Natural Cross-linker Research Articles

Influence of natural crosslinker and fibre weightage on waste kibisu fibre reinforced wheatgluten biocomposite

In this era of green and sustainable manufacturing, natural fibre-reinforced polymer composites (NFPC) have been widely accepted as the potential alternatives for polymer matrix composites (PMC) or any other non-biodegradable composites. Despite the increasing need to replace plastic bottles, bags, disposable plastic plates and trays, seedling pots used in our day to day life, not many studies have been made in this direction. The current work aims at developing a hundred percent biodegradable composite by reinforcing waste Kibisu silk fibre into wheat gluten as a possible replacement of plastic disposables. The developed composites are made up of different mass fractions of Kibisu silk fibre reinforced into plasticised wheat gluten. The prepared composites have been characterised to obtain the best combination. The developed composites were found to have adequate tensile property, mass degradation at a considerably high temperature and most importantly, the outstanding rate of biodegradation under normal atmospheric conditions. The soil quality test before and after degradation also showed no significant changes in the quality of the soil. FTIR studies revealed improved interaction between wheat gluten, glycerol and Kibisu fibres upon addition of natural lemon extract as crosslinker. Overall results indicate that the developed biocomposites have the potential to substitute harmful plastic disposables like plastic seedling pots and plates, disposable hospital tray, dustbin, etc.

Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild-type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched cross-linked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE–CotE and CotJA–CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat.

Chicken gelatin modification by caffeic acid: A response surface methodology investigation

Chemical modifications of gelatin from mechanically separated chicken meat (MSM) residue were practiced using caffeic acid as a cross-linker. The effects of oxidation period (OP), cross-linking temperature (CT), and caffeic acid (CA) concentration were investigated. Experiments were performed using Response Surface Methodology (RSM). The effects of 16 different cross-linking modifications on the physicochemical properties of chicken gelatin gels were investigated.Maximum gel strength was determined at 12.5 min OP, 50 °C CT and 2.5% CA concentration and this was 63% higher than the control (uncross-linked chicken gelatin). Temperature has an increasing effect on the degree of cross-linking value up to a certain degree. The highest degree of cross-linking was observed at between 50° and 55 °C. The color characteristics of gels were affected by cross-linking having more brown color. Overall this study demonstrated that caffeic acid has a potential to be an efficient natural cross-linking factor increasing the mechanical properties of chicken gelatin thermo-irreversibly.

Immobilization of laccase via cross-linked enzyme aggregates prepared using genipin as a natural cross-linker

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.

Genipin-cross-linked hydrogels based on biomaterials for drug delivery: a review.

Genipin is a naturally occurring nontoxic cross-linker, which has been widely used for drug delivery due to its excellent biocompatibility, admirable biodegradability and stable cross-linked attributes. These advantages led to its extensive application in the fabrication of hydrogels for drug delivery. This review describes the physicochemical characteristics and pharmacological activities of genipin and attempts to elucidate the detailed mechanisms of the cross-linking reaction between genipin and biomaterials. The current article entails a general review of the different biomaterials cross-linked by genipin: chitosan and its derivatives, collagen, gelatin, etc. The genipin-cross-linked hydrogels for various pharmaceutical applications, including ocular drug delivery, buccal drug delivery, oral drug delivery, anti-inflammatory drug delivery, and antibiotic and antifungal drug delivery, are reported. Finally, the future research directions and challenges of genipin-cross-linked hydrogels for pharmaceutical applications are also discussed in this review.

The mitochondrial basis of aging and related diseases

Aging is a programmed and spontaneous life course that organisms must undergo, and as an irreversible and relatively conservative process, several theories have tried to explain its causes. Among them, the somatic mutation theory, the free radical theory, the natural cross-linking of biomolecules, the immune theory, the telomere theory, the biological clock theory, and the toxic theory are among the most widely accepted hypotheses. However, no single doctrine can fully explain the aging process. Aging is mainly manifested in the structural and functional aging and decline of organisms, and as organisms continue to age, most organs will experience varying degrees of aging and irreversible damage, although it is directly related to many diseases, aging itself is generally not considered as a disease. Any organism ages slowly over time, with the most easily observable manifestations in external changes such as diminished hair color and sagging skin. Numerous studies have shown that mitochondria are instrumental in mitigating aging as the primary provider of cellular energy, providing energy for cell growth and development as well as being the center of cellular metabolism; therefore, once mitochondria become faulty, a large number of cells will lose function, age, and thus die. In this paper, we will discuss in detail the relationship between mitochondria and aging and how external interventions can promote the function of mitochondria in delaying aging.

Plant based cross-linkers for tissue engineering applications.

Utility of plant-based materials in tissue engineering has exponentially increased over the years. Recent efforts in this area have been focused on substituting synthetic cross-linkers with natural ones derived from biological sources. These cross-linkers are essentially derived from the vegetative components of plants therefore suitably categorised as 'green' and renewable materials. Utilization of plant based cross-linkers in scaffolds and hydrogels offers several advantages compared to the synthetic ones. Natural compounds, like ferulic acid and genipin, when incorporated into scaffolds can promote cellular proliferation and growth, by regulation of growth factors. They participate in crucial activities, thus providing impetus for cell growth, function, differentiation and angiogenesis. Several natural compounds inherently possess anti-microbial, antioxidant and anti-inflammatory effects, which enhance the inherent characteristics of the scaffolds. Versatility of natural cross-linkers can be exploited for diverse applications. Integrating such potent molecules can enable the scaffold to display relevant characteristics for each function. This review article focuses on the recent developments with plant based cross-linkers that are employed for scaffold synthesis and their applications, which may be explored to synthesize scaffolds suitable for diverse biomedical applications.

Biodegradable active food packaging structures based on hybrid cross-linked electrospun polyvinyl alcohol fibers containing essential oils and their application in the preservation of chicken breast fillets

Active food packaging materials produced through electrospinning of polyvinyl alcohol (PVOH) containing essential oils (EOs) from two broadly used spices (Laurus nobilis –LEO- and Rosmarinus officinalis -REO-) were developed and applied to chicken breast fillets with the aim of prolonging their shelf-life. Citric acid (CA) was successfully incorporated as a natural cross-linker and the developed electrospun structures were heat-treated to promote both the crosslinking and the crystallization of the PVOH matrix. Initially, the morphology, water solubility, physicochemical and thermal properties of the developed structures were evaluated. Then, the antioxidant and antibacterial efficiency of PVOH-EOs hybrid structures were evaluated when directly applied onto an inoculated side of chicken breast fillets. These annealed active food packaging structures containing EO and CA exhibited improved water resistant and thermal stability with respect to their non-crosslinked counterparts. Although part of the EOs content was degraded after the annealing process, the remaining amount in the PVOH samples inhibited the lipid oxidation process up to 68 % and displayed enhanced antimicrobial effectiveness when applied onto chicken breast fillets, having a beneficial effect on both the pH and color parameters during storage.

MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome.

Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5′-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.

Preparation and characterization of genipin cross-linked and lysozyme incorporated antimicrobial sodium caseinate edible films

Sodium caseinate (NaCas) possesses good film forming capacity, but mechanical strength of NaCas films is declined by hydrophilic residues in NaCas molecules. Genipin (GP), a low-toxicity natural cross-linking agent, was used at the level at 1 mg/g NaCas to improve the mechnical properties and lysozyme (LYS) was used at the levels of 42, 84 and 126 mg/g NaCas to confer antimicrobial activity of NaCas films. While films without GP and LYS were colorless, light blueness was observed for all types of NaCas films containing GP. The whiteness index and transparency of films decreased as LYS amount increased. The moisture content of films was not significanly affected by the additon of GP and LYS, but the water vapor permeability slightly increased as the amount of LYS increased. Compared with films without GP, significanly higher tensile strength and Young’s modulus and significantly lower break elongation were detected in NaCas films containing GP. The cross-linking reaction between NaCas and GP was confirmed by FTIR-ATR analysis. Incorporation of LYS into NaCas films exerted antimicobial activities against Staphylococcus aureus and Eschericha coli. In conclusion, a novel type of active edible food packaging film with antimicrobial activity was developed by cross-linked with GP and incoporated with LYS.

Using Genipin to Immobilize Bone Morphogenetic Protein-2 on Zirconia Surface for Enhancing Cell Adhesion and Mineralization in Dental Implant Applications.

Our objective in this study was to promote cell responses through the immobilization of bone morphogenetic protein-2 (BMP-2) on roughened zirconia (ZrO2) through using the natural cross-linker genipin in dental implant applications. Field emission scanning electron microscope, X-ray photoelectron spectroscopy, and attenuated total reflection-Fourier transform infrared spectroscopy were used to analyze the surface characterizations, including the topography, chemistry, and functional groups, respectively, of the test specimens. Human bone marrow mesenchymal stem cells (hMSCs) were used to detect cell responses (adhesion, proliferation, and mineralization). The surface characterizations analysis results revealed that genipin was effective in immobilizing BMP-2 on roughened zirconia surfaces. BMP-2 proved effective in promoting the adhesion and mineralization of hMSCs on roughened zirconia. The surface modification proposed has potential in zirconia dental implant applications.

Sponge-like processed D-periodic self-assembled atelocollagen supports bone formation in vivo

Fibrous biopolymeric collagen extracted from animal tissues has been widely used for fabricating matrices for bone tissue engineering (BTE). However, animal extracted collagens can trigger immune reactions when implanted in vivo and the presence of native crosslinks leads to batch-to-batch variability. Atelocollagen, a monomeric form of collagen, is free of telopeptides, which are mainly responsible for the immunogenicity of collagen, and can self-assemble in vitro to obtain fibrils with the characteristic D-periodic staining pattern of native collagen. However, atelocollagen-based biomaterials have not extensively been studied and, hence, their suitability for BTE remains relatively unexplored. Besides, to stabilize collagen biomaterials, chemical and physical crosslinking are used, although chemical agents are cytotoxic while the physical methods yield a less effective crosslinking. A combination of physical and chemical crosslinking is a suitable alternative that has rarely been tested in BTE programs. In this work, a sponge-like biomaterial (DCol-S) was processed from D-periodic self-assembled atelocollagen and its stabilization was studied using the combination of a dehydrothermal treatment (DHT) and minimal glutaraldehyde (GTA) exposition crosslinking, to increase the resistance to degradation of the scaffold without a major effect on the biomaterial structure. The microstructural features of the final sponges were characterised and compared to a commercial biomaterial processed from native bovine collagen (Helistat®, Integra Lifesciences, NJ, USA), demonstrating that a D-periodic nanostructure was obtained and maintained after processing of the sponges. MC3T3-E1 preosteoblast adhesion, proliferation and differentiation assays in vitro showed that DCol-S is biocompatible. Furthermore, intramuscular implantation of the biomaterials loaded with rhBMP-2 revealed that the double-crosslinked sponges were able to support ectopic bone formation, while sponges stabilised only with the DHT treatment were not. Altogether, these findings show that atelocollagen-based sponges stabilised with a DHT treatment followed by a mild GTA crosslinking are a suitable alternative to polymeric extracted collagen for BTE applications.

High strength and low swelling composite hydrogels from gelatin and delignified wood

A delignified wood template with hydrophilic characteristics and high porosity was obtained by removal of lignin. Gelatin was infiltrated into the delignified wood and further crosslinked with a natural crosslinker genipin to form hydrogels. The composite hydrogels showed high mechanical strength under compression and low swelling in physiological condition. The effect of genipin concentrations (1, 50 and 100 mM) on structure and properties of the composite hydrogels were studied. A porous honeycomb structure with tunable pore size and porosity was observed in the freeze-dried composite hydrogels. High elastic modulus of 11.82 ± 1.51 MPa and high compressive yield stress of 689.3 ± 34.9 kPa were achieved for the composite hydrogel with a water content as high as 81%. The equilibrium water uptake of the freeze-dried hydrogel in phosphate buffered saline at 37 °C was as low as 407.5%. These enables the delignified wood structure an excellent template in composite hydrogel preparation by using infiltration and in-situ synthesis, particularly when high mechanical strength and stiffness are desired.

Additive manufacturing of hydroxyapatite–chitosan–genipin composite scaffolds for bone tissue engineering applications

Additive manufacturing holds promise for the fabrication of three-dimensional scaffolds with precise geometry, to serve as substrates for the guided regeneration of natural tissue. In this work, a bioinspired approach is adopted for the synthesis of hybrid hydroxyapatite hydrogels, which were subsequently printed to form 3D scaffolds for bone tissue engineering applications. These hydrogels consist of hydroxyapatite nanocrystals, biomimetically synthesized in the presence of both chitosan and l-arginine. To improve their mechanical properties, chemical crosslinking was performed using a natural crosslinking agent (genipin), and their rheology was modified by employing an acetic acid/gelatin solution. Regarding the 3D printing process, several parameters (flow, infill and perimeter speed) were studied in order to accurately produce scaffolds with predesigned geometry and micro-architecture, while also applying low printing temperature (15 °C). Following the printing procedure, the 3D scaffolds were freeze dried in order to remove the entrapped solvents and therefore, obtain a porous interconnected network. Evaluation of porosity was performed using micro-computed tomography and nanomechanical properties were assessed through nanoindentation. Results of both characterization techniques, showed that the scaffolds' porosity as well as their modulus values, fall within the corresponding range of the respective values of cancellous bone. The biocompatibility of the 3D printed scaffolds was assessed using MG63 human osteosarcoma cells for 7 days of culturing. Cell viability was evaluated by MTT assay as well as double staining and visualized under fluorescence microscopy, while cell morphology was analyzed through scanning electron microscopy. Biocompatibility tests, revealed that the scaffolds constitute a cell-friendly environment, allowed them to adhere on the scaffolds' surface, increase their population and maintain high levels of viability.

Biocomposites from bamboo fibers and palm oil-based thermosets: Effects of natural phenolic cross-linkers

The development of highly biobased and robust thermosetting matrices using renewable feedstocks has become an interesting topic in the field of composites. A series of palm oil (PO)-based thermosets were prepared from a PO vinyl monomer and natural phenolic (NP) cross-linkers. The PO fatty acid-ethyl acrylamide (POFA-EA) was synthesized via a facile transesterification of PO with N-(2-hydroxyethyl)acrylamide (HEAA). NPs including eugenol, methyl gallate, and tannic acid were used to generate aromatic rings-containing cross-linkers via a solvent-free and ultrasonic-assisted esterification. The mechanical properties and processibility of the developed PO resins were closely associated with the chemical structures and unsaturation degrees of the incorporated NP cross-linkers. Besides, the thermosets were further used as the matrices for bamboo fibers-reinforced biocomposites. The resulting biocomposites showed comparable mechanical properties and Tgs toward those of petroleum-based counterparts. The biocomposites are anticipated to be fully decomposed because the PO matrices could be degraded in a mild alkali condition. This study provides insights to develop high performance and recyclable PO-based thermosets and their biocomposites.

In situ structural studies during denaturation of natural and synthetically crosslinked collagen using synchrotron SAXS

Collagen is an important biomacromolecule, making up the majority of the extracellular matrix in animal tissues. Naturally occurring crosslinks in collagen stabilize its intermolecular structure in vivo, whereas chemical treatments for introducing synthetic crosslinks are often carried out ex vivo to improve the physical properties or heat stability of the collagen fibres for applications in biomaterials or leather production. Effective protection of intrinsic natural crosslinks as well as allowing them to contribute to collagen stability together with synthetic crosslinks can reduce the need for chemical treatments. However, the contribution of these natural crosslinks to the heat stability of collagen fibres, especially in the presence of synthetic crosslinks, is as yet unknown. Using synchrotron small-angle X-ray scattering, the in situ role of natural and synthetic crosslinks on the stabilization of the intermolecular structure of collagen in skinswas studied. The results showed that, although natural crosslinks affected the denaturation temperature of collagen, they were largely weakened when crosslinked using chromium sulfate. The development of synergistic crosslinking chemistries could help retain the intrinsic chemical and physical properties of collagen-based biological materials.

TGase-Enhanced Microtissue Assembly in 3D-Printed-Template-Scaffold (3D-MAPS) for Large Tissue Defect Reparation.

Engineering large and functional tissue constructs with complex structures (e.g., external ear or nose) for reparation and reconstruction of tissue defects remains one of the major challenges in regenerative medicine, which demands abundant cell sources, advanced biofabrication schemes, and satisfactory integration with the host for long-term efficacy post implantation. Here the 'Microtissue Assembly in 3D-Printed-template-Scaffold' (3D-MAPS), as a platform technology to rapidly fabricate centimeter-sized functional tissue constructs with complex structures, is developed. 3D-MAPS facilitates bottom-up assembly of large-scale manufactured microtissues within the 3D-printed hollow polymeric templates with pre-defined architectures. The assembly and fusion of 2×106 mesenchymal stem cell-based microtissues within the defined 3D-printed template is further enhanced by addition of a natural protein crosslinker (i.e., transglutaminase (TGase)), and thereby achieves construction of centimeter-sized tissue with high cell viability and mechanical stability in vitro within 30 min. Further in vivo implantation of the 3D-MAPS-fabricated ear-like tissue construct in rabbit models assisted by flap prefabrication technique results in increased structural vascular support and strengthened functional survival. Thus, the TGase-enhanced 3D-MAPS demonstrates its potential and feasibility as a powerful biofabrication platform for tissue engineering application.

Crosslinking of Chitosan with Dialdehyde Chitosan as a New Approach for Biomedical Applications.

Materials based on natural high molecular compounds are particularly interesting for biomedical applications. It is known that the cross-linking agent used for preparation of biomacromolecule-based materials is as important as used biopolymer. Therefore, natural cross-linkers containing reactive carbonyl groups are of great interest especially for modifying properties of natural polysaccharides. One of the most popular cross-linking agents is glutaraldehyde. Nevertheless, the unreacted particles can be released from the cross-linked material and cause cytotoxic effects. This can be eliminated when using a cross-linker based e.g., on polysaccharides. This article describes quick and efficient synthesis of dialdehyde chitosan (DACS) and its application for the preparation of chitosan films. Materials obtained with different amount of DACS were fully characterized in terms of structure and surface morphology. Thermal and mechanical properties as well as hydrophilic character were also examined. The results obtained were compared with the materials obtained by cross-linking chitosan with low molecular weight glutaraldehyde and high molecular weight cross-linking agent based on polysaccharide–dialdehyde starch. Toxicity of all obtained materials was tested using the Microtox® test. It has been shown that due to better mechanical, thermal and surface properties as well as lower toxicity, dialdehyde chitosan is a very promising crosslinking agent.

Natural cross-linker-stabilized acellular porcine corneal stroma for lamellar keratoplasty

Acellular porcine corneal stroma (APCS) is a promising alternative to human donor cornea for lamellar keratoplasty (LKP). However, the detergents, enzymes and physical forces used during decellularization unavoidably alter the cornea's extracellular matrix composition and disrupt its ultrastructure, making it less mechanically stable and liable to degradation both in vitro and in vivo. Herein, we systematically analyzed the low biomechanics and easy degradability of APCS in terms of structure and protein composition. Then, we introduced natural cross-linkers, namely proanthocyanidin (PA), epigallocatechin-3-gallate and genipin, to stabilize the APCS that exhibited color variations during crosslinking. Then, we developed a protective crosslinking system by combining cross-linkers with bovine serum albumin (BSA) to reduce color change, maintain transparency and improve the mechanical property of APCS. PA/BSA-crosslinked APCS (PA/BSA-APCS) shows favorable corneal transparency and swelling property; the improved overall and surface corneal biomechanics were comparable to those of human cornea, revealing strong resistance to enzymatic degradation and good biocompatibility. Results from LKP in the rabbit model showed complete re-epithelialization without graft melting, the stitches were scarcely loosened after the operation and more host keratocytes had migrated in PA/BSA-APCS at six months post-operation. Therefore, PA/BSA-APCS could be useful as a corneal substitute for tissue regeneration and the protective crosslinking system could be applicable in other bioengineering fields.

Impact of Two Different Application Times of A Natural Collagen Crosslinker on The Bonding Quality to Caries Affected Dentin

Impact of Two Different Application Times of A Natural Collagen Crosslinker on The Bonding Quality to Caries Affected Dentin