Chitin nanocrystal-assisted 3D bioprinting of gelatin methacrylate scaffolds.

In recent years, there has been an increasing focus on the application of hydrogels in tissue engineering. The integration of 3D bioprinting technology has expanded the potential applications of hydrogels. However, few commercially available hydrogels used for 3D biological printing exhibit both excellent biocompatibility and mechanical properties. Gelatin methacrylate (GelMA) has good biocompatibility and is widely used in 3D bioprinting. However, its low mechanical properties limit its use as a standalone bioink for 3D bioprinting. In this work, we designed a biomaterial ink composed of GelMA and chitin nanocrystal (ChiNC). We explored fundamental printing properties of composite bioinks, including rheological properties, porosity, equilibrium swelling rate, mechanical properties, biocompatibility, effects on the secretion of angiogenic factors and fidelity of 3D bioprinting. The results showed that adding 1% (w/v) ChiNC to 10% (w/v) GelMA improved the mechanical properties and printability of the GelMA hydrogels, promoted cell adhesion, proliferation and vascularization and enabled the printing of complex 3D scaffolds. This strategy of incorporating ChiNC to enhance the performance of GelMA biomaterials could potentially be applied to other biomaterials, thereby expanding the range of materials available for use. Furthermore, in combination with 3D bioprinting technology, this approach could be leveraged to bioprint scaffolds with complex structures, further broadening the potential applications in tissue engineering.

Tuning liquid aggregation of zwitterionic chitin nanocrystals by graphene oxide planar catchers via electrostatic regulation.

As important structural units, biomass nanomaterials have exhibited great potentials to construct high-performance macroscopic materials for broad applications by liquid assembly. However, the liquid aggregation of nanomaterials was less investigated. Here, we demonstrate that the one-dimensional (1D) zwitterionic chitin nanocrystals (ZChNCs) can be reversibly captured and released by two-dimensional (2D) planar catchers of graphene oxide (GO) sheets. The dominant electrostatic regulation strategy by pH variation drives that there are three reversible changes for the liquid aggregation of ZChNCs and GO, which were the isolated dispersion state (pH > 7), homogeneous hybridization state (7 ≥ pH ≥ 5), and partially stacked hybridization state (pH < 5), respectively. We found there are no sedimentation during the change of liquid aggregation with the higher absolute Zeta potentials (almost>30 mV). Moreover, the ZChNCs-GO nanohybrids have reached a maximum Zeta potential up to -80 mV, which can be explained by the ionization of excess carboxyl groups on the surface of ZChNCs. Besides, the electrostatic regulation endows the nanohybrids with rheological behavior, which is beneficial to the macro assembly of liquid nanomaterials. This work provides a new class of hybrid colloidal nanomaterials, opens the structural design dimension of macro assembly and holds great potentials in high-performance biodegradable material.

Chitin nanocrystals assisted 3D printing of polycitrate thermoset bioelastomers.

Citrate-based thermoset bioelastomer has numerous tissue engineering applications. However, its insoluble and unmeltable features restricted processing techniques for fabricating complex scaffolds. Herein, direct ink writing (DIW) was explored for 3D printing of poly(1, 8-octanediol-co-Pluronic F127 citrate) (POFC) bioelastomer scaffolds considering that POFC prepolymer (pre-POFC) was waterborne and could form a stable emulsion. The pre-POFC emulsion couldn't be printed, however, chitin nanocrystal (ChiNC) could be as a rheological modifier to tune the flow behavior of pre-POFC emulsion, and thus DIW printing of POFC scaffolds was successfully realized; moreover, ChiNC was also as a supporting agent to prevent collapse of filaments during thermocuring, and simultaneously as a biobased nanofiller to reinforce scaffolds. The rheological analyses showed the pre-POFC/ChiNC inks fulfilled the requirements for DIW printing. The printed scaffolds exhibited low swelling, and good performances in strength and resilence. Furthermore, the entire process was easily performed and eco-friendly.

Chitin Pickering Emulsion for Oil Inclusion in Composite Films.

A film containing a stable and well-dispersed hydrophobic phase in a surfactant-free bio-based hydrophilic matrix is proposed. In this study, an aqueous suspension of rod-like chitin nanocrystals (ChiNCs), mixed with paraffin oil, form an oil-in-water Pickering emulsion with a droplet diameter of 3 µm. These emulsions mixed with a 5 wt% starch solution formed homogeneous composite films by solvent casting. Various amounts of emulsion were incorporated, leading to self-supported films with a volume of oil as high as 45 vol%, with less than 1% of ChiNCs. This model inclusion system leads to droplets homogeneously dispersed throughout the composite films, as revealed by microscopy (SEM and CLSM) with mechanical properties controlled by the matrix. Finally, the droplets were easily released from the matrix by enzymatic hydrolysis. This easy-to-implement transparent film proved to be a good candidate when it is desirable to disperse a poor water-soluble component in a hydrophilic edible matrix.

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.

Preparation of multifunctional carboxymethyl cellulose-based films incorporated with chitin nanocrystal and grapefruit seed extract.

Chitin nanocrystals (ChNC) were isolated from shrimp shells powder using acid hydrolysis and ammonium persulfate methods. Multifunctional carboxymethyl cellulose (CMC) composite films were prepared by adding ChNC and grapefruit seed extract (GSE), and their effects on the optical, mechanical, water vapor barrier, and antibacterial properties of CMC film were investigated. The isolated ChNC had a needle-like structure with a length of 340-370 nm and a diameter of 18-20 nm depending on the isolation method. The CMC films prepared with ChNC and GSE were transparent with high UV barrier properties. The addition of GSE reduced the strength (TS) and stiffness (EM) of CMC films by 10.4% and 30.3%, respectively, while the flexibility (EB) increased by 17.7%. However, when the ChNC was added, the TS and EM of CMC film increased by 19.7% and 58.7%, respectively, and the EB remained the same. The addition of ChNC reduced the water vapor permeability (WVP) of the CMC film by 27%. CMC films containing GSE also showed strong antibacterial activity against foodborne pathogenic bacteria, E. coli and L. monocytogenes.

Preparation, characterization and application of rod-like chitin nanocrystal by using p-toluenesulfonic acid/choline chloride deep eutectic solvent as a hydrolytic media.

Chitin nanocrystal (ChiNC) was fabricated based on p-toluenesulfonic acid -choline chloride deep eutectic solvent treatment. The obtained ChiNC was about 12-44 nm in width and 206-399 nm in length. The crystalline structure and the functional groups of ChiNC were maintained during the preparation process. Moreover, porcine pancreas lipase (PPL) was successfully immobilized onto the ChiNC to form the immobilized PPL (PPL@ChiNC). The resulting PPL@ChiNC has enzyme loading and activity recovery of 35.6 mg/g and 82.5%, respectively. The thermal stability, pH and temperature adaptabilities of PPL@ChiNC was improved, comparing with free PPL. The demonstrated DES treatment process was efficient for ChiNC preparation and the as-prepared ChiNC exhibited great potentials in biocatalysis and biomedical field.

Effect of isolation methods of chitin nanocrystals on the properties of chitin-silver hybrid nanoparticles.

Chitin nanocrystal (ChNC) was isolated using sulfuric acid hydrolysis (ChNCH2SO4), TEMPO-oxidation (ChNCTEMPO), and ammonium persulfate (ChNCAPS) methods, and used for the preparation of hybrid nanoparticles of ChNC/silver nanoparticles (AgNP). The ChNC exhibited a needle-shaped structure with a sulfate group content of 135 µmol/g for ChNCH2SO4 and carboxyl content of 0.71 and 1.42 mmol/g for ChNCTEMPO and ChNCAPS, respectively. ChNC worked as a reducing and stabilizing agent for the production of AgNP and reduced the size of AgNP from 23.9 nm to 6.3 nm in the ChNC/AgNP hybrid. The carboxyl content of ChNC played a significant role for the nucleation, size distribution, and antibacterial activity of ChNC/AgNP. ChNC/AgNP hybrid, especially ChNCAPS/AgNP, exhibited strong antibacterial activity against food-borne pathogenic Gram-negative (E. coli) and Gram-positive (L. monocytogenes) bacteria. The prepared ChNC/AgNP hybrid nanomaterials have a high potential for the application to be used as a nanofiller to improve the properties of food packaging materials to extend the shelf-life of packaged food.

Lyotropic liquid crystal self-assembly of H2O2-hydrolyzed chitin nanocrystals.

H2O2 hydrolysis of mechanically-defibrillated chitin nanofibrils was explored as a green way of fabricating rod-like chitin nanocrystals (H2O2-hydrolyzed CHNs) that have an average length of 350 nm and width of 40 nm. We investigated the structure and morphology of CHNs as well as the rheology and lyotropic self-assembly behavior of its colloidal dispersions. The results show that although H2O2-hydrolyzed CHNs maintained the crystalline structure of α-chitin, surface charge of the nanorods was switched from positive to negative. As a consequence, the colloidal nanocrystals were well-dispersed in neutral or alkaline aqueous media, and behaved as a lyotropic liquid crystal between two critical concentrations. It is interesting that lyotropic liquid crystal transition was a spontaneously self-assembly from well-aligned nanofibers, to nanobelts, and to multi-layered lamellae. At high critical concentration, H2O2-hydrolyzed CHN colloids exhibited a sol-gel transition, which was discovered to be highly dependent on the storage time, concentration, temperature, and surface charge density. It is also suggested that nematic mesophases rather than gel could be effectively maintained by improving the surface charge density or lowering the aging temperature and colloidal concentration of CHNs.

Chitin nanocrystal enhanced wet adhesion performance of mussel-inspired citrate-based soft-tissue adhesive.

Chitin nanocrystal (ChiNC) with its good biodegradability and biocompatibility as well as rod-like structure characteristic has become one of excellent nanofillers to enhance mechanical properties and bioactivity of biomedical polymers. For further extending its application fields, here, we dispersed ChiNC into a recently synthesized citrate-based tissue adhesive (POEC-d) and explored its effects on the adhesion and cytocompatibility of the adhesive. POEC-d, a mussel-inspired wet adhesive, was prepared by a one-pot melt polycondensation of 1, 8-octanediol, poly(ethylene oxide) (PEO), citric acid (CA) and dopamine (DA). The good water-solubility of POEC-d allowed facilely blending ChiNC and POEC-d to ultimately acquire POEC-d/ChiNC nanocomposite adhesives. The results showed the ChiNCs were finely dispersed in the POEC-d matrix and endowed the adhesive with extra crosslinks to enhance the bulk cohesion property. Synchronously, the lap-shear adhesion strengths measured by bonding wet porcine skins considerably improved with increasing the ChiNC content. In addition, the POEC-d/ChiNC nanocomposite adhesives exhibited lower swelling ratio as compared to the POEC-d adhesive as well as good cytocompatibility, revealing their superior applicability in soft-tissue adhesion.

Chitin nanocrystals prepared by oxidation of α-chitin using the O2/laccase/TEMPO system.

Laccase mediator oxidation was applied to chitin at pH 6.8 and 30 °C to prepare chitin nanocrystals with a catalytic amount of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO). When 40 mM TEMPO and a total of 500 U laccase were added to 1 g chitin, the yield of water-insoluble oxidized chitin was more than 95%, and the carboxylate content was 0.43 mmol/g. Adsorption of laccase molecules on chitin particles occurred in a buffer at pH 6.8, which may have been caused by electrostatic interactions between positively charged C2-ammonium groups of chitin and anionically charged groups of laccase. Rod-like chitin nanocrystals (ChNCs) were obtained with average lengths and widths of 480 ±â€¯200 nm and 24 ±â€¯17 nm, respectively, by sonication of the oxidized chitin/water suspensions. The O2/laccase/TEMPO oxidation caused no decrease in the degree of N-acetylation or the crystallinity of the original chitin based on FTIR and X-ray diffraction data.

A facile and green emulsion casting method to prepare chitin nanocrystal reinforced citrate-based bioelastomer.

Chitin nanocrystal (ChiNC) is a promising reinforcing nanofiller for biomedical polymers. However, its self-aggregation characteristics caused processing difficulty in developing ChiNC-based nanocomposites. Herein, a new degradable crosslinked bioelastomer, designated as poly(1,8-octanediol-co-Pluronic F127 citrate) (POFC) was synthesized by melt polycondensation of citric acid, 1,8-octanediol, and Pluronic F127. In comparison to poly(1,8-octanediol citrate) (POC), POFC pre-polymer exhibited self-emulsifying property. Once ChiNC was introduced into the emulsion, a ChiNC stabilized Pickering emulsion was formed. Coupled with a facile green emulsion casting/evaporation method, the ChiNC ultimately reinforced ChiNC/POFC nanocomposite elastomer was fabricated. The presence of F127 segments endowed POFC with better hydrophilicity and shorter degradation time relative to POC. The incorporation of ChiNC into POFC network led to highly increased tensile modulus and strength. In vitro cytotoxicity tests indicated that the ChiNC/POFC elastomer nanocomposite had a good cytocompatibility and it appeared as a potential biomaterial for tissue engineering application.

Chitosan-chitin nanocrystal composite scaffolds for tissue engineering.

Chitin nanocrystals (CNCs) with length and width of 300 and 20nm were uniformly dispersed in chitosan (CS) solution. The CS/CNCs composite scaffolds prepared utilizing a dispersion-based freeze dry approach exhibit significant enhancement in compressive strength and modulus compared with pure CS scaffold both in dry and wet state. A well-interconnected porous structure with size in the range of 100-200µm and over 80% porosity are found in the composite scaffolds. The crystal structure of CNCs is retained in the composite scaffolds. The incorporation of CNCs leads to increase in the scaffold density and decrease in the water swelling ratio. Moreover, the composite scaffolds are successfully applied as scaffolds for MC3T3-E1 osteoblast cells, showing their excellent biocompatibility and low cytotoxicity. The results of fluorescent micrographs images reveal that CNCs can markedly promote the cell adhesion and proliferation of the osteoblast on CS. The biocompatible composite scaffolds with enhanced mechanical properties have potential application in bone tissue engineering.

Prominent reinforcing effect of chitin nanocrystals on electrospun polydioxanone nanocomposite fiber mats.

The ultra-strong nanocomposite fiber mats based on biodegradable polydioxanone (PDO) and chitin nanocrystals (ChiNCs) were successfully prepared by means of electrospinning. The ChiNCs are uniformly dispersed in the PDO matrix and mostly oriented along fiber long axis, resulting in a significant improvement in mechanical property. Moreover, the introduction of ChiNCs led to the increase of the glass-transition temperature (Tg) and thermal decomposition temperature (Td) of PDO elucidated by thermal analyses. In addition, the loading of ChiNCs caused very different In vitro degradation behavior compared to neat PDO fiber mat. Furthermore, in vitro cell culture results indicated that the addition of ChiNCs improved the cellular adhesion and proliferation.

Impact of ionic strength on chitin nanocrystal-xyloglucan multilayer film growth.

The impact of the ionic strength on the film growth has been studied for the architectures composed of chitin nanocrystals (ChiNC) and xyloglucan (XG) to better understand the fabrication process of multilayer films. The formation of ChiNC-XG assemblies was monitored by quartz crystal microbalance with dissipation (QCM-D) and multilayer films were fabricated by the spin-coating assisted layer-by-layer (LbL) procedure. Films were prepared from 1 g L(-1) ChiNC dispersions at pH 4 without and with the addition of NaCl (0 and 5 mM, respectively) and 0.5 g L(-1) XG solutions in water. Distinct growth pattern and structural characteristics were found for the films prepared from ChiNC at 0 and 5 mM NaCl. Specifically, films assembled without salt exhibited lower mass deposition and film growth failed after 5 (ChiNC-XG) bilayers. Differently, at 5 mM NaCl higher amounts of both polymers (ChiNC and XG) were adsorbed; therefore, the films were thicker, and the deposition succeeded up to 10 bilayers. Atomic force microscopy (AFM) revealed an almost completely covered surface after the adsorption of ChiNC at 5 mM NaCl whereas salt-free ChiNC dispersions resulted in lower surface coverage. These results reliably concluded that the fabrication of (ChiNC-XG) films requires the screening of the charges to promote the layers stability.