In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Silver nanoparticle-loaded chitosan–starch based films: Fabrication and evaluation of tensile,barrier and antimicrobial properties

The fabrication of silver nanoparticles was accomplished by γ-ray irradiation reduction of silver nitrate in a chitosan solution. The obtained nanoparticles were stable in the solution for more than six months, and showed the characteristic surface plasmon band at 411 nm as well as a positively charged surface with 40.4 ± 2.0 mV. The silver nanoparticles presented a spherical shape with an average size of 20–25 nm, as observed by TEM. Minimum inhibitory concentration (MIC) against E. coli, S. aureus and B. cereus of the silver nanoparticles dispersed in the γ-ray irradiated chitosan solution was 5.64 µg/mL. The silver nanoparticle-loaded chitosan–starch based films were prepared by a solution casting method. The incorporation of silver nanoparticles led to a slight improvement of the tensile and oxygen gas barrier properties of the polysaccharide-based films, with diminished water vapor/moisture barrier properties. In addition, silver nanoparticle-loaded films exhibited enhanced antimicrobial activity against E. coli, S. aureus and B. cereus. The results suggest that silver nanoparticle-loaded chitosan–starch based films can be feasibly used as antimicrobial materials for food packaging and/or biomedical applications.     

Preparation and evaluation of chitosan–itraconazole co-precipitated nanosuspension for ocular delivery

The purpose of the present study was to prepare itraconazole-loaded chitosan nanosuspension and evaluate it for ocular delivery. Itraconazole-loaded chitosan nanosuspension was prepared by controlled co-precipitation of chitosan and itraconazole from aqueous acetate solution using a combination of pH change and non-solvent addition. The co-precipitated suspension was evaluated for particle size, zeta-potential, entrapment efficiency and solubility study. It was observed that co-precipitation of itraconazole and chitosan from chitosan–lysine system in the presence of Poloxamer-188 as stabiliser provided nanosuspension of the smallest particle size with a 12-fold increase in aqueous saturation solubility of itraconazole and the fastest in vitro release. Transmission electron micrographs of the nanosuspension showed ovoid-shaped particles. A comparative evaluation of the itraconazole (1%, w/v) nanosuspension with commercial itraconazole suspension (1%, w/v) revealed a significantly higher percentage cumulative permeation of itraconazole across the isolated goat cornea from the nanosuspension dosage form as compared to the commercial suspension (P < 0.01).     

Preparation and comparative characterization of keratin–chitosan and keratin–gelatin composite scaffolds for tissue engineering applications

We report fabrication of three dimensional scaffolds with well interconnected matrix of high porosity using keratin, chitosan and gelatin for tissue engineering and other biomedical applications. Scaffolds were fabricated using porous Keratin–Gelatin (KG), Keratin–Chitosan (KC) composites. The morphology of both KG and KC was investigated using SEM. The scaffolds showed high porosity with interconnected pores in the range of 20–100 μm. They were further tested by FTIR, DSC, CD, tensile strength measurement, water uptake and swelling behavior. In vitro cell adhesion and cell proliferation tests were carried out to study the biocompatibility behavior and their application as an artificial skin substitute. Both KG and KC composite scaffolds showed similar properties and patterns for cell proliferation. Due to rapid degradation of gelatin in KG, we found that it has limited application as compared to KC scaffold. We conclude that KC scaffold owing to its slow degradation and antibacterial properties would be a better substrate for tissue engineering and other biomedical application.     

Experimental evaluation of new chitin–chitosan graft for duraplasty

Natural materials such as collagen and alginate have promising applications as dural graft substitutes. These materials are able to restore the dural defect and create optimal conditions for the development of connective tissue at the site of injury. A promising material for biomedical applications is chitosan—a linear polysaccharide obtained by the deacetylation of chitin. It has been found to be nontoxic, biodegradable, biofunctional and biocompatible in addition to having antimicrobial characteristics. In this study we designed new chitin–chitosan substitutes for dura mater closure and evaluated their effectiveness and safety. Chitosan films were produced from 3 % of chitosan (molar mass—200, 500 or 700?kDa, deacetylation rate 80–90%) with addition of 20% of chitin. Antimicrobial effictively and cell viability were analysed for the different molar masses of chitosan. The film containing chitosan of molar mass 200?kDa, had the best antimicrobial and biological activity and was successfully used for experimental duraplasty in an in vivo model. In conclusion the chitin–chitosan membrane designed here met the requirements for a dura matter graft exhibiting the ability to support cell growth, inhibit microbial growth and biodegradade at an appropriate rate. Therefore this is a promising material for clinical duroplasty.     

Biomedical evaluation of polyvinyl alcohol–gelatin esterified hydrogel for wound dressing

The wound is a biosynthetic environment in which numerous cellular processes are interlinked in the process of repair. Modern dressings are designed to facilitate wound healing rather than just to cover it. Hydrogel dressing can protect injured skin and keep it appropriately moist to speed the healing process by absorbing exudates while maintaining the products of tissue repair, including growth factor and lysosomes, in contact with the wound. The design and development of novel membrane of hydrogels prepared by esterification of polyvinyl alcohol with gelatin were attempted. Contact angle of goat blood was determined. The hydrogel was characterized by hemolysis test and water vapor transmission rate. Diffusion coefficient of salicylic acid (SA) and gatifloxacin, a fourth generation fluoroquinolone, through the membrane was determined. Both the drugs were used as model drug. Methyl tetrazolium dye assay of the membrane was done using L929 fibroblast cell line and mice splenocytes to establish the biocompatibility of the membrane. The equilibrium goat blood-in-air contact angles of measured ester films ranged from 56 to 60°. The hydrogel was found to be hemocompatible and moisture retentive indicating its possible use in moist wound care. The diffusion coefficient of SA and gatifloxacin through the membrane was found to be 1.49 × 10⁻⁵ and 3.97 × 10⁻⁶ cm²/s respectively. The membrane was found to be compatible with the L929 fibroblast cell line and mice splenocytes.     

Nanohydroxyapatite–chitosan–gelatin polyelectrolyte complex with enhanced mechanical and bioactivity

A wet chemical approach was used to synthesis HAp–chitosan–gelatin nanocomposites at different wt.% of chitosan–gelatin (CG) ratios such as CG-00, CG-40, CG-31, CG-22, CG-13 and CG-04. XRD analysis confirms the formation of HAp on nanocomposite matrix and the decreased crystallite size was found with the decrease in chitosan and increase in gelatin compositions. FTIR study reveals that the presence of characteristic bands of HAp and CG. The decrease in chitosan in CG leads to band shift of organic phase towards a higher wave number side due to the intimate bonding with an inorganic phase. FE-SEM images show that the particles had a short nanorod-like morphology. The bioresorbability and microhardness test indicate that the composites have better resorbability and hardness than pure HAp. It was inferred that particle size, morphology, resorbability and hardness of the composite can be altered by tuning the composition.     

Fabrication of gelatin–siloxane fibrous mats via sol–gel and electrospinning procedure and its application for bone tissue engineering

Our strategy is to design and fabricate biomimetic and bioactive scaffolds that resemble the native extracellular matrix as closely as possible so as to create conducive living milieu that will induce cell to function naturally. In the present study, gelatin/siloxane (GS) hybrids were prepared by a sol–gel processing, and electrospinning technique was used to fabricate GS fibrous mats to support the growth of bone marrow-derived mesenchymal stem cells (BMSCs) for tissue engineering of bone. The results indicate that the porous structure and fiber size of the GS fibrous mats can be fine tuned by varying the viscosity of GS precursor solution. Additionally, the Ca²⁺-containing GS fibrous mats biomimetically deposited apatite in a simulated body fluid (SBF), as well as stimulating its BMSCs proliferation and differentiation in vitro, thereby dignifying its in vitro bioactivity.     

Fabrication and mechanical evaluation of SiC–TiC nanocomposites by SPS

In the present study, the composites of SiC–TiC are prepared by spark plasma sintering (SPS) in vacuum without additive. The relationship of density and temperature of SiC–TiC composites with different content of TiC is studied. The maximum relative density reached was 98%. The mechanical properties of SiC–TiC composites with different content of TiC, which were sintered at 1800 °C, have been evaluated. From the fracture surface observation, two models of fracture mechanisms of the composites existed: transgranular and intergranular.     

In vitro evaluation of electrospun gelatin–glutaraldehyde nanofibers

The gelatin–glutaraldehyde (gelatin–GA) nanofibers were electrospun in order to overcome the defects of ex-situ crosslinking process such as complex process, destruction of fiber morphology and decrease of porosity. The morphological structure, porosity, thermal property, moisture absorption and moisture retention performance, hydrolytic resistance, mechanical property and biocompatibility of nanofiber scaffolds were tested and characterized. The gelatin–GA nanofiber has nice uniform diameter and more than 80% porosity. The hydrolytic resistance and mechanical property of the gelatin–GA nanofiber scaffolds are greatly improved compared with that of gelatin nanofibers. The contact angle, moisture absorption, hydrolysis resistance, thermal resistance and mechanical property of gelatin–GA nanofiber scaffolds could be adjustable by varying the gelatin solution concentration and GA content. The gelatin–GA nanofibers had excellent properties, which are expected to be an ideal scaffold for biomedical and tissue engineering applications.     

Biomimetic design and fabrication of porous chitosan–gelatin liver scaffolds with hierarchical channel network

The presence of a hierarchical channel network in tissue engineering scaffold is essential to construct metabolically demanding liver tissue with thick and complex structures. In this research, chitosan–gelatin (C/G) scaffolds with fine three-dimensional channels were fabricated using indirect solid freeform fabrication and freeze-drying techniques. Fabrication processes were studied to create predesigned hierarchical channel network inside C/G scaffolds and achieve desired porous structure. Static in-vitro cell culture test showed that HepG2 cells attached on both micro-pores and micro-channels in C/G scaffolds successfully. HepG2 proliferated at much higher rates on C/G scaffolds with channel network, compared with those without channels. This approach demonstrated a promising way to engineer liver scaffolds with hierarchical channel network, and may lead to the development of thick and complex liver tissue equivalent in the future.     

The effect of cross-linking on the microstructure,mechanical properties and biocompatibility of electrospun polycaprolactone–gelatin/PLGA–gelatin/PLGA–chitosan hybrid composite

Abstract

In this study, multilayered scaffolds composed of polycaprolactone (PCL)–gelatin/poly(lactic-co-glycolic acid) (PLGA)–gelatin/PLGA–chitosan artificial blood vessels were fabricated using a double-ejection electrospinning system. The mixed fibers from individual materials were observed by scanning electron microscopy. The effects of the cross-linking process on the microstructure, mechanical properties and biocompatibility of the fibers were examined. The tensile stress and liquid strength of the cross-linked artificial blood vessels were 2.3 MPa and 340 mmHg, respectively, and were significantly higher than for the non-cross-linked vessel (2.0 MPa and 120 mmHg). The biocompatibility of the cross-linked artificial blood vessel scaffold was examined using the MTT assay and by evaluating cell attachment and cell proliferation. The cross-linked PCL–gelatin/PLGA–gelatin/PLGA–chitosan artificial blood vessel scaffold displayed excellent flexibility, was able to withstand high pressures and promoted cell growth; thus, this novel material holds great promise for eventual use in artificial blood vessels.     

The fabrication of nanocomposites via calcium phosphate formation on gelatin–chitosan network and the gelatin influence on the properties of biphasic composites

A novel biodegradable polymer–ceramic nanocomposite which consisted of gelatin (Gel), chitosan (CS), and calcium phosphate (CaP) nanoparticles was prepared based on in situ preparation method. The fabricated biocomposites were characterized by FTIR, X-ray diffraction (XRD), transmission electron microscopy (TEM) as well as scanning electron microscope with X-ray elemental analysis (SEM-EDX). The characterization results confirmed that the crystalline calcium phosphate nanoparticles were mineralized in polymeric matrix and the interaction between Ca2+ in calcium phosphate and functional groups in polymers molecular chains was formed. XRD result showed that in addition to hydroxyapatite (HA), Brushite (BR) and tricalcium phosphate (β-TCP) particles also were formed due to lack of complete penetration of the basic solution into the polymeric matrix. However, SEM image indicated that the polymeric matrix has the controlling role in the particle size of calcium phosphate. The size of particles in three component composites was about 100 nm while in two component composites proved to be more in μm size. TEM observation supported SEM results and showed that the three component composites have calcium phosphate nanoparticles. The elastic modulus and compressive strength of the composites were also improved by the employment of gelatin and chitosan together, which can make them more beneficial for surgical applications.     

Supermacroprous chitosan–agarose–gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering

The study focuses on the synthesis of a novel polymeric scaffold having good porosity and mechanical characteristics synthesized by using natural polymers and their optimization for application in cartilage tissue engineering. The scaffolds were synthesized via cryogelation technology using an optimized ratio of the polymer solutions (chitosan, agarose and gelatin) and cross-linker followed by the incubation at sub-zero temperature (−12°C). Microstructure examination of the chitosan–agarose–gelatine (CAG) cryogels was done using scanning electron microscopy (SEM) and fluorescent microscopy. Mechanical analysis, such as the unconfined compression test, demonstrated that cryogels with varying chitosan concentrations, i.e. 0.5–1% have a high compression modulus. In addition, fatigue tests revealed that scaffolds are suitable for bioreactor studies where gels are subjected to continuous cyclic strain. In order to confirm the stability, cryogels were subjected to high frequency (5 Hz) with 30 per cent compression of their original length up to 1 × 10⁵ cycles, gels did not show any significant changes in their mass and dimensions during the experiment. These cryogels have exhibited degradation capacity under aseptic conditions. CAG cryogels showed good cell adhesion of primary goat chondrocytes examined by SEM. Cytotoxicity of the material was checked by MTT assay and results confirmed the biocompatibility of the material. In vivo biocompatibility of the scaffolds was checked by the implantation of the scaffolds in laboratory animals. These results suggest the potential of CAG cryogels as a good three-dimensional scaffold for cartilage tissue engineering.     

Fabrication of an rhBMP-2 loaded porous β-TCP microsphere-hyaluronic acid-based powder gel composite and evaluation of implant osseointegration

Methods to improve osseointegration that include implantation of rhBMP-2 with various kinds of carriers are currently of considerable interest. The present study was conducted to evaluate if the rhBMP-2 loaded β-TCP microsphere-hyaluronic acid-based powder-like hydrogel composite (powder gel) can act as an effective rhBMP-2 carrier for implantation in host bone with a bone defect or poor bone quality. The release pattern for rhBMP-2 was then evaluated against an rhBMP-2-loaded collagen sponge as a control group. Dental implants were also inserted into the tibias of three groups of rabbits: an rhBMP-2 (200 µg) loaded powder gel composite implanted group, an implant only group, and a powder gel implanted group. Micro-CT and histology of the implanted areas were carried out four weeks later. The rhBMP-2 powder gel released less rhBMP-2 than the collagen sponge, but it continued a slow release for more than 7 days. The rhBMP-2 powder gel composite improved osseointegration of the dental implant by increasing the amount of new bone formation in the implant pitch and it improved the bone quality and bone quantity of new bone. The histology results indicated that the rhBMP-2 powder gel composite improved the osseointegration in the cortical bone as well as the marrow space along the fixture. The bone-to-implant contact ratio of the rhBMP-2 (200 µg) loaded powder gel composite implanted group was significantly higher than those of the implant only group and the powder gel implanted group. The powder gel appeared to be a good carrier and could release rhBMP-2 slowly to promote the formation of new bone following implantation in a bone defect, thereby improving implant osseointegration.     

Fabrication and evaluation of biomimetic scaffolds by using collagen–alginate fibrillar gels for potential tissue engineering applications

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.     

Fabrication of Pd–DNA and Pd–CNT hybrid nanostructures for hydrogen sensors

This review reports fabrication methods for ordered metallic nanostructures such as nanowires and nanoparticles based on deoxyribonucleic acid (DNA) templates. The phosphate groups in DNA are negatively charged; consequently, the DNA conformation may mineralize metals, e.g., palladium (Pd) at a relatively high metal concentration. We successfully form unique spherically shaped moss-like hybrid Pd nanoparticles using the small compacted globular state of DNA by controlling the reductive reaction. Pd can absorb hydrogen to become PdH_x, and hydrogen storage increases the electrical resistance and volume of Pd materials. Hence, the use of this material is attracting growing interest as a reliable, cheap, ultracompact, and safe hydrogen sensor. Pd–DNA hybrid nanoparticles can be used as highly sensitive hydrogen sensors, which exhibit a switch response that depends on the volume expansion in a cyclic atmosphere exchange. This paper also shows the fabrications of Pd–carbon nanotube (CNT) hybrid nanostructures.     

Fabrication of hollow core–shell conductive nanoparticles based on nanocrystalline cellulose for conductive adhesive

A novel environmentally friendly method was developed to fabricate hollow core–shell conductive nanoparticles using a natural and nontoxic material, nanocrystalline cellulose (NCC), as the template. The NCC used in this study has nano-scale rod-like structure. After the oxidization to dialdehyde cellulose, the insulated NCC was functionalized by poly(dopamine) (PDA) in weakly alkaline conditions through Schiff base reaction and self-polymerization. The Schiff base can be hydrolyzed in an aqueous acetone solution via ultraviolet radiation so that the hollow structure constructed. This structure not only strengthened the mechanical properties but also provided more active sites for silver deposition. Utilizing the chelating ability of the catechol groups in PDA, electroless plating method was used to form the silver coating layer. Scanning electron microscope and Dynamic Light Scattering measurements indicated that these nanoparticles (NPs) had well-defined morphology and a mean diameter of 100–120 nm. Moreover, these prepared Ag–DA–NCC₀ NPs exhibited excellent conductivity. Their electrical resistivity reached 0.2 mΩ·cm, which is much higher than that of many other conductive particles used in conductive adhesive.     

Novel and simple route for the synthesis of core–shell chitosan–gold nanocomposites

This work presents a novel and simple route for the synthesis of water-soluble core–shell chitosan–gold nanocomposites. The experimental procedure can be summarized by the following steps: (i) chitosan deacetylation, (ii) chitosan depolymerization, (iii) chitosan nanoparticles’ formation and (iv) chitosan–gold nanocomposite formation. FT-IR spectroscopic results indicate that the formation of chitosan nanoparticles (ChtNPs) occurs via NH₃⁺ and PO⁻ groups electrostatic interactions, while UV–vis spectra points to a possible embedding of gold nanoparticles into the ChtNPs. This feature was confirmed by electronic transmission microscopy measurements. Chitosan and gold are biocompatible materials. Added to this, the obtained chitosan–gold nanocomposites presented thermal and absorbance properties which strongly point to their potential use in phototherapeutic processes.     

Formulation and performance evaluation of a novel coconut oil–based metalworking fluid

Metalworking fluid (MWF) supplies a film of lubricant to abate friction, acts as a cooling media to rebate induced heat, and prevents metal pick-ups by flushing away the chips. Hence a liquid used as a cutting fluid reduces wear on the tool, reduces the energy consumption, and produces a better surface quality on the work piece. This paper describes the formulation of a novel water-soluble MWF and its performance evaluation during straight turning and end milling experiments carried out with AISI 304 stainless steel, mild steel, and cast iron as work piece materials. The MWF was prepared by mixing water with white coconut oil as the base oil and food-grade additives as surfactants. Viscosity, pH value, and biodegradability were measured and compared with a commercially available non-vegetable oil–based MWF. The surface roughness and tool surface temperature were measured throughout the machining experiments, and better performances were observed with the coconut oil–based MWF. Tool tip geometry and flank wear for straight turning machining operation were identified by observing scanning electron microscope (SEM) images.