRS) was also applied.

In situ UV-crosslinking gelatin electrospun fibers for tissue engineering applications

Electrospun fibers of natural polymers are desirable for biomedical applications such as tissue engineering. Crosslinking of electrospun fibers of natural polymers is needed to prevent dissolution in water and to enhance mechanical strength. In this study, an in situ UV-crosslinking method was developed for crosslinking of gelatin electrospun fibers (GESFs) and water-soluble synthetic polymers. A mixture of gelatin and phenylazide-conjugated poly(acrylic acids) was electrospun under UV irradiation. The UV-crosslinked GESFs were not dissolved in water with improved mechanical strength. Compared to traditional crosslinking by glutaraldehyde vapor, the GESFs crosslinked by our method are superior in terms of retention of GESF morphology, uniform crosslinking throughout the fibers, low cytotoxic and retention of biofunctionality. L929 cells grew better on the UV-crosslinked GESF scaffolds compared to glutaraldehyde-crosslinked ones. Furthermore, bioactive nanoparticles, e.g. hydroxyapatite, could be incorporated into GESFs for enhancing osteoconductivity, which possess a great potential in bone tissue engineering.

ChemInform Abstract: Photo‐Induced Covalent Cross‐Linking for the Analysis of Biomolecular Interactions

AbstractReview: 34 refs.

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.

Synthesis and characterization of addition-type silicone rubbers (ASR) using a novel cross linking agent PH prepared by vinyl-POSS and PMHS

A novel cross-linker (denoted as PH) prepared using vinyl-POSS and poly(methylhydrogensiloxane) (PMHS), was introduced to vinyl terminated polydimethylsiloxane (VPDMS) matrix to prepare a series of addition-type silicone rubbers (ASR) for the first time. Morphology, thermal properties and mechanical properties of these ASR were respectively studied by means of scanning electron microscope (SEM), thermal gravimetric analysis (TGA) and universal tensile testing machine. The results revealed that the thermal stabilities and mechanical properties of ASR were significantly enhanced by the addition of PH compared with the traditional cross-linker PMHS. It was found that the tensile strength of ASR prepared using PH as cross-linker was strikingly increased by 43.8% compared with the one prepared using PMHS as cross-linker. The striking enhancements in thermal properties and improvements on mechanical properties of ASR were likely attributed to the increase of dimensionality of novel cross-linked networks in novel ASR.

Photo-induced covalent cross-linking for the analysis of biomolecular interactions

Photo-induced cross-linking (PIC) is a powerful strategy for generating information on biomolecular interactions. In PIC, the utility of traditional cross-linking methods is supplemented by the temporal control of photo-activation, enabling the study of non-covalent kinetic intermediates and heterogeneous mixtures. This tutorial review will introduce the photochemistry of activation, reactive intermediates, methods for the functionalisation of biomolecules and the installation of additional functionalities (e.g., affinity tags). In doing so, we shall illustrate the wealth of data that can be obtained using this approach, ranging from the identification of interacting partners and structural data to temporal information. Alongside a discussion of the strengths and weaknesses of the various approaches, their applicability to different types of biological system will be described.

Defined high protein content surfaces for stem cell culture

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent ( N-hydroxysulfosuccinimide/ N-(3-Dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.