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.     

Fabrication of open‐porous PCL/PLA tissue engineering scaffolds and the relationship of foaming process,morphology, and mechanical behavior

Tissue engineering scaffolds should provide a suitable porous structure and proper mechanical strength, which is beneficial for the delivery of growth factor and regulation of cells. In this study, the open‐porous polycaprolactone (PCL)/poly (lactic acid) (PLA) tissue engineering scaffolds with suitable porous scale were fabricated using different ratios of PCL/PLA blends. At the same time, the relationship of foaming process, morphology, and mechanical behavior in the optimized batch microcellular foaming process were studied based on the single‐factor experiment method. The porous structures and mechanical strength of the scaffolds were optimized by adjusting foaming parameters, including the temperature, pressure, and CO₂ dissolution time. The results indicated that the foaming parameters influence the cell morphology, further determine the mechanical behavior of PCL/PLA blends. When the PCL content is high, with the increase of temperature and time, the cell diameter and the elastic modulus increased, and the tensile strength and elastic modulus increased with the increase of the average cell size, and decreased as the increase of the cell density. While when the PLA content was high, the cell diameter showed the same trend, and the tensile strength and elastic modulus were higher, and the elongation at break was lower, and tensile strength and elastic modulus decreased with the increase of the average cell size and increased with the increase of cell density. This work successfully fabricated optimized porous PCL/PLA scaffolds with excellent suitable mechanical properties, pore sizes, and high interconnectivity, indicating the effectiveness of modulating the batch foaming process parameters.     

Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing

The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water‐based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well‐defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass‐transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme‐laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.     

Porous-conductive chitosan scaffolds for tissue engineering, 1. Preparation and characterization

Novel porous-conductive chitosan scaffolds were fabricated by incorporating conductive polypyrrole (PPy) particles into a chitosan matrix and employing a phase separation technique to build pores inside the scaffolds. Conductive polypyrrole particles were prepared with a microemulsion method using FeCl3 as a dopant. The preparation conditions were optimized to obtain scaffolds with controlled pore size and porosity. The conductivity of the scaffolds was investigated using a standard four-point probe technique. It was found that several kinds of scaffolds showed a conductivity close to 10(-3) S.cm(-1) with a low polypyrrole loading of around 2 wt.-%. The main mechanical properties, such as tensile strength, breaking elongation and Young's modulus of the scaffolds, were examined both in the dry and in the hydrated states. The results indicated that a few different kinds of scaffolds exhibited the desired mechanical strength for some tissue engineering applications. The miscibility of polypyrrole and chitosan was also evaluated using a dynamic mechanical method. The presence of significant phase separation was detected in non-porous PPy/chitosan scaffolds but enhanced miscibility in porous PPy/chitosan scaffolds was observed.     

Production and characterization of chitosan fibers and 3-D fiber mesh scaffolds for tissue engineering applications

This study reports on the production of chitosan fibers and 3-D fiber meshes for the use as tissue engineering scaffolds. Both structures were produced by means of a wet spinning technique. Maximum strain at break and tensile strength of the developed fibers were found to be 8.5% and 204.9 MPa, respectively. After 14 d of immersion in simulated body fluid (SBF), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and inductively coupled plasma emission (ICP) spectroscopy analyses showed that a bioactive Ca-P layer was formed on the surface of the fibers, meaning that they exhibit a bioactive behavior. The samples showed around 120% max. swelling in physiological conditions. The pore sizes of 3-D chitosan fiber mesh scaffolds were observed to be in the range of 100-500 microm by SEM. The equilibrium-swelling ratio of the developed scaffolds was found to be around 170% (w/w) in NaCl solution at 37 degrees C. Besides that, the limit swelling strain was less than 30%, as obtained by mechanical spectroscopy measurements in the same conditions. The viscoelastic properties of the scaffolds were also evaluated by both creep and dynamic mechanical tests. By means of using short-term MEM extraction test, both types of structures (fibers and scaffolds) were found to be non-cytotoxic to fibroblasts. Furthermore, osteoblasts directly cultured over chitosan fiber mesh scaffolds presented good morphology and no inhibition of cell proliferation could be observed.Osteoblast-like cells proliferating over chitosan based fibers after 7 d of culture.     

Microstructure and mechanical properties of bacterial cellulose/chitosan porous scaffold

A family of polysaccharide based scaffold materials, bacterial cellulose/chitosan (BC/CTS) porous scaffolds with various weight ratios (from 20/80 to 60/40 w/w%) were prepared by freezing (−30 and −80 °C) and lyophilization of a mixture of microfibrillated BC suspension and chitosan solution. The microfibrillated BC (MFC) was subjected to 2,2,6,6-tetramethylpyperidine-1-oxyl radical (TEMPO)-mediated oxidation to introduce surface carboxyl groups before mixing. The integration of MFC within chitosan matrix was performed by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-mediated cross-linking. The covalent amide bond formation was determined by ATR-FTIR. Because of this covalent coupling, the scaffolds retain their original shapes during autoclave sterilization. The composite scaffolds are three-dimensional open pore microstructure with pore size ranging from 120 to 280 μm. The freezing temperature and mean pore size take less effect on scaffold mechanical properties. The compressive modulus and strength increased with increase in MFC content. The results show that the scaffolds of higher MFC content contribute to overall better mechanical properties.     

Blending chitosan with polycaprolactone: porous scaffolds and toxicity

The preparation and characterization of porous scaffolds from chitosan-PCL blends by freeze extraction, freeze gelation and freeze drying is reported. Using freeze extraction, stable structures were obtained only from PCL, but these were not porous. No stable scaffolds were obtained using the freeze gelation process. Stable scaffolds of chitosan/PCL mixtures could not be obtained using 77% acetic acid by any of these techniques. With 25% aqueous acetic acid, stable scaffolds of chitosan/PCL mixtures were obtained by the freeze drying technique. The stability and pore morphology of freeze dried scaffolds were dependent on the relative mass ratio of chitosan and PCL. A chorioallantoic membrane assay showed that formed 3D chitosan/PCL mixtures were not toxic to vasculature.     

Preparation and mechanical properties of poly(chitosan‐g‐DL‐lactic acid) fibrous mesh scaffolds

DL ‐lactic acid was grafted onto chitosan to produce poly(chitosan‐g‐DL ‐lactic acid)(PCLA) without using a catalyst. These PCLAs were then spun into filaments and further fabricated into fibrous mesh scaffolds using an improved wet‐spinning technique. The diameter of filaments in different scaffolds could vary from a few micrometers to several tens of micrometers. The scaffolds exhibited various pore sizes ranging from about 20 µm to more than 200 µm and different porosities up to 80%. The several main processing conditions were optimized for obtaining the desired scaffolds with well‐controlled structures. The tensile and compressive mechanical properties of the mesh scaffolds in both dry and hydrated states were mainly examined. Significantly improved tensile strength and modulus, enhanced compressive modulus, and stress as well as the dimensional stability for these mesh scaffolds in their hydrated state were observed. Copyright © 2007 John Wiley & Sons, Ltd.     

3D printed biodegradable composites: An insight into mechanical properties of PLA/chitosan scaffold

Three-dimensional (3D) printing is a frontier manufacturing approach with great potential to benefit biomedical and patient care sectors. In the last decades, different types of biomedical materials were investigated in purpose of developing medical tools and devices. The present study attempts to assess mechanical performances (namely: tensile, compression, and flexural) of the newly developed chitosan-reinforced poly-lactic-acid (PLA) scaffolds by using fused filament fabrication (FFF) based 3D printing technology. Specifically, the effects of chitosan loading, infill density and annealing temperature on mechanical behavior of PLA composite scaffolds are investigated via design of experiments. Moreover, fracture behavior under various load types is studied with the help of selective electron microscopy. It is found that the strength of the produced composite samples depends significantly on the loading of chitosan and infill density, while annealing temperature does not affect mechanical response. Overall, the developed PLA composite scaffolds are mechanically efficient and they appear suitable for clinical purposes.     

Porous 3‐D scaffolds from regenerated Antheraea pernyi silk fibroin

In this study, porous three‐dimensional (3‐D) materials were prepared with the regenerated Antheraea pernyi (A. pernyi) silk fibroin by freeze‐drying from a lithium thiocyanate solution of its fibers. The relationship between preparation conditions and morphological structures of 3‐D materials was also studied. We concluded that with the decrease in A. pernyi silk fibroin solution concentration and the increase in the freezing temperature, the porosity and the average pore diameter of the 3‐D materials were increased while the pore density was decreased. By adjusting the freezing temperature and the silk fibroin solution concentration, the 3‐D materials having the average pore diameter of 75–260 µm and the porosity of 70–90% can efficiently be produced. As a kind of new material with excellent biocompatibility and bioactivity, the material is expected to be applied to tissue regeneration scaffolds. Copyright © 2007 John Wiley & Sons, Ltd.     

Thermally produced biodegradable scaffolds for cartilage tissue engineering

A novel process was developed to fabricate biodegradable polymer scaffolds for tissue engineering applications, without using organic solvents. Solvent residues in scaffolds fabricated by processes involving organic solvents may damage cells transplanted onto the scaffolds or tissue near the transplantation site. Poly(L-lactic acid) (PLLA) powder and NaCl particles in a mold were compressed and subsequently heated at 180 degrees C (near the PLLA melting temperature) for 3 min. The heat treatment caused the polymer particles to fuse and form a continuous matrix containing entrapped NaCl particles. After dissolving the NaCl salts, which served as a porogen, porous biodegradable PLLA scaffolds were formed. The scaffold porosity and pore size were controlled by adjusting the NaCl/PLLA weight ratio and the NaCl particle size. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting/particulate leaching (SC/PL) process, in terms of pore structure, pore-size distribution, and mechanical properties. A scanning electron microscopic examination showed highly interconnected and open pore structures in the scaffolds fabricated using the thermal process, whereas the SC/PL process yielded scaffolds with less interconnected and closed pore structures. Mercury intrusion porosimetry revealed that the thermally produced scaffolds had a much more uniform distribution of pore sizes than the SC/PL process. The utility of the thermally produced scaffolds was demonstrated by engineering cartilaginous tissues in vivo. In summary, the thermal process developed in this study yields tissue-engineering scaffolds with more favorable characteristics, with respect to, freedom from organic solvents, pore structure, and size distribution than the SC/PL process. Moreover, the thermal process could also be used to fabricate scaffolds from polymers that are insoluble in organic solvents, such as poly(glycolic acid). Cartilage tissue regenerated from thermally produced PLLA scaffold.     

Compressive mechanical properties and biodegradability of porous poly(caprolactone)/chitosan scaffolds

Porous polycaprolactone(PCL)/chitosan(CH) scaffolds with large pore sizes and high porosities were fabricated via a particle-leaching technique using hexafluoro-2-propanol as a shared solvent and salt (sodium chloride) particles as porogen. By optimizing processing conditions, numerous PCL/CH scaffolds with CH proportion lower than 50 wt% and similar pore parameters were built. These scaffolds were further evaluated for their compressive mechanical properties and biodegradation behaviors. It was found that their compressive modulus and stress at 10% strain were basically maintained in their dry state in contrast to their individual components, and these scaffolds still showed well-defined compressive characteristics and dimension stability even in their hydrated state compared with pure chitosan scaffolds. After being exposed to PBS or enzymatic degradation systems in vitro for various periods up to 10 weeks, it was observed that degradation of the PCL component could be accelerated at various rates depending on the compositions of the scaffolds and the media, and the chiosan component could effectively buffer the acidic degradation products of the PCL component.     

Large-Scale Production of 3D Bioactive Glass Macroporous Scaffolds for Tissue Engineering

For tissue engineering applications, a scaffold is required that can act as a template and guide for cell proliferation, cell differentiation and tissue growth. Interconnected pores with diameters greater than 100 m are required for tissue ingrowth, vascularisation and nutrient delivery to the centre of the scaffold. 3D bioactive glass scaffolds have been produced, by foaming sol-gel derived bioactive glasses. The method to produce foams with a modal macropore diameter of 100 m, and a handling strength suitable for cell culture, was to foam 50 ml batches of sol with the aid of a surfactant and gelling agent. In vitro and in vivo tests show that the scaffolds have high potential to be used in bone tissue engineering applications. Larger batches are required to produce scaffolds commercially. The aim of this work was to investigate how the process could be up-scaled for commercial use. This study shows that foaming larger aliquots of sol decreased the scaffold porosity and interconnectivity and investigates methods of modifying the process to obtain large quantities of foam scaffolds with pores in excess of 100 m. The optimum method to produce foams of similar pore structure from 200 ml sol to those produced from 50 ml sol comprised of adding 3 ml surfactant and 12 ml dionised water to the sol to start foaming and injecting a gas mixture (70% helium, 30% nitrogen) at 0.2 bar while applying vigorous agitation.     

Preparation and characterization of collagen/chitosan poly (ethylene glycol)/nanohydroxyapatite composite scaffolds

Porous three‐dimensional collagen/chitosan scaffolds combined with poly (ethylene glycol) (PEG) and hydroxyapatite were obtained through a freeze‐drying method. Physical cross‐linking was examined by dehydrothermal treatment. The prepared materials were characterized by different analyses, eg, scanning electron microscopy (SEM), measurements of porosity and swelling, mechanical properties, and resistance to enzymatic degradation. The porosity of scaffolds and their swelling ratio decreased with the addition of hydroxyapatite. Moreover, after exposure to collagenase, the collagen/chitosan matrices containing PEG showed much faster degradation rate than matrices with the addition of hydroxyapatite. The results indicated that the addition of hydroxyapatite led to improvement of stiffness. The highest degree of porosity and swelling were demonstrated by collagen/chitosan/PEG matrices without hydroxyapatite.     

超临界CO_2发泡法制备PLGA多孔组织工程支架

利用超临界CO2(SC-CO2)发泡法制备了一系列聚(乳酸-乙醇酸)共聚物(PLGA)多孔支架材料,研究了PLGA分子量和组成、发泡过程温度、压力以及泄压速率等对泡孔尺寸及形态的影响.结果表明,随着PLGA组成中乳酸含量的增加,泡孔平均孔径增大且连通性增强;提高过程压力易形成孔径小且泡孔密度大的微孔结构材料;降低泄压速率,泡孔易合并形成大孔.聚合物处于高弹态时,较低的发泡温度易导致特殊的多面体结构大孔的形成;而当温度较高时,泡孔塌缩形成球形微孔结构,且泡孔尺寸随着温度升高而增大.SC-CO2发泡法能有效地去除有机溶剂,避免在高温条件下操作,可以实现5～500μm范围内孔径可控的PLGA多孔支架材料的制备.     

Effects of polymer concentration and coagulation temperature on the properties of regenerated cellulose films prepared from LiOH/urea solution

Aqueous 5 wt% LiOH/12 wt% urea solution pre-cooled to −12 °C has a more powerful ability to dissolve cellulose compared to that of NaOH/urea and NaOH/thiourea solution system. The influences of the cellulose concentration and coagulation temperature on the structure, pore size and mechanical properties of the cellulose films prepared from LiOH/urea system were investigated. The cellulose films exhibited good mechanical properties either at wet or dry state and their pore size and water permeability at wet state can be controlled by changing the cellulose concentration or coagulation temperature. With a decrease of the coagulation temperature, the mechanical properties and optical transmittance of the cellulose films enhanced, as a result of the formation of relative smaller pore size and denser structures. This work provided a promising way to prepare cellulose films with different pore sizes at wet state and good physical properties at dry state.     

Nano-Structured Ridged Micro-Filaments (≥100 µm Diameter) Produced Using a Single Step Strategy for Improved Bone Cell Adhesion and Proliferation in Textile Scaffolds

Textile scaffolds that are either 2D or 3D with tunable shapes and pore sizes can be made through textile processing (weaving, knitting, braiding, nonwovens) using microfilaments. However, these filaments lack nano-topographical features to improve bone cell adhesion and proliferation. Moreover, the diameter of such filaments should be higher than that used for classical textiles (10–30 µm) to enable adhesion and the efficient spreading of the osteoblast cell (>30 µm diameter). We report, for the first time, the fabrication of biodegradable nanostructured cylindrical PLLA (poly-L-Lactic acid) microfilaments of diameters 100 µm and 230 µm, using a single step melt-spinning process for straightforward integration of nano-scale ridge-like structures oriented in the fiber length direction. Appropriate drawing speed and temperature used during the filament spinning allowed for the creation of instabilities giving rise to nanofibrillar ridges, as observed by AFM (Atomic Force Microscopy). These micro-filaments were hydrophobic, and had reduced crystallinity and mechanical strength, but could still be processed into 2D/3D textile scaffolds of various shapes. Biological tests carried out on the woven scaffolds made from these nano-structured micro filaments showed excellent human bone cell MG 63 adhesion and proliferation, better than on smooth 30 µm- diameter fibers. Elongated filopodia of the osteoblast, intimately anchored to the nano-structured filaments, was observed. The filaments also induced in vitro osteogenic expression, as shown by the expression of osteocalcin and bone sialoprotein after 21 days of culture. This work deals with the fabrication of a new generation of nano-structured micro-filament for use as scaffolds of different shapes suited for bone cell engineering.     

Using NaCl particles as porogen to prepare a highly adsorbent chitosan membranes

Macroporous chitosan membranes were prepared by using NaCl particles porogen and genipin as cross-linking agent. For characterization and sorption behavior comparison, other genipin cross-linked chitosan membranes were prepared by either freeze drying or by using silica particles as porogen. The mean pore diameter, the porosity, the crystallinity index (CrI) as well as the effect of the drying procedures of these chitosan membranes were examined. NaCl reduced the CrI of the chitosan membrane. The oven drying (OD) procedure decreased the mean pore diameter, the porosity, and increased the CrI of the chitosan membranes when compared with the vacuum drying (VD) procedure. The heat treatment of chitosan membrane in aqueous NaOH to attract silica porogen increased the CrI of the membrane. Under the same conditions, the membranes prepared with NaCl had better sorption performance on RR 189 and Cu²⁺ than other membranes. The maximum sorption capacity (q_e) reached 1836.17 mg RR 189/g chitosan and 151.98 mg Cu²⁺/g chitosan. The pore diameter (d_pore) of the membranes was much larger than the diameter of the adsorbate molecules (d_adsorbate), such that the ratio of d_pore/d_adsorbate had little influence on q_e. The porosity and the amorphous extent of the membranes were almost the same on q_e. When using tyrosinase catalyzing, the hydrocaffeic acid (HCA) grafted on the NaCl treated chitosan membrane was almost 10 times more than on chitosan beads. The chitosan membrane prepared with NaCl can be used as a good adsorbent with high loading capacity for implanting molecules (such as ligands, enzymes, etc.) on.     

Chitosan-based nanocomposites for medical applications

Chitosan as a biobased polymer is gaining increasing attention due to its extraordinary physico-chemical characteristics and properties. While a primary use of chitosan has been in horticultural and agricultural applications for plant defense and to increase crop yield, recent research reports display various new utilizations in the field of advanced biomedical devices, targeted drug delivery, and as bioimaging sensors. Chitosan possesses multiple characteristics such as antimicrobial properties, stimuli-responsiveness, tunable mechanical strength, biocompatibility, biodegradability, and water-solubility. Further, chitosan can be processed into nanoparticles, nano-vehicles, nanocapsules, scaffolds, fiber meshes, and 3D printed scaffolds for a variety of applications. In recent times, nanoparticles incorporated in chitosan matrices have been identified to show superior biological activity, as cells tend to proliferate/differentiate faster when they interact with nanocomposites rather than bulk or micron size substrates/scaffolds. The present article intents to cover chitosan-based nanocomposites used for regenerative medicine, wound dressings, drug delivery, and biosensing applications.     

Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.