Tetsuya Furuike

Wet chemical synthesis of chitosan hydrogel–hydroxyapatite composite membranes for tissue engineering applications

Chitosan, a deacetylated derivative of chitin is a commonly studied biomaterial for tissue-engineering applications due to its biocompatibility, biodegradability, low toxicity, antibacterial activity, wound healing ability and haemostatic properties. However, chitosan has poor mechanical strength due to which its applications in orthopedics are limited. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has mechanical strength and osteoconductive property. In this work, HAp was deposited on the surface of chitosan hydrogel membranes by a wet chemical synthesis method by alternatively soaking the membranes in CaCl(2) (pH 7.4) and Na(2)HPO(4) solutions for different time intervals. These chitosan hydrogel-HAp membranes were characterized using SEM, AFM, EDS, FT-IR and XRD analyses. MTT assay was done to evaluate the biocompatibility of these membranes using MG-63 osteosarcoma cells. The biocompatibility studies suggest that chitosan hydrogel-HAp composite membranes can be useful for tissue-engineering applications.

The Mechanical and Biological Properties of Chitosan Scaffolds for Tissue Regeneration Templates Are Significantly Enhanced by Chitosan from Gongronella butleri

Chitosan with a molecular weight (MW) of 104 Da and 13% degree of acetylation (DA) was extracted from the mycelia of the fungus Gongronella butleri USDB 0201 grown in solid substrate fermentation and used to prepare scaffolds by the freeze-drying method. The mechanical and biological properties of the fungal chitosan scaffolds were evaluated and compared with those of scaffolds prepared using chitosans obtained from shrimp and crab shells and squid bone plates (MW 105-106 Da and DA 10-20%). Under scanning electron microscopy, it was observed that all scaffolds had average pore sizes of approximately 60-90 μm in diameter. Elongated pores were observed in shrimp chitosan scaffolds and polygonal pores were found in crab, squid and fungal chitosan scaffolds. The physico-chemical properties of the chitosans had an effect on the formation of pores in the scaffolds, that consequently influenced the mechanical and biological properties of the scaffolds. Fungal chitosan scaffolds showed excellent mechanical, water absorption and lysozyme degradation properties, whereas shrimp chitosan scaffolds (MW 106Da and DA 12%) exhibited the lowest water absorption properties and lysozyme degradation rate. In the evaluation of biocompatibility of chitosan scaffolds, the ability of fibroblast NIH/3T3 cells to attach on all chitosan scaffolds was similar, but the proliferation of cells with polygonal morphology was faster on crab, squid and fungal chitosan scaffolds than on shrimp chitosan scaffolds. Therefore fungal chitosan scaffold, which has excellent mechanical and biological properties, is the most suitable scaffold to use as a template for tissue regeneration.

Chitin Scaffolds in Tissue Engineering

Tissue engineering/regeneration is based on the hypothesis that healthy stem/progenitor cells either recruited or delivered to an injured site, can eventually regenerate lost or damaged tissue. Most of the researchers working in tissue engineering and regenerative technology attempt to create tissue replacements by culturing cells onto synthetic porous three-dimensional polymeric scaffolds, which is currently regarded as an ideal approach to enhance functional tissue regeneration by creating and maintaining channels that facilitate progenitor cell migration, proliferation and differentiation. The requirements that must be satisfied by such scaffolds include providing a space with the proper size, shape and porosity for tissue development and permitting cells from the surrounding tissue to migrate into the matrix. Recently, chitin scaffolds have been widely used in tissue engineering due to their non-toxic, biodegradable and biocompatible nature. The advantage of chitin as a tissue engineering biomaterial lies in that it can be easily processed into gel and scaffold forms for a variety of biomedical applications. Moreover, chitin has been shown to enhance some biological activities such as immunological, antibacterial, drug delivery and have been shown to promote better healing at a faster rate and exhibit greater compatibility with humans. This review provides an overview of the current status of tissue engineering/regenerative medicine research using chitin scaffolds for bone, cartilage and wound healing applications. We also outline the key challenges in this field and the most likely directions for future development and we hope that this review will be helpful to the researchers working in the field of tissue engineering and regenerative medicine.

Novel chitin/nanosilica composite scaffolds for bone tissue engineering applications

Biopolymers like chitin are widely investigated as scaffolds in bone tissue engineering. Its properties like biocompatibility, biodegradability, non-toxicity, wound healing ability, antibacterial activity, hemostatic property, etc., are widely known. However, these materials are not much bioactive. Addition of material like silica can improve the bioactivity and biocompatibility of chitin. In this work, chitin composite scaffolds containing nanosilica were prepared using chitin hydrogel and their bioactivity, swelling ability and cytotoxicity was analyzed in vitro. These scaffolds were found to be bioactive in simulated body fluid (SBF) and biocompatible when tested with MG 63 cell line. These results suggest that chitin/nanosilica composite scaffolds can be useful for bone tissue engineering applications.

Synthesis of phosphorylated chitosan by novel method and its characterization

Chitosan a natural based polymer is non-toxic, biocompatible and biodegradable. Chemical modification of chitosan to generate new bifunctional materials and finally would bring new properties depending on the nature of the group introduced. In our present study, we prepared phosphorylated chitosan (P-chitosan) by using H(3)PO(4)/P(2)O(5)/Et(3)PO(4)/hexanol method. From our present method, we got high yield and high degree of substitution (DS). The prepared P-chitosan (DS-1.18) was characterized by FT IR, (13)C NMR, (31)P NMR, elemental, XRD, TGA, DTA and SEM studies. After the phosphorylation, the solubility of the polymer was improved. The P-chitosan showed less thermal stability and crystallinity than the chitosan. It was due to the phosphorylation.

A Highly Practical Synthesis of Cyclodextrin-Based Glycoclusters Having Enhanced Affinity with Lectins

Abstract A simple and highly practical method for the synthesis of cyclodextrin-scaffolded glycoclusters recognized specifically by lectins is described. Nucleophilic displacement of iodide from heptakis 6-deoxy-6-iodo-β-cyclodextrin by unprotected sodium thiolates derived from 3-(3-thioacetyl propionamido)propyl glycosides proceeded smoothly in mild condition and gave novel per-glycosylated cyclodextrins (glycocyclodextrins, glycoCDs) having d -galactose, N -acetyl- d -glucosamine, lactose or N -acetyllactosamine residues in high yields (78–88%). It was demonstrated that all these per-glycosylated β-cyclodextrins showed amplified inhibitory effects on the erythrocytes agglutination induced by wheat germ ( Triticum vulgaris ) agglutinin (WGA) or Erythrina corallodendron lectin (ECorL).

Bioactive and osteoblast cell attachment studies of novel α- and β-chitin membranes for tissue-engineering applications

Chitin is a novel biopolymer and has excellent biological properties such as biodegradation in the human body and biocompatible, bioabsorable, antibacterial and wound healing activities. In this work, alpha- and beta-chitin membranes were prepared using alpha- and beta-chitin hydrogel. The bioactivity studies were carried out using these chitin membranes with the simulated body fluid solution (SBF) for 7, 14 and 21 days. After 7, 14 and 21 days the membranes were characterized using SEM, EDS and FT-IR. The SEM, EDS and FT-IR studies confirmed the formation of calcium phosphate layer on the surface of the both chitin membranes. These results indicate that the prepared chitin membranes were bioactive. Cell adhesion studies were also carried out using MG-63 osteoblast-like cells. The cells were adhered and spread over the membrane after 24h of incubation. These results indicated that the chitin membranes could be used for tissue-engineering applications.

Novel Chitosan-Spotted Alginate Fibers from Wet-Spinning of Alginate Solutions Containing Emulsified Chitosan−Citrate Complex and their Characterization

The major problem associated with the production of alginate/chitosan hybridized fibers by wet spinning is the formation of gels due to ionic interactions of the oppositely charged molecules of alginate and chitosan when these two polymers are directly mixed. Here, we proposed a novel method of using chitosan in the form of an emulsion. The emulsion was prepared by adding a primary emulsion of olive oil in a sodium dodecyl sulfate (SDS) aqueous solution into a chitosan-citrate complex. The complexation of chitosan with citric acid is the key of this method. The citrate ions neutralize the positive charges of chitosan, rendering the chitosan-citrate complex to readily penetrate into the core of the SDS/olive oil micelles. The obtained emulsified chitosan-citrate complex (hereafter, the chitosan-citrate emulsion) of varying amount was then added into an alginate aqueous solution to prepare the alginate/chitosan spinning dope suspensions. The alginate/chitosan hybridized fibers showed spotty features of the emulsified chitosan-citrate complex particles locating close to the surface and the inside of the hybridized fibers. At the lowest content of incorporated chitosan (i.e., 0.5% w/w chitosan), both the tenacity and the elongation at break of the obtained chitosan-spotted alginate fibers were the greatest. Further increase in the chitosan content resulted in a monotonous decrease in the property values. Lastly, preliminary studies demonstrated that the obtained chitosan-spotted alginate fibers showed great promises as carriers for drug delivery.

In vitro evaluation of paclitaxel loaded amorphous chitin nanoparticles for colon cancer drug delivery.

Chitin and its derivatives have been widely used in drug delivery applications due to its biocompatible, biodegradable and non-toxic nature. In this study, we have developed amorphous chitin nanoparticles (150±50 nm) and evaluated its potential as a drug delivery system. Paclitaxel (PTX), a major chemotherapeutic agent was loaded into amorphous chitin nanoparticles (AC NPs) through ionic cross-linking reaction using TPP. The prepared PTX loaded AC NPs had an average diameter of 200±50 nm. Physico-chemical characterization of the prepared nanoparticles was carried out. These nanoparticles were proven to be hemocompatible and in vitro drug release studies showed a sustained release of PTX. Cellular internalization of the NPs was confirmed by fluorescent microscopy as well as by flow cytometry. Anticancer activity studies proved the toxicity of PTX-AC NPs toward colon cancer cells. These preliminary results indicate the potential of PTX-AC NPs in colon cancer drug delivery.

Bioactive and metal uptake studies of carboxymethyl chitosan-graft-d-glucuronic acid membranes for tissue engineering and environmental applications

Carboxymethyl chitosan-graft-D-glucuronic acid (CMCS-g-D-GA) was prepared by grafting D-GA onto CMCS in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and then the membranes were made from it. In this work, the bioactivity studies of CMCS-g-D-GA membranes were carried out and then characterized by SEM, CLSM, XRD and FT-IR. The CMCS-g-D-GA membranes were found to be bioactive. The adsorption of Ni2+, Zn2+ and Cu2+ ions onto CMCS-g-D-GA membranes has also been investigated. The maximum adsorption capacity of CMCS-g-D-GA for Ni2+, Zn2+ and Cu2+ was found to be 57, 56.4 and 70.2 mg/g, respectively. Hence, these membranes were useful for tissue engineering, environmental and water purification applications.