Oriented crystallization of octacalcium phosphate into beta-chitin scaffold

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.     

Crystallization of calcium carbonate salts into beta-chitin scaffold

Composites of beta-chitin with calcium carbonate polymorphs were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. The three main polymorphs of calcium carbonate: aragonite, calcite and vaterite, were observed. Their location within the matrix is a function of the polymorph. The supersaturation inside the compartmentalized space in the chitin governs the location and polymorphism of the crystals.     

Utilization of commercial non-chitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy-D-glucose from beta-chitin

Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma viride were investigated for potential utilization in the preparation of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine, GlcNAc) from chitin. Among the tested enzymes, cellulase A. cellulolyticus exhibited highest chitinolytic activity per weight toward amorphous chitin and beta-chitin from squid pen. The optimum pH of the enzyme was 3 where it produced two major hydrolytic products, GlcNAc and N,N'-diacetylchitobiose ([GlcNAc](2)). The product ratio, GlcNAc:[GlcNAc](2), increased while the total yield decreased as the pH was raised from 3. All of the [GlcNAc](2) produced at pH 3 can be converted in situ to GlcNAc by mixing cellulase A. cellulolyticus with one of several other enzymes from A. niger resulting in a higher yield of GlcNAc. An appropriate mixing ratio of cellulase A. cellulolyticus to another enzyme was 9:1 (w/w) and an optimum substrate concentration was 20 mg/mL.     

Beta-chitin from the pens of Loligo sp.: extraction and characterization

Recently the squid pens, a rich source of beta-chitin containing low contents of inorganic compounds, have become available in considerable amounts as a refuse of the fishery industries in Brazil. Thus, the aim of this work is to use squid pens from Loligo sanpaulensis and Loligo plei, species found in the Brazilian coast, as the raw material for the extraction of beta-chitin. The squid pens were submitted to the usual sequence of treatments used for chitin extraction - demineralization and deproteinization - but due to its low content of inorganic compounds a two-step alkaline treatment was enough to produce beta-chitin with low contents of ash (< or = 0.7%). Indeed, the low contents of ash and metals, such as Ca (< or = 10.4 ppm), Mg (< or = 2.5 ppm), Mn (< or = 3.1 ppm) and Fe (< or = 1.8 ppm), are lower than those reported in most of the papers found in the literature. Also, the beta-chitin extracted by employing only the alkaline treatment was more acetylated than the other samples prepared in this work. Regardless of the treatment employed for the extraction of the beta-chitin from the squid pens, its infrared spectra and X-ray diffraction pattern presented only minor differences, however they were clearly distinguished from commercial alpha-chitin.     

Preparation and characterization of novel beta-chitin-hydroxyapatite composite membranes for tissue engineering applications

Beta-chitin is a biopolymer principally found in shells of squid pen. It has the properties of biodegradability, biocompatibility, chemical inertness, wound healing, antibacterial and anti-inflammatory activities. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has osteoconductive property. In this work, beta-chitin-HAp composite membranes were prepared by alternate soaking of beta-chitin membranes in CaCl2 (pH 7.4) and Na2HPO4 solutions for 2 h in each solution. After 1, 3 and 5 cycles of immersion, beta-chitin membranes were characterized using the SEM, FT-IR, EDS and XRD analyses. The results showed the presence of apatite layer on surface of beta-chitin membranes, and the amounts of size and deposition of apatite layers were increased with increasing number of immersion cycles. Human mesenchymal stem cells (hMSCs) were used for evaluation of the biocompatibility of pristine as well as composite membranes for tissue engineering applications. The presence of apatite layers on the surface of beta-chitin membranes increased the cell attachment and spreading suggesting that beta-chitin-HAp composite membranes can be used for tissue engineering applications.     

Two-dimensional magic angle spinning NMR investigation of naturally occurring chitins: precise 1H and 13C resonance assignment of alpha- and beta-chitin

13C homonuclear through-bond correlations of alpha- and beta-chitin were determined by using two-dimensional (2D) INADEQUATE spectra of these allomorphs purified from crab shell and squid pen, respectively. The 2D (13)C-(13)C correlation spectra where two directly bonded carbons share a common double-quantum frequency (DQ) enabled us to precisely assign all (13)C resonances of the chitin allomorphs for the first time. Following the complete (13)C assignment, (1)H chemical shifts of protons attached to each carbon nuclei were assigned by 2D frequency-switched Lee-Goldberg (FSLG) (1)H-(13)C heteronuclear correlation (HETCOR) spectra of the chitin allomorphs, recorded with a short mixing time (60 micros) to provide isotropic (1)H-(13)C chemical shift correlations between bonded pairs proton and carbon nuclei. From the (13)C and (1)H chemical shifts of chitin allomorphs, all 2-deoxy-2-acetamide-D-glucose (N-acetyl-D-glucosamine) monomer units in each allomorph were revealed to be an identical (13)C-(13)C backbone conformation and magnetically equivalent. In addition, it was strongly suggested that there are two different hydrogen-bonding patterns at the hydroxyl groups of alpha-chitin by comparing (1)H chemical shifts at the C6 site of alpha-chitin with those at the same site of beta-chitin.     

Chitosan functional properties

Chitosan is a partially deacetylated polymer of N-acetyl glucosamine. It is essentially a natural, water-soluble, derivative of cellulose with unique properties. Chitosan is usually prepared from chitin (2 acetamido-2-deoxy β-1,4-D-glucan) and chitin has been found in a wide range of natural sources (crustaceans, fungi, insects, annelids, molluscs, coelenterata etc.) However chitosan is only manufactured from crustaceans (crab and crayfish) primarily because a large amount of the crustacean exoskeleton is available as a by product of food processing. Squid pens (a waste byproduct of New Zealand squid processing) are a novel, renewable source of chitin and chitosan. Squid pens are currently regarded as waste and so the raw material is relatively cheap. This study was intended to assess the functional properties of squid pen chitosan. Chitosan was extracted from squid pens and assessed for composition, rheology, flocculation, film formation and antimicrobial properties. Crustacean chitosans were also assessed for comparison. Squid chitosan was colourless, had a low ash content and had significantly improved thickening and suspending properties. The flocculation capacity of squid chitosan was low in comparison with the crustacean sourced chitosans. However it should be possible to increase the flocculation capacity of squid pen chitosan by decreasing the degree of acetylation. Films made with squid chitosan were more elastic than crustacean chitosan with improved functional properties. This high quality chitosan could prove particularly suitable for medical/analytical applications. This revised version was published online in November 2006 with corrections to the Cover Date.     

Ultrafine polysaccharide nanofibrous membranes for water purification

Ultrafine polysaccharide nanofibers (i.e., cellulose and chitin) with 5-10 nm diameters were employed as barrier layers in a new class of thin-film nanofibrous composite (TFNC) membranes for water purification. In addition to concentration, the viscosity of the polysaccharide nanofiber coating suspension was also found to be affected by the pH value and ionic strength. When compared with two commercial UF membranes (PAN10 and PAN400), 10-fold higher permeation flux with above 99.5% rejection ratio were achieved by using ultrafine cellulose nanofibers-based TFNC membranes for ultrafiltration of oil/water emulsions. The very high surface-to-volume ratio and negatively charged surface of cellulose nanofibers, which lead to a high virus adsorption capacity as verified by MS2 bacteriophage testing, offer further opportunities in drinking water applications. The low cost of raw cellulose/chitin materials, the environmentally friendly fabrication process, and the impressive high-flux performance indicate that such ultrafine polysaccharide nanofibers-based TFNC membranes can surpass conventional membrane systems in many different water applications.     

TEMPO-mediated oxidation of β-chitin to prepare individual nanofibrils

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation was applied to β-chitins, originating from tubeworm and squid pen, to prepare chitin nanofibrils. Water-insoluble fractions of the TEMPO-oxidized β-chitins were obtained in various ratios by controlling the addition level of NaClO used as the primary oxidant in the oxidation. The water-insoluble fractions of the TEMPO-oxidized tubeworm β-chitins, when they had carboxylate groups of 0.18–0.25 mmol/g, were successfully converted to highly viscous and translucent gels by disintegration in water. The gels consisted of mostly individual nanofibrils 20–50 nm in width and at least several microns in length. On the other hand, the water-insoluble fractions of the TEMPO-oxidized squid pen β-chitins could be transformed to neither transparent nor translucent dispersions under any oxidation or disintegration conditions used. When sufficient amounts of NaClO were used, both β-chitins were completely oxidized to the corresponding water-soluble polyuronic acids by oxidation of all C6 primary hydroxyls to carboxylate groups.     

β-Chitin nanofibrils for self-sustaining hydrogels preparation via hydrothermal treatment

A transparent nanofibril suspension could be readily obtained by treating purified squid pen powder in water with ultrasonic irradiation. The obtained suspension is consisted of β-chitin nanofibrils (CNF) with 3-10nm in width and several micrometers in length. The degree of acetylation (DA) of CNF was found to be 84% which is about 10% lower than that of untreated sample. The CNF suspension could be transformed into a durable 3-D hydrogels (CH) by simply heating to 180°C for 1-4h in an autoclave. Hydrophobic interaction between CNF was believed to play the major role for CNF self-assembling into hydrogels, since the as-prepared chitin hydrogels readily dissolved in a typical chaotropic solution (8M urea) under room temperature. The hydrothermal duration and CNF concentration (0.3-2% (w/v)) strongly affected the physical properties of CH. The suspension of 1% (w/v) CNF treated with 4h, 180°C hydrothermal heating generated a CH with 99.3% water content, CNF with 87% crystallinity and an mechanical strength of 0.7N breaking force.     

Esterification of beta-chitin via intercalation by carboxylic anhydrides

beta-chitin is known to form intercalation complexes with aliphatic alcohols and amines. We found that it also forms complexes with carboxylic anhydrides. When the beta-chitin-acetic anhydride complex was heated to 105 degrees C, the hydroxyl groups of chitin were acetylated by a host-guest reaction, maintaining the host's crystal structure. Structures of complex and acetylated products were analyzed by X-ray diffraction, (13)C CP/MAS NMR, and infrared spectroscopy. The maximum degree of substitution (DS) was close to 1.0, suggesting regioselective esterification at the C6 position of chitin. Partially acetylated beta-chitin with a DS of 0.4 could incorporate various guest species that are difficult to be incorporated by original beta-chitin. In contrast, beta-chitin acetate with a DS of 1 lost the ability to form a complex. Intercalation complexes of beta-chitin with cyclic anhydrides (succinic and maleic) also underwent esterification by heating, and the products with a DS of approximately 1 dissolved in aqueous alkali, apparently as the result of the dissociation of introduced carboxyl groups. These phenomena are potentially useful in controlling the complexation ability of beta-chitin and the preparation of regioselectively esterified chitin derivatives.     

Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials

The main purpose of this research was to investigate the antitumor and antimicrobial activities of the chitooligosaccharides containing hydrolyzates obtained from the hydrolysis of chitinous materials (such as chitin, chitosan, and squid pen) by bromelain. The optimum preparations were gained in the hydrolysates of squid pen powder hydrolyzed at pH 5, 37 degrees C for 2 days by 0.1% bromelain. The hydrolysates had an 80% inhibitory activity on phyto-pathogenic mold Fusarium oxysporum. Chitooligosaccharides were recovered from the hydrolysates and were used for tumor cell surviving test. Surviving rate of the human leukemic U937 cells was reduced to 69% by the chitooligosaccharides. The solution of 0.1% of water-soluble chitosan was also hydrolyzed for 1 day at pH 5, 37 degrees C by bromelain. The resultant hydrolysates contained the highest chitooligosaccharides, which had inhibitory effect on Bacillus subtilis and also had 40% inhibitory activity on human pathogenic mold Aspergillus fumigatus. Surviving rate of mouse CT26 colorectal adenocarcinoma cells was reduced to 57% by the chitooligosaccharides. This is the first publication of enzymatic reclamation of squid pen (fishery processing waste) for the preparation of antitumor and antimicrobial materials.     

XRD studies of beta-chitin from squid pen with calcium solvent

The crystalline structure of β-chitin from squid pen was investigated by X-ray diffraction (XRD). The purified β-chitin was prepared from bigfin reefsquid pen. β-Chitin was treated with saturated calcium chloride dihydrate/alchohol (CaCl₂·2H₂O/MeOH) solvent system at different conditions for XRD studies. The change of crystallinity of β-chitin from squid pen was studied by using the fiber photographs on imaging plates. The results showed that the diffraction peak (0 1 0) was shifted. It means that the lattice plane (0 1 0) interplanarilly spreaded to 3.4 Å, when the squid pen was washed with water after treatment of Ca solvent. Furthermore, when the squid pen was dried after treatment of Ca solvent and washing with water, interplanar spacing of (0 1 0) inversely shrank to 1.1 Å. These results suggested that Ca solvent especially influences the plane (0 1 0) of β-chitin structure.     

Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose

Never-dried and once-dried hardwood celluloses were oxidized by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated system, and highly crystalline and individualized cellulose nanofibers, dispersed in water, were prepared by mechanical treatment of the oxidized cellulose/water slurries. When carboxylate contents formed from the primary hydroxyl groups of the celluloses reached approximately 1.5 mmol/g, the oxidized cellulose/water slurries were mostly converted to transparent and highly viscous dispersions by mechanical treatment. Transmission electron microscopic observation showed that the dispersions consisted of individualized cellulose nanofibers 3-4 nm in width and a few microns in length. No intrinsic differences between never-dried and once-dried celluloses were found for preparing the dispersion, as long as carboxylate contents in the TEMPO-oxidized celluloses reached approximately 1.5 mmol/g. Changes in viscosity of the dispersions during the mechanical treatment corresponded with those in the dispersed states of the cellulose nanofibers in water.     

Inclusion complex of beta-chitin and aliphatic amines

Inclusion complexation of beta-chitin with linear aliphatic amines was studied by X-ray diffraction. All tested amines, C3 to C8 monoamines and C2 to C7 diamines with terminal amino groups, reversibly formed crystalline complexes with beta-chitin by immersion of dry chitin in pure liquid. Complex formation caused linear increase in the 010 sheet spacing of beta-chitin depending on the carbon number of amine. The complexes could be classified as type I and type II according to the increment of sheet spacing against carbon number. All monoamines formed type II complexes. In dry conditions, diamine formed a type I complex though the type of diamine complex differed for guest species in wet conditions. Based on the unit cell dimension and thermogravimetry, type II and type I are likely to correspond to guest-host (amine-chitobiose) ratios of 2:1 and 1:1, respectively. These differences seem to arise from varied interactions between functional groups of chitin and amines.     

Fabrication and characterisation of α-chitin nanofibers and highly transparent chitin films by pulsed ultrasonication

α-Chitin nanofibers were fabricated with dried shrimp shells via a simple high-intensity ultrasonic treatment under neutral conditions (60 KHz, 300 W, pH = 7). The diameter of the obtained chitin nanofibers could be controlled within 20–200 nm by simply adjusting the ultrasonication time. The pulsed ultrasound disassembled natural chitin into high-aspect-ratio nanofibers with a uniform width (19.4 nm after 30 min sonication). The EDS, FTIR, and XRD characterisation results verified that α-chitin crystalline structure and molecular structure were maintained after the chemical purification and ultrasonic treatments. Interestingly, ultrasonication can slightly increase the degree of crystallinity of chitin (from 60.1 to 65.8). Furthermore, highly transparent chitin films (the transmittance was 90.2% at a 600 nm) and flexible ultralight chitin foams were prepared from chitin nanofiber hydrogels.     

Structural data on the intra-crystalline swelling of beta-chitin

The intra-crystalline swelling of the highly crystalline beta-chitin from Tevnia jerichonana was investigated by X-ray crystallography and Fourier transform infrared (FTIR) spectroscopy, using hydrogenated and deuterated hydrochloric acids as swelling agents. Three levels of swelling were identified that could be defined as inter- and intra-sheet swelling. A moderate and reversible swelling in water and methanol gave crystalline beta-chitin cystallosolvates, namely dihydrate and methanolate, respectively. In these, an inter-sheet swelling was observed, corresponding to an expansion of only the b parameter of the unit cell of beta-chitin. Under these swelling conditions, the use of deuterated reagents had no effect on the amide N&z.sbnd;H⋯O&z.dbnd6;C hydrogen bonds that hold the structure of beta-chitin together, but only induced a partial and reversible deuteration of the chitin hydroxymethyl groups. A more severe swelling - but still reversible - occurred with 6 N HCl or DCl, which converted the crystals of beta-chitin into a paracrystalline gel-like product resulting from inter-sheet+intra-sheet swelling. With this acid strength, the deuteration pattern indicated that a fraction of the amide hydrogen bonds was broken and became susceptible to an irreversible deuteration. A very severe and irreversible swelling occurred with 8 N HCl or DCl. In that case, the inter- and intra-sheet swelling was extensive to the point where all memory of the parallel-chain beta-chitin was lost. In addition, this swelling was accompanied by a drastic and rapid depolymerization. The treatment with 8 N HCl led invariably to crystalline alpha-chitin when the samples were neutralized.     

Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

Fine structure and enzymatic degradation of poly[(R)-3-hydroxybutyrate] and stereocomplexed poly(lactide) nanofibers

Fiber morphology and crystalline structure of poly[(R)-3-hydroxybutyrate] (P(3HB)) and stereocomplexed poly(lactide) (PLA) nanofibers were investigated by using scanning and transmission electron microscopies and X-ray and electron diffractions. In the P(3HB) nanofibers spun from less than 1 wt% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution, planar zigzag conformation (beta-form) as well as 2(1) helix conformation (alpha-form) structure was formed. Based on the electron diffraction measurement of single P(3HB) nanofiber, it was revealed that the molecular chains of P(3HB) align parallel to the fiber direction. From the enzymatic degradation test of P(3HB) nanofiber, it was shown that beta-form molecular chains are degraded more preferentially than alpha-form chains. Stereocomplexed PLA nanofibers were electrospun from 1 wt% poly(l-lactide)/poly(d-lactide) (PLLA/PDLA) solution in HFIP, which contains equal amounts of PLLA and PDLA. While as-spun stereocomplexed PLA nanofiber was amorphous, PLA nanofiber annealed at 100 degrees C contained only racemic crystal. It was supposed that the crystallization behavior of stereocomplexed PLA in the nanofiber is affected by the electrospinning process, which forcibly exerts the strain onto the polymer chains.     

Modification of hydrophilic and hydrophobic surfaces using an ionic-complementary peptide

Ionic-complementary peptides are novel nano-biomaterials with a variety of biomedical applications including potential biosurface engineering. This study presents evidence that a model ionic-complementary peptide EAK16-II is capable of assembling/coating on hydrophilic mica as well as hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces with different nano-patterns. EAK16-II forms randomly oriented nanofibers or nanofiber networks on mica, while ordered nanofibers parallel or oriented 60 degrees or 120 degrees to each other on HOPG, reflecting the crystallographic symmetry of graphite (0001). The density of coated nanofibers on both surfaces can be controlled by adjusting the peptide concentration and the contact time of the peptide solution with the surface. The coated EAK16-II nanofibers alter the wettability of the two surfaces differently: the water contact angle of bare mica surface is measured to be <10 degrees , while it increases to 20.3+/-2.9 degrees upon 2 h modification of the surface using a 29 microM EAK16-II solution. In contrast, the water contact angle decreases significantly from 71.2+/-11.1 degrees to 39.4+/-4.3 degrees after the HOPG surface is coated with a 29 microM peptide solution for 2 h. The stability of the EAK16-II nanofibers on both surfaces is further evaluated by immersing the surface into acidic and basic solutions and analyzing the changes in the nanofiber surface coverage. The EAK16-II nanofibers on mica remain stable in acidic solution but not in alkaline solution, while they are stable on the HOPG surface regardless of the solution pH. This work demonstrates the possibility of using self-assembling peptides for surface modification applications.