Design and fabrication of tubular scaffolds via direct writing in a melt electrospinning mode

Flexible tubular structures fabricated from solution electrospun fibers are finding increasing use in tissue engineering applications. However it is difficult to control the deposition of fibers due to the chaotic nature of the solution electrospinning jet. By using non-conductive polymer melts instead of polymer solutions the path and collection of the fiber becomes predictable. In this work we demonstrate the melt electrospinning of polycaprolactone in a direct writing mode onto a rotating cylinder. This allows the design and fabrication of tubes using 20 μm diameter fibers with controllable micropatterns and mechanical properties. A key design parameter is the fiber winding angle, where it allows control over scaffold pore morphology (e.g. size, shape, number and porosity). Furthermore, the establishment of a finite element model as a predictive design tool is validated against mechanical testing results of melt electrospun tubes to show that a lesser winding angle provides improved mechanical response to uniaxial tension and compression. In addition, we show that melt electrospun tubes support the growth of three different cell types in vitro and are therefore promising scaffolds for tissue engineering applications.     

Mechanism of silk fibroin scaffolds with oriented multichannels and its cytocompatibility

As a biomaterial, besides excellent biocompatibility and biodegradability, suitable macropores and pores structure should be provided to guide cell extension and migration. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional temperature field freezing technique. The average pore diameter, pore density and porosity of the scaffold with oriented channels are ～128.7 µm, ～158 mm^?2 and ～91.4 %, respectively. By controlling of the temperature gradient direction, the oriented multichannels of the scaffolds were formed in longitudinal easily. In process of the scaffolds fabrication, the directional growth of ice crystal could shear and draft to the silk fibroin molecule segments, which resulted in the new crystal nucleus formation in new zone and increase of β-sheet components in the scaffolds. In vitro, L929 cells were seeded on the scaffolds with oriented channels to evaluate the effect on cell behavior. Cell viability, adhesion and morphology were determined by methyl thiazolyl tetrazolium, confocal microscope and scanning electron microscope. The results showed that the cells anchored on the oriented channels, spread along the direction of the channels and hold a higher viability on the scaffolds with oriented channels. These new oriented multichannel scaffold could guide the adhesion and proliferation of L929 cells, which hold a potential in tissue engineering.     

Fabrication of Biocompatible PLGA/PCL/PANI Nanofibrous Scaffolds with Electrical Excitability

Application of electrospun nanofibrous scaffolds has received immense attention in tissue engineering. Fabrication of scaffolds with appropriate electrical properties plays a key role in neural tissue engineering. Since fibers orientation in the scaffolds affects the growth and proliferation of the cells, this study aimed to prepare aligned electrospun conductive nanofibers by mixing 1 %, 10 % and 18 % (w/v) doped polyaniline (PANI) with polycaprolactone (PCL)/poly lactic-coglycolic acid (PLGA) (25/75) solution through the electrospinning process. The fibers diameter, hydrophilicity and conductivity were measured. In addition, the shape and proliferation of the nerve cells seeded on fibers were evaluated by MTT cytotoxicity assay and scanning electron microscopy. The results revealed that the conductive nanofibrous scaffolds were appropriate substrates for the attachment and proliferation of nerve cells. The electrical stimulation enhanced neurite outgrowth compared to those PLGA/PCL/PANI scaffolds that were not subjected to electrical stimulation. As polyaniline ratio increases, electric stimulation through nanofibrous PLGA/PCL/PANI scaffolds results in cell proliferation enhancement. However, a raise more than 10 % in polyaniline will result in cell toxicity. It was concluded that conductive scaffolds with appropriate ratio of PANI along with electrical stimulation have potential applications in treatment of spinal cord injuries.     

Functionalizing cellulose scaffold prepared by ionic liquid with bovine serum albumin for biomedical application

The present study reports the preparation of a cellulose scaffold for tissue engineering directly from cellulose fiber using ionic liquid (IL) by the NaCl leaching method with bovine serum albumin (BSA), which is well known protein utilized for biomedical applications like degradation of polymer, cell attachment and proliferation on scaffold. The 1-n-allyl-3-methylimidazolium chloride (AmimCl) IL was used as a solvent for cellulose. The morphology of the scaffold was studied by scanning electron microscopy (SEM) and the images showed that the pore sizes of the scaffolds were about 200 µm. In addition, the water uptake (WU) and degree of degradation of the cellulose scaffold were measured. Meanwhile, the biocompatibility and bioactivity of the scaffold were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay and the Live/Dead viability test. The various results demonstrated the ability of the Mesenchymal stem cells (MSC) to attach to the surface of the scaffolds amplified as percentage of BSA increased in cellulose scaffold.     

Electrospinning and mechanical properties of polymeric fibers using a novel gap-spinning collector

Tissue engineering is an interdisciplinary field which combines the basic principles of life sciences and engineering. One promising idea is the combination of scaffolds and living cells in order to produce new functional tissue. The scaffolds play the role of a microenvironment that guides the cells towards tissue formation and regeneration. One of the most frequently used techniques to produce scaffolds is electrospinning. Tissue engineered constructs have to exhibit physiological and mechanical properties comparable to the native tissue they are intended to replace. To create polymeric fibers with controlled orientation, a cylindrical collector that rotates at a certain speed could be used, creating fibers that run longitudinally. The process of gap-spinning enables the production of specifically aligned fibers. Aim of this study was to develop a novel setup capable of producing multilayered structures with controlled fiber angle. The structural, morphological and mechanical characteristics of the fibers were accessed using scanning electron microscopy and uniaxial tensile tests. Longer pre-stretching led to thinner (in the sub-micron scale), more brittle and less elastic fibers. In a nutshell, the results indicated that fiber mats of desired orientation, fiber diameter and mechanical properties could be produced by controlled gap-spinning with a translational collector.     

Effect of fiber diameter on surface morphology,mechanical property,and cell behavior of electrospun poly(ε-caprolactone) mat

In this study, electrospinning of poly(ε-caprolactone) (PCL) and its optimum preparation conditions were examined in detail using various solvent systems, such as formic acid, dichloromethane/dimethyl formamide (DMF), chloroform/DMF, and dichloroethane. The average fiber diameter of the electrospun PCL mat was controlled by the solvent used with a proper concentration of PCL dope solution. Different fiber sizes (0.1, 0.8, 1.9, and 3.4 μm) of uniform PCL mats were fabricated and the effects of fiber size on surface morphology, tensile properties and cell behavior were investigated. Here, we manipulated much broader range of average fiber diameter of the mats, from nano to several micron size of fiber. It was found that the fiber diameter greatly affected topology (surface roughness) and mechanical properties of the electrospun PCL mat and consequently, they influenced the cell behavior (adhesion and proliferation) significantly. We expect that these results will provide more feasible application of electrospun PCL scaffold in tissue engineering through the co-relations in structure and property of PCL fiber mat on cell behavior.     

Highly porous three-dimensional poly(lactide-co-glycolide) (PLGA) microfibrous scaffold prepared by electrospinning method: A comparison study with other PLGA type scaffolds on its biological evaluation

In this study, a three-dimensional (3D) poly(lactide-co-glycolide) (PLGA) microfibrous scaffold with high porosity (ca. 90 % porosity) was developed for evaluating its performance in tissue engineering application. A dope solution of PLGA/polyethylene oxide (PEO) blend was electrospun into a methanol coagulation bath for fabricating highly porous 3D PLGA scaffold and a salt leaching method was used for making interconnected pores of 100?C200 ??m size inside the scaffold. The morphological structure, pore size and porosity of the microfibrous scaffold were determined, and compared with twodimensional (2D) mat-type and 3D sponge-type of PLGA scaffold. Also, swelling ratio, water uptake and compressive strength were compared in order to elucidate the structure-property relationships of different types of the scaffolds, especially in a wet condition. As a result of scanning electron microscopy (SEM) observation, normal human dermal fibroblasts (nHDF) were migrated, attached, and proliferated well inside the 3D scaffold. MTT assay confirmed that the highly porous 3D PLGA microfibrous scaffold had superior cell adhesion and proliferation abilities due to fibrous structure of large specific surface area, and interconnected pore structure. Therefore, this high performance 3D PLGA scaffold can have a high potentiality for application in tissue engineering in comparison with conventional PLGA scaffolds.     

Electrospun PU-PEG and PU-PC hybrid scaffolds for vascular tissue engineering

Produced via electrospinning, polyurethane (PU) scaffolds have always attracted the interest of medical applications due of their unique properties such as good adhesion, biocompatibility and excellent mechanical strength. However, the poor hydrophilicity and hemocompatibility of PU presented a problem during PU’s application in the manufacturing of biomedical materials. We hypothesized that the incorporation of polyethylene glycol (PEG) and phosphatidylcholine (PC) into electrospinning solution of PU could improve the cell affinity and hemocompatibility. This research focused on fabricating hybrid PU-PEG and PU-PC random/aligned scaffolds through electrospinning technique and comparing their properties as a potential biocompatible scaffold for vascular tissue engineering. PC was doped into a PU solution in order to prepare an electrospun scaffold through the electrospinning technology while crosslinked electrospun PUPEG hybrid scaffolds were fabricated by photoinduced polymerization. The contact angle dramatically decreased from 122.3±0.8° to 39.1±0.8° with doping of PC in electrospinning solution while it decreased from 122.3±0.8° to 41.6±0.8° with doping of PEG. Furthermore, the mechanical properties of PU scaffolds were altered significantly by the addition of PC. The hemolysis and cytocompatibility assays demonstrated that these composite scaffolds could potentially be used as a smalldiameter vascular graft.     

Fabrication of Gelatin-Based Electrospun Composite Fibers for Anti-Bacterial Properties and Protein Adsorption

A major goal of biomimetics is the development of chemical compositions and structures that simulate the extracellular matrix. In this study, gelatin-based electrospun composite fibrous membranes were prepared by electrospinning to generate bone scaffold materials. The gelatin-based multicomponent composite fibers were fabricated using co-electrospinning, and the composite fibers of chitosan (CS), gelatin (Gel), hydroxyapatite (HA), and graphene oxide (GO) were successfully fabricated for multi-function characteristics of biomimetic scaffolds. The effect of component concentration on composite fiber morphology, antibacterial properties, and protein adsorption were investigated. Composite fibers exhibited effective antibacterial activity against Staphylococcus aureus and Escherichia coli. The study observed that the composite fibers have higher adsorption capacities of bovine serum albumin (BSA) at pH 5.32–6.00 than at pH 3.90–4.50 or 7.35. The protein adsorption on the surface of the composite fiber increased as the initial BSA concentration increased. The surface of the composite reached adsorption equilibrium at 20 min. These results have specific applications for the development of bone scaffold materials, and broad implications in the field of tissue engineering.     

Growth of primary hippocampal neurons on multichannel silk fibroin scaffold

Particular attention has been given to axonal outgrowth of neurons to understand how topographical surface cues influence attachment and subsequent directional migration and growth. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional freeze-drying processes. The average pore diameter, the porosity, and pore density of the scaffold are 120 µm, 88 %, and 203 mm^?2, respectively. Further, hippocampal neurons were seeded onto the scaffold and the hippocampal neurons morphology was investigated. Cell-cell networks and cell-matrix interactions had been established by newly formed axons and the diversity of neurons was much higher after culturing 7 days. The neurons expressed β-III-tubulin and nerve filament, while glial fibrillary acidic protein immunofluorescence was barely above background. These results indicated that the SF scaffolds with uniaxial multichannels could be guided axons of neurons spread along the channels. SF scaffolds with oriented pores have a potential for nerve tissue regeneration.     

Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering

Over the last few years, significant research has been conducted in the construction of artificial bone scaffolds. In the present study, different types of polymer scaffolds, such as chitosan-alginate (Chi-Alg) and chitosan-alginate with fucoidan (Chi-Alg-fucoidan), were developed by a freeze-drying method, and each was characterized as a bone graft substitute. The porosity, water uptake and retention ability of the prepared scaffolds showed similar efficacy. The pore size of the Chi-Alg and Chi-Alg-fucoidan scaffolds were measured from scanning electron microscopy and found to be 62–490 and 56–437 µm, respectively. In vitro studies using the MG-63 cell line revealed profound cytocompatibility, increased cell proliferation and enhanced alkaline phosphatase secretion in the Chi-Alg-fucoidan scaffold compared to the Chi-Alg scaffold. Further, protein adsorption and mineralization were about two times greater in the Chi-Alg-fucoidan scaffold than the Chi-Alg scaffold. Hence, we suggest that Chi-Alg-fucoidan will be a promising biomaterial for bone tissue regeneration.     

Conductive 3D structure nanofibrous scaffolds for spinal cord regeneration

The complex nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials and scaffolds which are capable of stimulating neural tissue repair strategies. Recently, conductive polymers have gained much attention for improving the nerve regeneration. In our previous study, a three-dimensional (3D) structure with reliable performance was achieved for electrospun scaffolds. The main purpose in the current study is formation of electrical excitable 3D scaffolds by appending polyaniline (PANI) to biocompatible polymers. In this paper, an attempt was made to develop conductive nanofibrous scaffolds, which can simultaneously present both electrical and topographical cues to cells. By using a proper 3D structure, two kinds of conductive scaffolds are compared with a non-conductive scaffold. The 3D nanofibrous core-sheath scaffolds, which are conductive, were prepared with nanorough sheath and aligned core. Two different sheath polymers, including poly(lactic-co-glycolic acid) PLGA and PLGA/PANI, with identical PCL/PANI cores were fabricated. Nanofibers of PCL and PLGA blends with PANI have fiber diameters of 234±60.8 nm and 770±166.6 nm, and conductivity of 3.17×10^-5 S/cm and 4.29×10^-5 S/cm, respectively. The cell proliferation evaluation of nerve cells on these two conductive scaffolds and previous non-conductive scaffolds (PLGA) indicate that the first conductive scaffold (PCL/ PANI-PLGA) could be more effective for nerve tissue regeneration. Locomotor scores of grafted animals by developed scaffolds showed significant performance of non-conductive 3D scaffolds. Moreover, the animal studies indicated the ability of two new types of conductive scaffolds as spinal cord regeneration candidates.     

Fabrication of silk fibroin based three dimensional scaffolds for tissue engineering

Herein we report successful synthesis of silk fibroin (SF) three dimensional scaffolds (SF 3D-scaffold) from SF sponge and SF nanofibers. Both the nanofibers and sponge were prepared from Bombyx mori fibroin. The SF 3D-scaffold was prepared by electrospinning the fibroin nanofibers over the sponge. Surface morphology was determined by scanning electron microscopy (SEM), while nanofiber diameter and pore size were measured using imageJ software. Effect of spinning time on the pore size and cell adhesion was determined. Average diameter of the SF nanofibers was measured to be 320 nm and pore size was found to reduce with increasing spinning time, such that, for 1 h spinning time pore size was 231 µm and the same for 3.5 h was 4.1 µm. However, the number of pores increased with spinning time. The results confirmed adhesion of MC3T3-E1 cells on the SF sponge, SF nanofibers and SF three dimensional scaffolds. Higher cell adhesion was found on the three dimensional scaffold in comparison to the nanofibers and sponge, possibly due to highly porous structure with very small and numerous pores in the resultant composite; hence more cell adhesion sites. The cell adhesion result confirmed biocompatibility of the SF 3D-scaffold and hence its suitability for applications in tissue engineering.     

Surface-modified nanofibrous biomaterial bridge for the enhancement and control of neurite outgrowth

Biomaterial bridges constructed from electrospun fibers offer a promising alternative to traditional nerve tissue regeneration substrates. Aligned and unaligned polycaprolactone (PCL) electrospun fibers were prepared and functionalized with the extracellular matrix proteins collagen and laminin using covalent and physical adsorption attachment chemistries. The effect of the protein modified and native PCL nanofiber scaffolds on cell proliferation, neurite outgrowth rate, and orientation was examined with neuronlike PC12 cells. All protein modified scaffolds showed enhanced cellular adhesion and neurite outgrowth compared to unmodified PCL scaffolds. Neurite orientation was found to be in near perfect alignment with the fiber axis for cells grown on aligned fibers, with difference angles of less than 7° from the fiber axis, regardless of the surface chemistry. The bioavailability of PCL fibers with covalently attached laminin was found to be identical to that of PCL fibers with physically adsorbed laminin, indicating that the covalent chemistry did not change the protein conformation into a less active form and the covalent attachment of protein is a suitable method for enhancing the biocompatibility of tissue engineering scaffolds.     

Preparation and characterization of blended <Emphasis Type="Italic">Bombyx mori</Emphasis> silk fibroin scaffolds

The aim of this study was to compare physical, mechanical and biological properties of 3-dimensional scaffolds prepared from Bombyx mori silk fibroin (SF), fibroin blended with collagen (SF/C), and fibroin blended with gelatin (SF/G) using a freeze-drying technique. The prepared scaffolds were sponge-like structure that exhibited homogeneous porosity with highly interconnected pores. Average pore size of these scaffolds ranged from 65–147 μm. All biodegradable scaffolds were capable of water absorption of 90 %. The degradation behavior of these scaffolds could be controlled by varying the amount of blended polymer. The SF/C and SF/G scaffolds showed higher compressive modulus than that of SF scaffolds which could be attributed to the thicker pore wall observed in the blended constructs. The less crystalline SF structure was observed in SF/G scaffolds as compared to SF/C scaffolds. Thus, the highest compressive modulus was observed on SF/C matrix. To investigate the feasibility of the scaffolds for cartilage tissue engineering application, rat articular chondrocytes were seeded onto the scaffolds. The MTT assay demonstrated that blending collagen or gelatin into SF sponge facilitated cell attachment and proliferation better than SF scaffolds. The blended SF scaffolds possessed superior physical, mechanical and biological properties in comparison to SF scaffolds and showed high potential for application in cartilage tissue engineering.     

Fabrication and surface modification of melt-electrospun poly(D,L-lactic-co-glycolic acid) microfibers

In this study, biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) fibers were prepared by a melt-electrospinning and treated with plasma in the presence of either oxygen or ammonia gas to modify the surface of the fibers. The effects of processing parameters on the melt-electrospinning of PLGA were examined in terms of fiber morphology and diameter. Among the processing parameters, the spinning temperature and mass flow rate had a significant effect on the average fiber diameter and its distribution. The water contact angle of melt-electrospun PLGA fibers decreased significantly from 123 ° to 55 ° (oxygen plasma treatment) or to 0 ° (ammonia plasma treatment) by plasma treatment for 180 sec, while their water content increased significantly from 2.4 % to 123 % (oxygen plasma treatment) or to 189 % (ammonia plasma treatment). Ammonia gas-plasma enhanced the surface hydrophilicity of PLGA fibers more effectively compared to oxygen gas-plasma. X-ray photoelectron spectroscopy analysis supported that the number of polar groups, such as hydroxyl and amino groups, on the surface of PLGA fibers increased after plasma treatment. Overall, the microfibrous PLGA scaffolds with appropriate surface hydrophilicity and fiber diameter could be fabricated by melt electrospinning and subsequent plasma treatment, without a significant deterioration of fiber structure and dimensional stability. This approach of controlling the surface properties and structures of fibers could be useful in the design and tailoring of novel scaffolds for tissue engineering.     

Silk fibroin/hyaluronic acid porous scaffold for dermal wound healing

The use of silk protein as a biomaterial has been studied for decades. In this study, silk fibroin (SF)/hyaluronic acid (HA) blend scaffolds were prepared by freeze-drying technique. The structure and properties of the blend scaffolds were examined and analyzed. The results demonstrated that the secondary structures of the SF/HA scaffolds were mainly amorphous and β-sheet structures. The pore radius and porosity of the scaffolds decreased with a decrease in the freezing temperature decrease and an increase in the HA ratio. The pore radius and porosity were regulated from 32.22 μm to 290.76 μm and from 74.1 % to 91.15 %, respectively. In vitro, the SF/HA scaffolds could support the fibroblast cell adhesion and proliferation and showed good cytocompatibility. In vivo, the SF/HA scaffolds were implanted into the dorsum of Sprague Dawley rats to evaluate their bioactivity for dermal tissue reconstruction. The vascular-like structures appeared more rapidly in SF/HA scaffolds than that in the PVA group, and a new dermal layer was formed, as determined by histological analysis. The SF/HA porous scaffolds have promise as a dermal substitute.     

The Effect of Melanocyte Stimulating Hormone and Hydroxyapatite on Osteogenesis in Pulp Stem Cells of Human Teeth Transferred into Polyester Scaffolds

Presently, tissue engineering is employed in the restoration and repair of tissue defects. Degradable scaffolds, stem cells and stimulating factors are employed in this method. In this study, the effect of melanocyte-stimulating hormone (MSH) and/or hydroxyapatite (HA) on proliferation, osteoblast differentiation, and mineralization of human dental pulp stem cells (hDPSCs) seeded on PLLA-PCL nanofibrous scaffolds was evaluated. For this aim, PLLA-PCL-HA nanofibrous scaffolds were fabricated using electrospinning method. FE-SEM images exhibited that all nanofibers had bead-free morphologies with average diameters ranging from 150–205 nm. Human DPSCs seeded into PLLA-PCL nanofibers were treated with MSH. Cell viability, proliferation, morphology, osteogenic potential, and the expression of tissue-specific genes were assessed by means of MTT assay, FE-SEM, alizarin red S staining, and RT-PCR analysis. hDPSCs exhibited improved adhesion and proliferation capacity on the PLLA-PCL-HA nanofibers treated with MSH compared to other groups (p<0.05). Additionally, PLLA-PCL-HA nanofibers treated with MSH exhibited significantly higher mineralization and alkaline phosphatase activity than other groups. RT-PCR results confirmed that PLLA-PCL-HA nanofibers enriched with MSH could significantly unregulated the gene expression of BMP2, osteocalcin, RUNX2 and DSPP that correlated to osteogenic differentiation (p<0.05). Based on results, incorporation of HA nanoparticles in PLLA-PCL nanofibers and addition of MSH in media exhibited synergistic effects on the adhesion, proliferation, and osteogenesis differentiation of hDPSCs, and therefore assumed to be a favorable scaffold for bone tissue engineering applications.     

Effect of Dioxane and N-Methyl-2-pyrrolidone as a Solvent on Biocompatibility and Degradation Performance of PLGA/nHA Scaffolds

Background:Solvent casting/particulate leaching is one of the most conventional methods for fabricating polymer/ceramic composite scaffolds. In this method, the solvent generally affects resulting scaffold properties, including porosity and degradation rate. Methods:Herein, composite scaffolds of PLGA/nHA with different percentages of nHA (25, 35, and 45 wt. %) were prepared by the solvent casting/particle leaching combined with freeze drying. The effects of two different solvents, DIO and NMP, on morphology, porosity, bioactivity, degradation rate, and biocompatibility of the resulting scaffolds were investigated. Results:The results revealed that increasing the nHA percentages had no significant effect on the porosity and interconectivity of scaffolds (p > 0.05), whereas altering the solvent from DIO into NMP decreased the porosity from about 87% into 71%, respectively. Moreover, scaffolds of DIO illustrated the high results of cell proliferation compared to those of NMP; the cell viability of GD25 decreased from 85% to 65% for GN25. The findings also indicated that scaffolds prepared by NMP had a higher rate of losing weight in comparison to DIO. Adding nHA to PLGA had a significant effect on the bioactivity of scaffolds (p < 0.05), composite scaffolds with 45 wt % nHA had at least 30% more weight gain compared to the neat polymer scaffolds. Conclusion:The DIO scaffolds have higher rates of porosity, interconnectivity, bioactivity, and biocompatibility than NMP scaffolds due to its high evaporation rate. Key Words: Freeze drying, Porosity, Solvents