The role of nanochitin in biologically-active matrices for tissue engineering-where do we stand?

Our regard to the use of chitin as a material has drastically changed since its discovery, 210 years ago. From an intractable material because of its insolubility in common solvents, it became one of the most important raw materials serving as a source of chitosan (its main derivative), and more recently, as source of nanometric forms: nanocrystals and nanofibers. Nanoscale chitin forms are remarkable high-value compounds for nanomaterials' development, due to their intrinsic biological and mechanical properties, as well as their potential as eco-friendly components to valorize the plentiful by-products of the seafood industry. Lately, these nanochitin forms have been widely used as nanofillers in polymer nanocomposites, and in particular, in natural biologically-active matrices for the development of biomaterials. The recent progresses achieved in the last two decades concerning the use of nanoscale chitin in biologically-active matrices for tissue engineering is highlighted in this review. First, an overview on the use of nanochitin in the different biomedical fields is presented and discussed. Then, the state-of-the-art regarding the development of biomaterials based on chitin nanocrystals or nanofibers is described in the context of the role of nanochitin in biologically-active matrices namely polysaccharides (chitin, chitosan, cellulose, hyaluronic acid, alginate), proteins (silk, collagen, gelatin) and others (lignin). Finally, major conclusions and perspectives on the use of nanochitin as an increasingly important raw material are described.

Aero Grade Epoxy Vitrimer towards Commercialization.

Traditional crosslinked aero grade epoxy resins have excellent thermal-mechanical properties and solvent resistance, but they cannot be remolded, recycled, or repaired. Vitrimers can be topologically rearranged via an associative exchange mechanism, endowing them with thermoplasticity. Introducing dynamic bonds into crosslinked networks to obtain more sustainable thermosets is currently an interesting research topic. While recent research into vitrimers has indicated many advantages over traditional thermosets, an important shortcoming has been identified: susceptibility to creep at service temperature due to the dynamic bonds present in the network. In addition, designing aero grade epoxy vitrimers (similar to RTM6 resin) still remains a challenge. Herein, low creep aero grade epoxy vitrimer with thermal and mechanical properties similar to those of aero grade epoxy resins and with the ability to be recyclable, repairable, and reprocessable, has been prepared. In this manuscript, we demonstrate that aero grade epoxy vitrimer with reduced creep can be easily designed by the introduction of a certain fraction of permanent crosslinks, without having a negative effect on the stress relaxation of the material. Subsequently, the mechanical and relaxation properties were investigated and compared with those of classical aero grade epoxy resin. A high Tg (175 °C) epoxy vitrimer was obtained which fulfilled all mechanical and thermal specifications of the aero sector. This work provides a simple network design to obtain aero grade epoxy resins with excellent creep resistance at elevated temperatures while being sustainable.

Study into the Mechanical Properties of a New Aeronautic-Grade Epoxy-Based Carbon-Fiber-Reinforced Vitrimer.

The current drive for sustainability demands recyclable matrices for composite materials. Vitrimers combine thermoset properties with reprocessability, but their mechanical performance in highly loaded applications, for instance, composites for aeronautics, is still to be demonstrated. This work presents the complete mechanical characterization of a new vitrimer reinforced with carbon fiber. This vitrimer formulation consists of functional epoxy groups and a new dynamic disulfide crosslinks-based hardener. The testing campaign for the vitrimer composites encompassed tension, compression, interlaminar shear strength (ILSS), in-plane shear (IPS), open-hole tension (OHT) and compression (OHC), filled-hole compression (FHC) and interlaminar fracture toughness tests under mode I and II. Test conditions included room temperature and high temperature of 70 °C and 120 °C, respectively, after moisture saturation. Tension and flexural tests also were applied on the neat vitrimer resin. The results compared well with those obtained for current aeronautic materials manufactured by Resin Transfer Molding (RTM). The lower values observed in compression and ILSS derived from the thermoplastic veils included as a toughening material. This work demonstrates that the vitrimer formulation presented meets the requirements of current matrices for aeronautic-grade carbon-reinforced composites.

Eco-friendly isolation and characterization of nanochitin from different origins by microwave irradiation: Optimization using response surface methodology.

The extraction of nanochitin from marine waste has attracted great industrial interest due to its unique properties, namely biodegradability, biocompatibility and as a functional reinforcing agent. Conventional acid hydrolysis isolation of nanochitin requires high temperatures and acid concentration, time and energy. Herein, for the first time, microwave irradiation method was used as an eco-friendly approach to isolate nanochitin from different sources. The isolation conditions were optimized through an experimental Box-Behnken design using surface response methodology. The data showed optimal conditions of 1 M HCl, 10.00 min and 124.75 W to obtain lobster nanocrystals; 1 M HCl, 14.34 min and 50.21 W to obtain shrimp nanocrystals; and 1 M HCl, 29.08 min and 54.08 W to obtain squid pen nanofibres, reducing time and HCl concentration. The obtained isolation yields where of 85.30, 79.92 and 80.59 % for lobster, shrimp and squid, respectively. The morphology of the nanochitins was dependent of the chitin origin, and the lengths of the nanochitins were of 314.74, 386.12 and > 900 nm for lobster, shrimp and squid pen, respectively. The thermal stability of the ensuing nanochitins was maintained after treatment. The results showed that nanochitin could be obtained by using an eco-friendly approach like microwave irradiation.

Adipose-Derived Mesenchymal Stem Cell Chondrospheroids Cultured in Hypoxia and a 3D Porous Chitosan/Chitin Nanocrystal Scaffold as a Platform for Cartilage Tissue Engineering.

Articular cartilage degeneration is one of the most common causes of pain and disability in middle-aged and older people. Tissue engineering (TE) has shown great therapeutic promise for this condition. The design of cartilage regeneration constructs must take into account the specific characteristics of the cartilaginous matrix, as well as the avascular nature of cartilage and its cells' peculiar arrangement in isogenic groups. Keeping these factors in mind, we have designed a 3D porous scaffold based on genipin-crosslinked chitosan/chitin nanocrystals for spheroid chondral differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) induced in hypoxic conditions. First, we demonstrated that, under low oxygen conditions, the chondrospheroids obtained express cartilage-specific markers including collagen type II (COL2A1) and aggrecan, lacking expression of osteogenic differentiation marker collagen type I (COL1A2). These results were associated with an increased expression of hypoxia-inducible factor 1α, which positively directs COL2A1 and aggrecan expression. Finally, we determined the most suitable chondrogenic differentiation pattern when hASC spheroids were seeded in the 3D porous scaffold under hypoxia and obtained a chondral extracellular matrix with a high sulphated glycosaminoglycan content, which is characteristic of articular cartilage. These findings highlight the potential use of such templates in cartilage tissue engineering.

Physicochemical and in vitro cytotoxic properties of chitosan from mushroom species (Boletus bovinus and Laccaria laccata).

Chitosan samples from two mushroom species (Boletus bovinus, Laccaria laccata) were obtained and characterized by viscosimetry, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), elemental analyses (EA), nuclear magnetic resonance spectroscopy (13C NMR), X-ray diffraction (XRD) and thermogravimetric (TGA) analyses. Properties of the fungal chitosan samples were compared to commercial low-molecular weight chitosan, crustacean chitosan (Cervimunida johni) and chitosan obtained from an insect (Hilobius abietis). Additionally, the cytotoxic properties of chitosan in vitro on cancerous hepatoma and non-cancerous ovary cells cultivated on films with different chitosan concentrations was evaluated. As a conclusion, this study clearly revealed that low-molecular weight chitosan films and solutions with high degree of deacetylation can act cytotoxically on both tumor MH-22A and normal CHO cells in vitro. Consequently, this work may be useful for further investigations of natural anticancer products in medical areas.

Functional Chitosan Derivative and Chitin as Decolorization Materials for Methylene Blue and Methyl Orange from Aqueous Solution.

Dyes are classified as one of the major pollutants of water. They have negative impacts not only on environment but also on human health. In fact, wastewater that contains these harmful substances requires many types of treatments. Therefore, alternative methods and adsorption agents are needed. Herein, we propose to evaluate the decolorization of methylene blue (MB) and methyl orange (MO) as two models of soluble dyes from water using chitin and chitosan-graft-polyacrylamide. Furthermore, the applicability of these biomacromolecules as alternative adsorption agents, their sticking probability and desorption were also examined. Experimental parameters such as dye concentration, contact time, pH solution, adsorbent dosage and temperature were thoroughly examined for the grafted chitosan and chitin. The activation energy ( E a ) and the thermodynamic variables (i.e., standard Gibb's free energy ( Δ G 0 ), standard enthalpy ( Δ H 0 ), and standard entropy ( Δ S 0 )) were determined using the Van't Hoff and Arrhenius equations. The sticking probability ( S *) model for MB and MO removal by chitin and the chitosan derivative demonstrated that both dyes were successfully removed under the proposed conditions. Desorption studies of MB and MO showed the reusability of both materials, suggesting their application for removing dyes from aqueous solution.

Chitin Nanoforms Provide Mechanical and Topological Cues to Support Growth of Human Adipose Stem Cells in Chitosan Matrices.

The precise role and value of incorporating nanoforms in biologically active matrices for medical applications is not known. In our current work, we incorporate two chitin nanoforms (i.e., nanocrystals or nanofibers) into Genipin-chitosan crosslinked matrices. These materials were studied as 2D films and 3D porous scaffolds to assess their potential as primary support and guidance for stem cells in tissue engineering and regenerative medicine applications. The incorporation of either nanoforms in these 2D and 3D materials reveals significantly better swelling properties and robust mechanical performance in contrast to nanoform-free chitosan matrices. Furthermore, our data shows that these materials, in particular, incorporation of low concentration chitin nanoforms provide specific topological cues to guide the survival, adhesion, and proliferation of human adipose-derived stem cells. These findings demonstrate the potential of Genipin-chitosan crosslinked matrices impregnated with chitin nanoforms as value added materials for stem cell-based biomedical applications.

Supplementing capsaicin with chitosan-based films enhanced the anti-quorum sensing, antimicrobial, antioxidant, transparency, elasticity and hydrophobicity.

In the current study, capsaicin, a plant alkaloid with high antioxidative, anti-inflammatory, antiobesity, anticancer and analgesic properties, was used in the film technology for the first time. In the same regard, chitosan (as a versatile animal-based polymer) was blended with capsaicin at three different concentrations to obtain edible films. The produced films were characterized by FT-IR, SEM, and DSC. Mechanical, transmittance, hydrophobicity, anti-quorum sensing, antimicrobial and antioxidant properties were also examined. Incorporation of 0.6 mg of capsaicin into the chitosan matrix (200 mg dissolved in 1% acetic acid solution) was observed as an optimal concentration for boosting up three film properties including mechanical, optical and surface morphology. A continuous improvement was recorded in anti-quorum sensing and antimicrobial activities, antioxidative and hydrophobicity with increasing concentration of capsaicin in the film. In further studies, chitosan-capsaicin blend films can be used as a food packaging material as well dermal and wound healing patches.

False flax (Camelina sativa) seed oil as suitable ingredient for the enhancement of physicochemical and biological properties of chitosan films.

To overcome the drawbacks of synthetic films in food packaging industry, researchers are turned to natural bio-based edible films enriched with various plant additives. In current study chitosan blend films were produced by incorporating Camelina sativa seed oil at varying concentrations to chitosan matrix. The chitosan blend films were characterized both physicochemically (structural, morphological, thermal, optical and mechanical) and biologically (antimicrobial and antioxidant activity). The incorporation of C. sativa seed oil notably enhanced thermal stability, antioxidative, anti-quorum sensing and antimicrobial activity. Except elongation at break, other mechanical properties of the blend films were not affected by incorporation of C. sativa seed oil. The surface morphology of blend films was recorded as slightly rough, non-porous and fibre-free surface. As it was expected the optical transmittance in visible region was gradually decreased with increasing fraction of seed oil. Interestingly the hydrophilicity of the blend films revealed a swift increase which can be explained by the formation of micelle between glycerol and Tween 40 in blend films. This study provides valuable information for C. sativa seed oil to be used as a blending ingredient in chitosan film technology.

Effect of different animal fat and plant oil additives on physicochemical, mechanical, antimicrobial and antioxidant properties of chitosan films.

Practical application of chitosan-essential oil blend films is limited due to the uneconomical extraction procedure of essential oils from plants. This study aimed to produce chitosan films blended with low cost and commercially available oils and fats consumed in daily human diet (olive, corn and sunflower oils, butter and animal fats). The study also focused on how physicochemical, biological and mechanical properties of chitosan blend films were influenced by the incorporation of oils and fats with varying unsaturation degrees. Possible interactions of chitosan film matrix with incorporated oils or fats were investigated. Chitosan-olive oil film showed better surface morphology and higher thermal stability than the films with other unsaturated oils. Tensile strength, Young's modulus and elongation at break were improved by 57.2%, 25.1% and 31.7% for chitosan-olive oil film, respectively. Chitosan-olive oil blend film had the highest antibacterial activity (almost equal to that of commercial antibiotic gentamicin). Edible films obtained from by incorporation of natural oils and fats into chitosan can help produce an environmentally friendly packaging material that is low cost and easily manufactured.

An inclusive physicochemical comparison of natural and synthetic chitin films.

Natural and synthetic chitin films, obtained from the same source were produced and their physicochemical properties were examined comparatively. Firstly, natural chitin film was obtained from elytra of an insect (Oryctes nasicornis L.) and purity of the obtained chitin film (degree of acetylation: 79±2%) was demonstrated by solid state 13C nuclear magnetic resonance (13C NMR). Then, the synthetic film was produced by dissolving of natural chitin film in LiCl-DMAc. The obtained natural and synthetic films were characterized by AFM, TGA, DSC, FTIR, mechanical properties, light transmission and contact angle. The analyses result demonstrated that natural chitin film lost very important properties such as high thermal stability, transparency, nanofibrous nature, tensile strength, Young's modulus and hydrophobicity after transforming the synthetic film.

Diatomite as a novel composite ingredient for chitosan film with enhanced physicochemical properties.

Practical applications of biopolymers in different industries are gaining considerable increase day by day. But still, these biopolymers lack important properties in order to meet the industrial demands. In the same regard, in the current study, chitosan composite films are produced by incorporating diatomite soil at two different concentrations. In order to obtain a homogeneous film, glutaraldehyde was supplemented to chitosan solution as a cross-linker. Compositing diatomaceous earth to chitosan film resulted in improvement of various important physicochemical properties compared to control such as; enhanced film wettability, increase elongation at break and improved thermal stability (264-277°C). The microstructure of the film was observed to haveconsisted of homogeneously distributed blister-shaped structures arised due to the incorporation of diatomite. The incorporation of diatomite did not influence the overall antioxidant activity of the composite films, which can be ascribe to the difficulty radicals formation. Chitosan film incorporated with increasing fraction of diatomite revealed a notable enhancement in the antimicrobial activity. Additionally with the present study, for the first time possible interactions between chitosan/diatomite were determined via quantum chemical calculations. Current study will be helpful in giving a new biotechnological perspective to diatom in terms of its successful application in hydrophobic composite film production.

On chemistry of γ-chitin.

The biological material, chitin, is present in nature in three allomorphic forms: α, ß and γ. Whereas most studies have dealt with α- and ß-chitin, only few investigations have focused on γ-chitin, whose structural and physicochemical properties have not been well delineated. In this study, chitin obtained for the first time from the cocoon of the moth (Orgyia dubia) was subjected to extensive physicochemical analyses and examined, in parallel, with α-chitin from exoskeleton of a freshwater crab and ß-chitin from cuttlebone of the common cuttlefish. Our results, which are supported by13C CP-MAS NMR, XRD, FT-IR, Raman spectroscopy, TGA, DSC, SEM, AFM, chitinase digestive test and elemental analysis, verify the authenticity of γ-chitin. Further, quantum chemical calculations were conducted on all three allomorphic forms, and, together with our physicochemical analyses, demonstrate that γ-chitin is distinct, yet closer in structure to α-chitin than ß-chitin.

The Antifungal Activity of Functionalized Chitin Nanocrystals in Poly (Lactid Acid) Films.

As, in the market, poly (lactic acid) (PLA) is the most used polymer as an alternative to conventional plastics, and as functionalized chitin nanocrystals (CHNC) can provide structural and bioactive properties, their combination sounds promising in the preparation of functional nanocomposite films for sustainable packaging. Chitin nanocrystals were successfully modified via acylation using anhydride acetic and dodecanoyl chloride acid to improve their compatibility with the matrix, PLA. The nanocomposite films were prepared by extrusion/compression approach using different concentrations of both sets of functionalized CHNC. This investigation brings forward that both sets of modified CHNC act as functional agents, i.e., they slightly improved the hydrophobic character of the PLA nanocomposite films, and, very importantly, they also enhanced their antifungal activity. Nonetheless, the nanocomposite films prepared with the CHNC modified with dodecanoyl chloride acid presented the best properties.

Utilization of flax (Linum usitatissimum) cellulose nanocrystals as reinforcing material for chitosan films.

Use of plastic based packaging tools is causing both health and economic problems. To overcome this situation, researchers are focusing on the use of different biomaterials such as chitosan and cellulose. The current study was conducted to check the effect of flax (Linum usitatissimum) cellulose nanocrystals (CNC) on mechanical and barrier properties of chitosan-based films. CNC was incorporated in different concentrations (5, 10, 20 and 30%). CNC was isolated from flax fiber using acid hydrolysis method. Tensile strength (TS) and young modulus (YM) values increased with the increase of CNC concentration. Chitosan film with 20% CNC revealed the highest YM value as 52.35MPa. No significant improvement was recorded in water vapor permeability due to overall lower film crystallinity. All the films were observed to be transparent up to an acceptable level. SEM and AFM analysis confirmed the homogeneity of films. A gradual enhancement was recorded in the antimicrobial activity of chitosan/CNC composite films. No significant improvement revealed in the thermal stability of composites.

Biological, mechanical, optical and physicochemical properties of natural chitin films obtained from the dorsal pronotum and the wing of cockroach.

In previous studies, chitin-based films were produced from chitin nanofibers in dust form and fully characterized. However, chitin films naturally present in many organisms have not been isolated and characterized. Herein, structurally intact chitin films were successfully extracted from the dorsal pronotum and the wing of cockroach. Despite using the same extraction procedure, important differences were observed. Especially, hydrophobicity, transparency, antifungal and antibacterial biofilm activities of wing chitin film were recorded notably higher than those of chitin film from the dorsal pronotum. However, better mechanical properties were observed for chitin film from the dorsal pronotum. Notably, among the tested bacteria, two common pathogens could not form biofilms on the surface of the films. This study clearly demonstrated natural chitin films obtained from an insect can provide a new perspective to chitin-based applications where chitin films with high thermal stability, transparency, resistance to bacterial biofilm formation and antifungal activity are needed.

Triethyl Citrate (TEC) as a Dispersing Aid in Polylactic Acid/Chitin Nanocomposites Prepared via Liquid-Assisted Extrusion.

The production of fully bio-based and biodegradable nanocomposites has gained attention during recent years due to environmental reasons; however, the production of these nanocomposites on the large-scale is challenging. Polylactic acid/chitin nanocrystal (PLA/ChNC) nanocomposites with triethyl citrate (TEC) at varied concentrations (2.5, 5.0, and 7.5 wt %) were prepared using liquid-assisted extrusion. The goal was to find the minimum amount of the TEC plasticizer needed to enhance the ChNC dispersion. The microscopy study showed that the dispersion and distribution of the ChNC into PLA improved with the increasing TEC content. Hence, the nanocomposite with the highest plasticizer content (7.5 wt %) showed the highest optical transparency and improved thermal and mechanical properties compared with its counterpart without the ChNC. Gel permeation chromatography confirmed that the water and ethanol used during the extrusion did not degrade PLA. Further, Fourier transform infrared spectroscopy showed improved interaction between PLA and ChNC through hydrogen bonding when TEC was added. All results confirmed that the plasticizer plays an important role as a dispersing aid in the processing of PLA/ChNC nanocomposites.

Self-bonded composite films based on cellulose nanofibers and chitin nanocrystals as antifungal materials.

Cellulose nanofibers and chitin nanocrystals, two main components of agricultural and aquacultural by-products, were obtained from blue agave and yellow squat lobster industrial residues. Cellulose nanofibers were obtained using high pressure homogenization, while chitin nanocrystals were obtained by hydrolysis in acid medium. Cellulose nanofibers and chitin nanocrystals were characterized by X-ray diffraction, Atomic Force Microscopy and Infrared spectroscopy. Self-bonded composite films with different composition were fabricated by hot pressing and their properties were evaluated. Antifungal activity of chitin nanocrystals was studied using a Cellometer(®) cell count device, mechanical properties at tension were measured with a universal testing machine, water vapor permeability was evaluated with a thermohygrometer and surface tension with sessile drop contact angle method. The addition of chitin nanocrystals reduced slightly the mechanical properties of the composite. Presence of chitin nanocrystals influenced the growth of Aspergillus sp fungus in the surface of the composites as expected.

Processing of α-chitin nanofibers by dynamic high pressure homogenization: characterization and antifungal activity against A. niger.

Chitin nano-objects become more interesting and attractive material than native chitin because of their usable form, low density, high surface area and promising mechanical properties. This work suggests a straightforward and environmentally friendly method for processing chitin nanofibers using dynamic high pressure homogenization. This technique proved to be a remarkably simple way to get α-chitin into α-chitin nanofibers from yellow lobster wastes with a uniform width (bellow 100 nm) and high aspect ratio; and may contributes to a major breakthrough in chitin applications. Moreover, the resulting α-chitin nanofibers were characterized and compared with native α-chitin in terms of chemical and crystal structure, thermal degradation and antifungal activity. The biological assays highlighted that the nano nature of chitin nanofibers plays an important role in the antifungal activity against Aspergillus niger.