A nanofibrous composite membrane of PLGA-chitosan/PVA prepared by electrospinning

Tissue engineering scaffolds produced by electrospinning feature a structural similarity to the natural extracellular matrix. In this study, poly(lactide-co-glycolide) (PLGA) and chitosan/poly(vinyl alcohol) (PVA) were simultaneously electrospun from two different syringes and mixed on the rotating drum to prepare the nanofibrous composite membrane. The composite membrane was crosslinked by glutaraldehyde vapor to maintain its mechanical properties and fiber morphology in wet stage. Morphology, shrinkage, absorption in phosphate buffered solution (PBS) and mechanical properties of the electrospun membranes were characterized. Fibroblast viability on electrospun membranes was discussed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and cell morphology after 7 days of culture. Results indicated that the PBS absorption of the composite membranes, no matter crosslinked or not, was higher than the electrospun PLGA membrane due to the introduction of hydrophilic components, chitosan and PVA. After crosslinking, the composite membrane had a little shrinkage after incubating in PBS. The crosslinked composite membrane also showed moderate tensile properties. Cell culture suggested that electrospun PLGA-chitosan/PVA membrane tended to promote fibroblast attachment and proliferation. It was assumed that the nanofibrous composite membrane of electrospun PLGA-chitosan/PVA could be potentially used for skin reconstruction.     

Fabrication and properties of mineralized collagen‐chitosan/hydroxyapatite scaffolds

A hydroxyapatite (HAp)/biopolymer composite scaffold was fabricated by mineralizing a crosslinked collagen/chitosan, which was pre‐mineralized with Ca²⁺ and phosphate salts, in simulated body fluid (SBF) for only 24 hr. A self‐organized structure similar to bone is expected. Microstructures of the crosslinked collagen/chitosan scaffold, the pre‐mineralized collagen–chitosan scaffold (CCS), and the mineralized collagen‐chitosan/HAp scaffolds (MCCHS) were characterized by scanning electron microscopy (SEM), revealing non‐alteration of the porous structure and formation of the HAp particles. X‐ray diffractometer (XRD) confirmed the crystalline structure of the HAp. Thermal gravimetric analysis found that more HAp particles were formed when the CCSs were pre‐mineralized in a higher concentration of Ca²⁺. Water‐uptake ratio of the crosslinked CCS was ～160, decreased to ～120 after incubating in Ca²⁺ solution, and further decreased to ～20 after mineralization. Mechanical strength of the CCS was improved significantly after the in situ mineralization too. The method introduced here may be potentially applied to obtain other biopolymer/HAp composite in a short period. Copyright © 2008 John Wiley & Sons, Ltd.     

Photocrosslinked methacrylated chitosan-based nanofibrous scaffolds as potential skin substitute

Nanofibers based on natural polymers have recently been attracting research interest as promising materials for use as skin substitutes. Here, we prepared photocrosslinked nanofibrous scaffolds based on methacrylated chitosan (MACS) by photocrosslinking electrospun methacrylated chitosan/poly (vinyl alcohol) (PVA) mats and subsequently removing PVA from the nanofibers. We comprehensively investigated the solution properties of MACS/PVA precursors, the intermolecular action between MACS and PVA components, and the morphology of MACS/PVA nanofibers. Results indicated that the fiber diameter and morphology of the photocrosslinked methacrylated chitosan-based nanofibrous scaffolds were controlled by the MACS/PVA mass ratio and showed highly micro-porous structures with many fibrils. In vitro cytotoxicity evaluation and cell culture experiments confirmed that MACS-based mats with micro-pore structure were biocompatible with L929 cells and facilitated cellular migration into the 3D matrix, demonstrating their potential application as skin replacements for wound repair.     

Improved cellular response on multiwalled carbon nanotube-incorporated electrospun polyvinyl alcohol/chitosan nanofibrous scaffolds

We report the fabrication of multiwalled carbon nanotube (MWCNT)-incorporated electrospun polyvinyl alcohol (PVA)/chitosan (CS) nanofibers with improved cellular response for potential tissue engineering applications. In this study, smooth and uniform PVA/CS and PVA/CS/MWCNTs nanofibers with water stability were formed by electrospinning, followed by crosslinking with glutaraldehyde vapor. The morphology, structure, and mechanical properties of the formed electrospun fibrous mats were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and mechanical testing, respectively. We showed that the incorporation of MWCNTs did not appreciably affect the morphology of the PVA/CS nanofibers; importantly the protein adsorption ability of the nanofibers was significantly improved. In vitro cell culture of mouse fibroblasts (L929) seeded onto the electrospun scaffolds showed that the incorporation of MWCNTs into the PVA/CS nanofibers significantly promoted cell proliferation. Results from this study hence suggest that MWCNT-incorporated PVA/CS nanofibrous scaffolds with small diameters (around 160 nm) and high porosity can mimic the natural extracellular matrix well, and potentially provide many possibilities for applications in the fields of tissue engineering and regenerative medicine.     

Dual-syringe reactive electrospinning of cross-linked hyaluronic acid hydrogel nanofibers for tissue engineering applications

A facile fabrication of a cross-linked hyaluronic acid (HA) hydrogel nanofibers by a reactive electrospinning method is described. A thiolated HA derivative, 3,3'-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), and poly(ethylene glycol) diacrylate (PEGDA) are selected as the cross-linking system. The cross-linking reaction occurs simultaneously during the electrospinning process using a dual-syringe mixing technique. Poly(ethylene oxide) (PEO) is added into the spinning solution as a viscosity modifier to facilitate the fiber formation and is selectively removed with water after the electrospinning process. The nanofibrous structure of the electrospun HA scaffold is well preserved after hydration with an average fiber diameter of 110 nm. A cell morphology study on fibronectin (FN)-adsorbed HA nanofibrous scaffolds shows that the NIH 3T3 fibroblasts migrate into the scaffold through the nanofibrous network, and demonstrate an elaborate three-dimensional dendritic morphology within the scaffold, which reflects the dimensions of the electrospun HA nanofibers. These results suggest the application of electrospun HA nanofibrous scaffolds as a potential material for wound healing and tissue regeneration. [image: see text] Laser scanning confocal microscopy demonstrates that the NIH3T3 fibroblast develops an extended 3D dendritic morphology within the fibronectin-adsorbed electrospun HA nanofibrous scaffold.     

Enhancing the stiffness of electrospun nanofiber scaffolds with a controlled surface coating and mineralization

A new method was developed to coat hydroxyapatite (HAp) onto electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers for tendon-to-bone insertion site repair applications. Prior to mineralization, chitosan and heparin were covalently immobilized onto the surface of the fibers to accelerate the nucleation of bone-like HAp crystals. Uniform coatings of HAp were obtained by immersing the nanofiber scaffolds into a modified, 10-fold-concentrated simulated body fluid (m10SBF) for different periods of time. The new method resulted in thicker and denser coatings of mineral on the fibers compared to those produced by previously reported methods. Scanning electron microscopy measurements confirmed the formation of nanoscale HAp particles on the fibers. A mechanical property assessment demonstrated a higher stiffness with respect to previous coating methods. A combination of the nanoscale fibrous structure and bonelike mineral coating could mimic the structure, composition, and function of mineralized tissues.     

Biocomposites of nanohydroxyapatite with collagen and poly(vinyl alcohol)

Biocomposites of hydroxyapatite, HAp, in conjunction with various binders including poly(vinyl alcohol), PVA, and collagen have the potential of serving in various tissue engineering applications, such as in bone repair and reconstruction tasks, especially if the nanoparticles of hydroxyapatite are used. Here, hydroxyapatite nanoparticles (n-HAp) were synthesized at the ultimate size range of 10-50 nm and then incorporated into PVA or in situ synthesized in collagen/PVA. The biocomposites of HAp with PVA exhibited relatively high elasticity (as revealed by the linear viscoelastic material functions, characterized upon small-amplitude oscillatory shear) especially upon cryogenic treatment. The incorporation of the collagen into the PVA/HAp biocomposite provided internal porosity to the biocomposite with the pores in the 50-100 nm range for collagen/HAp and 50-500 nm for the collagen/HAp/PVA.     

Cell adhesive and growth behavior on electrospun nanofibrous scaffolds by designed multifunctional composites

Nanostructured biocomposite scaffolds of poly(l-lactide) (PLLA) blended with collagen (coll) or hydroxyapatite (HA), or both for tissue engineering application, were fabricated by electrospinning. The electrospun scaffolds were characterized for the morphology, chemical and tensile properties by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), Fourier transform infrared (FTIR) measurement, and tensile testing. Electrospun biocomposite scaffolds of PLLA and collagen or (and) HA in the diameter range of 200-700 nm mimic the nanoscale structure of the extracellular matrix (ECM) with a well-interconnection pore network structure. The presence of collagen in the scaffolds increased their hydrophility, and enhanced cell attachment and proliferation, while HA improved the tensile properties of the scaffolds. The biocompatibility of the electrospun scaffolds and the viability of contacting cells were evaluated by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and by fluorescein diacetate (FDA) and propidium iodide (PI) double staining methods. The results support the conclusion that 293T cells grew well on composite scaffolds. Compared with pure PLLA scaffolds a greater density of viable cells was seen on the composites, especially the PLLA/HA/collagen scaffolds.     

Microstructural and mechanical properties of porous biocomposite scaffolds based on polyvinyl alcohol,nano-hydroxyapatite and cellulose nanocrystals

In this study, in situ synthesis of polyvinyl alcohol (PVA)/nano-hydroxyapatite (n-HA)/cellulose nanocrystals (CNC) organic–inorganic biocomposite porous scaffolds is reported. The effect of the CNC content on the properties of the biocomposite scaffold was investigated and characterized using field-emission scanning electron microscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis, porosity and compressive strength measurements, thermal studies, and in vitro biomineralization and degradation studies. The morphological study showed highly porous structures with good pore interconnectivity in which n-HA was homogeneously dispersed. XRD analysis showed a decrease in the crystalline fraction and crystallite size of nano-hydroxyapatite with introduction of PVA and with increasing content of CNC. It was observed that the porosity decreased to some extent with increasing CNC content, while increases in the compressive strength (from 0.85 to 2.09 MPa) and elastic modulus (from 4.68 to 16.01 MPa) were found as the CNC content was increased. In vitro biomineralization study revealed the formation of apatite on PVA/n-HA/CNC biocomposite scaffolds when soaked for 7 and 14 days in simulated body fluid (SBF) solution. The obtained porous scaffolds offering good mechanical performance may provide a promising alternative scaffolding matrix for use in the field of bone tissue engineering.     

Development of nano-tricalcium phosphate/polycaprolactone/platelet-rich plasma biocomposite for bone defect regeneration

Nano-tricalcium phosphate (n-TCP) is an osteoconductive substance which, like polycaprolactone (PCL), has been used for clinical purposes for many years; It has now been licensed for a range of products for clinical and medication distribution. This research aimed to examine the effects of platelet-rich plasma on mesenchymal stem cell proliferation and osteogenic differentiation. Thus, we decided to examine the in vitro and in vivo actions of PRP-treated porous biocomposite scaffolds based on nano-tricalcium phosphate- polycaprolactone (n-TCP-PCL/PRP). The prepared samples were described utilizing FTIR, XRD, and SEM. MTT has measured the cytotoxicity of the biocomposite scaffolds. After two weeks of cell seeding, Alizarin red staining confirmed bone mineral formation by MSCs cells. Moreover, from day 4 to day 7, n-TCP-PCL/PRP biocomposite scaffold improved the expresses of bone marker genes. Platelet-rich plasma (PRP) in conjunction with nano-tricalcium phosphate- polycaprolactone (n-TCP-PCL) biocomposite scaffold is beneficial for the regeneration and stability of the freshly developed bone tissue.     

Preparation and modification of collagen-based porous scaffold for tissue engineering

In the effort to generate cartilage tissues using mesenchymal stem cells, porous scaffolds with prescribed biomechanical properties were prepared. Scaffolds with interconnected pores were prepared via lyophilisation of frozen hydrogels made from collagen modified with chitosan nanofibres, hyaluronic acid, copolymers based on poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), and itaconic acid (ITA), and hydroxyapatite nanoparticles. The modified collagen compositions were cross-linked using N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) combined with N-hydroxysuccinimide (NHS) in water solution. Basic physicochemical and mechanical properties were measured and an attempt to relate these properties to the molecular and supermolecular structure of the modified collagen compositions was carried out. Scaffolds containing hydrophilic chitosan nanofibres showed the highest swelling ratio (SR = 20–25) of all the materials investigated, while collagen modified with an amphiphilic PLGA-PEG-PLGA copolymer or functionalised with ITA exhibited the lowest swelling ratio (SR = 5–8). The best resistance to hydrolytic degradation was obtained for hydroxyapatite containing scaffolds. On the other hand, the fastest degradation rate was observed for synthetic copolymer-containing scaffolds. The results showed that the addition of hydroxyapatite or hyaluronic acid to the collagen matrix increases the rigidity in comparison to the collagen-chitosan scaffold. Collagen scaffold modified with hyaluronic acid presented reduced deformation at break while the presence of hydroxypatatite enhanced the scaffold deformation under tensile loading. The tensile elastic modulus of chitosan nanofibre collagen scaffold was the lowest but closest to the articular cartilage; however, the strength and deformation to failure increased up to 200 %. Presented at the 1st Bratislava Young Polymer Scientists Workshop, Bratislava, 20–23 August 2007.     

Synthesis,characterization, and swelling behaviors of chitosan‐g‐poly(acrylic acid)/poly(vinyl alcohol) semi‐IPN superabsorbent hydrogels

A series of granulated semi‐interpenetrating polymer network (semi‐IPN) superabsorbent hydrogels composed of chitosan‐g‐poly(acrylic acid) (CTS‐g‐PAA) and poly(vinyl alcohol) (PVA) were prepared by solution polymerization using ammonium persulfate (APS) as an initiator and N,N′‐methylenebisacrylamide (MBA) as a crosslinker. The effects of reaction conditions such as the concentration of MBA, the weight ratio of AA to CTS, and the content of PVA on water absorbency were investigated. Infrared (IR) spectra and differential scanning calorimetry (DSC) analyses confirmed that AA had been grafted onto CTS backbone, and PVA semi‐interpenetrating into CTS‐g‐PAA networks. SEM analyses indicated that CTS‐g‐PAA/PVA has improved porous surface and PVA was uniformly dispersed in CTS‐g‐PAA network. The semi‐IPN hydrogel containing 10 wt% PVA shows the highest water absorbency of 353 and 53 g g^?1 in distilled water and 0.9 wt% NaCl solution, respectively. Swelling behaviors revealed that the introduction of PVA could improve the swelling rate and enhance the pH stability of the superabsorbent hydrogel. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospinning of Chitosan

Summary: An electrospun nonwoven fabric of a cationic polysaccharide, chitosan, was successfully prepared. The present study focuses on the effect of the electrospinning solvent and the chitosan concentration on the morphology of the resulting nonwoven fabrics. The solvents tested were dilute hydrochloric acid, acetic acid, neat formic acid and trifluoroacetic acid. As the chitosan concentration was increased, the morphology of the deposition on the collector changed from spherical beads to interconnected fibrous networks. The addition of dichloromethane to the chitosan‐TFA solution improved the homogeneity of the electrospun chitosan fiber. Under optimized conditions, homogenous (not interconnected) chitosan fibers with a mean diameter of 330 nm were prepared.

Effects of the coexisting dichloromethane (MC) in the prespun chitosan‐TFA solution on the morphology of the electrospun chitosan fibers. The volume ratio of TFA:MC was 70:30 (×5 000).     

Effect of microcrystal cellulose and cellulose whisker on biocompatibility of cellulose-based electrospun scaffolds

To investigate the potential application of microcrystal cellulose (MCC) and cellulose whisker (CW) in the electrospun vascular tissue scaffolds, cellulose acetate (CA) and cellulose composite scaffolds containing MCC and CW were electrospun from CA solutions and deacetylation. Structure and morphology of MCC, CW and the fibrous composite scaffolds were investigated using FT-IR, SEM, TEM and AFM. The wettability of the scaffolds was evaluated by water contact angle analysis. The effect of MCC and CW on the biocompatibility of the scaffolds for vascular smooth muscle cells (VSMC) was assayed by MTT test, fluorescent imaging and SEM. The biocomposite scaffolds displayed multi-scaled structure and morphology. The scaffolds containing MCC and CW simultaneously exhibited significantly higher cell viability compared to those with only MCC or CW and no filler. Cell viability and morphology within the scaffolds become better with increasing content of MCC and CW. The composite scaffolds with both micro- and nano-scale organization could mimic the native extracellular matrix more closely, and further produce synergistic enhancement on VSMC viability, adhesion and proliferation. This study provides the potential applications of renewable cellulose-based particulates in biomedical field.     

The potential of biomimetic nanofibrous electrospun scaffold comprising dual component for bone tissue engineering

Oils play a putative choice for alleviating various symptoms associated with bone-related disorders. In this present study, polyurethane (PU) scaffold encompassing with Mahua oil (MO) and propolis (PP) were developed using the electrospinning technique. Morphological analysis showed the reduction in the diameter of the electrospun scaffold with blending of MO and MO/PP into the PU matrix. The strong interactions between PU, MO, and PP were evident through the infrared spectrum and thermal analysis. The wettability results showed the hydrophobic nature in electrospun PU/MO scaffold and hydrophilic behavior in electrospun PU/MO/PP scaffold. Mechanical testing indicated the enhancement in the strength of the PU due to the addition of MO and PP. Moreover, the fabricated scaffolds exhibited nontoxicity, low hemoglobin release and improved blood clotting time as evident in the coagulation studies. The cell proliferation studies showed the enhanced fibroblast cell adhesion in the developed nanocomposites than the pristine PU. Hence, the fabricated PU scaffolds blended with MO and PP having desirable properties can serve as a valuable candidate for bone tissue repair.     

壳聚糖不饱和羧酸盐聚集行为的研究

以半干法制备了一系列壳聚糖不饱和羧酸盐--壳聚糖水杨酸盐(a1)、壳聚糖苯甲酸盐(a2)、壳聚糖肉桂酸盐(a3)壳聚糖丙烯酸盐(a4)和壳聚糖衣康酸盐(a5).用红外光谱和紫外光谱表征了该产品的结构,以凯氏定氮法测定了羧酸的结合量.结果表明壳聚糖和不饱和羧酸盐是通过壳聚糖上的氨基和羧酸中的羧基发生了成盐反应,羧酸的结合...     

Evaluation of a novel nanocrystalline hydroxyapatite powder and a solid hydroxyapatite/Chitosan-Gelatin bioceramic for scaffold preparation used as a bone substitute material

Artificially fabricated hydroxyapatite (HAP) shows excellent biocompatibility with various kinds of cells and tissues which makes it an ideal candidate for a bone substitute material. In this study, hydroxyapatite nanoparticles have been prepared by using the wet chemical precipitation method using calcium nitrate tetra-hydrate [Ca(NO₃)₂.4H₂O] and di-ammonium hydrogen phosphate [(NH₄)₂ HPO₄] as precursors. The composite scaffolds have been prepared by a freeze-drying method with hydroxyapatite, chitosan, and gelatin which form a 3D network of interconnected pores. Glutaraldehyde solution has been used in the scaffolds to crosslink the amino groups (|NH₂) of gelatin with the aldehyde groups (|CHO) of chitosan. The X-ray diffraction (XRD) performed on different scaffolds indicates that the incorporation of a certain amount of hydroxyapatite has no influence on the chitosan/gelatin network and at the same time, the organic matrix does not affect the crystallinity of hydroxyapatite. Transmission electron microscope (TEM) images show the needle-like crystal structure of hydroxyapatite nanoparticle. Scanning Electron Microscope (SEM) analysis shows an interconnected porous network in the scaffold where HAP nanoparticles are found to be dispersed in the biopolymer matrix. Fourier transforms infrared spectroscopy (FTIR) confirms the presence of hydroxyl group (OH^-) , phosphate group (PO^3-₄) , carbonate group (CO^2-₃) , imine group (C=N), etc. TGA reveals the thermal stability of the scaffolds. The cytotoxicity of the scaffolds is examined qualitatively by VERO (animal cell) cell and quantitatively by MTTassay. The MTT-assay suggests keeping the weight percentage of glutaraldehyde solution lower than 0.2%. The result found from this study demonstrated that a proper bone replacing scaffold can be made up by controlling the amount of hydroxyapatite, gelatin, and chitosan which will be biocompatible, biodegradable, and biofriendly for any living organism.     

Controllable synthesis and characterization of porous polyvinyl alcohol/hydroxyapatite nanocomposite scaffolds via an in situ colloidal technique

During the last decades, there have been several attempts to combine bioactive materials with biocompatible and biodegradable polymers to create nanocomposite scaffolds with excellent biocompatibility, bioactivity, biodegradability and mechanical properties. In this research, the nanocomposite scaffolds with compositions based on PVA and HAp nanoparticles were successfully prepared using colloidal HAp nanoparticles combined with freeze-drying technique for tissue engineering applications. In addition, the effect of the pH value of the reactive solution and different percentages of PVA and HAp on the synthesis of PVA/HAp nanocomposites were investigated. The SEM observations revealed that the prepared scaffolds were porous with three dimensional microstructures, and in vitro experiments with osteoblast cells indicated an appropriate penetration of the cells into the scaffold's pores, and also the continuous increase in cell aggregation on the scaffolds with increase in the incubation time demonstrated the ability of the scaffolds to support cell growth. According to the obtained results, the nanocomposite scaffolds could be considered as highly bioactive and potential bone tissue engineering implants.     

Preparation of electrospun chitosan/poly(vinyl alcohol) membranes

Electrospinning of chitosan from its solutions in 2% aqueous acetic acid was studied by adding poly(vinyl alcohol) (PVA) as a “guest” polymer. Properties of the chitosan/PVA solutions including viscosity, conductivity, and surface tension were measured, and effects of the polymer concentration, chitosan/PVA mass ratio and processing parameters (applied voltage, flow rate, capillary-to-collector distance) on the electrospinnability of chitosan/PVA were investigated. Analyses of scanning electron micrographs and transmission electron micrographs suggested that the chitosan/PVA ultrafine fibers were often obtained along with beads, and chitosan was located in the elctrospun fibers as well as in the beads. Uniform chitosan/PVA fibers with an average diameter of 99 ± 21 nm could be prepared from a 7% chitosan/PVA solution in 40:60 mass ratio. Results of Fourier transform infrared spectroscopy and X-ray diffraction demonstrated that there were possible hydrogen bonds between chitosan and PVA molecules, which could weaken the strong interaction in chitosan itself and facilitate chitosan/PVA electrospinnability. The electrospun chitosan/PVA membranes showed higher water uptake and would have potential applications in wound dressings.     

Stabilization of electrospun poly(vinyl alcohol) nanofibrous mats in aqueous solutions

<正>The stability ofpoly(vinyl alcohol)(PVA) nanofibrous mats in water media was improved by post-electrospinning treatments.Bifunctional glutaraldehyde(GA) in methanol was used as a crosslinking agent to stabilize PVA nanofiber,but fiber twinning was observed frequently,and the highly porous structure of PVA nanofibrous mats was destroyed when the crosslinked fiber was soaked in water.To overcome this shortcoming,chitosan(CS) was introduced into the PVA spinning solution to prepare PVA/CS composite nanofibers.Their treatment in GA/methanol solution could retain the fiber morphology of PVA/CS nanofibers and porous structure of PVA/CS nanofibrous mats even if they were soaked in aqueous solutions for 1 month.Scanning electron microscopy(SEM),X-ray diffraction(XRD),thermal gravimetric analysis(TGA) and differential scanning calorimetry(DSC) were applied to characterize the physicochemical structure and thermal properties of PVA nanofibers.It was found that the water resistance of PVA nanofibrous mats was enhanced because of the improvement of the degree of crosslinking and crystallinity in the electrospun PVA fibers after soaking in GA/methanol solution.