Preparation and in vitro characterization of electrospun PVA scaffolds coated with bioactive glass for bone regeneration

An important objective in bone tissue engineering is to fabricate biomimetic three-dimensional scaffolds that stimulate mineralization for rapid regeneration of bone. In this work, scaffolds of electrospun poly(vinyl alcohol) (PVA) fibers (diameter = 286 ± 14 nm) were coated with a sol-gel derived bioactive glass (BG) and evaluated in vitro for potential applications in bone repair. Structural and chemical analyses showed that the BG coating was homogeneously deposited on the PVA fibers. In vitro cell culture studies showed that the BG-coated PVA scaffold had a greater capacity to support proliferation of osteogenic MC3T3-E1 cells, alkaline phosphatase activity, and mineralization than the uncoated PVA scaffold. The BG coating improved the tensile strength of the PVA scaffold from 18 ± 2 MPa to 21 ± 2 MPa, but reduced the elongation to failure from 94 ± 4% to 64 ± 5%. However, immersion of the BG-coated PVA scaffolds in a simulated body fluid for 5 days resulted in an increase in the tensile strength (24 ± 2 MPa) and elongation to failure (159 ± 4%). Together, the results show that these BG-coated PVA scaffolds could be considered as candidate materials for bone tissue engineering applications.     

Fabrication and material properties of fibrous PHBV scaffolds depending on the cross-ply angle for tissue engineering

Fibrous PHBV cross-ply scaffolds were fabricated using the electrospinning technique. The electrospun fibers were arranged depending on angles of alignment, which were 180°, 90°, 60°, and 45°. The stress and strain values of the fibrous PHBV cross-ply scaffolds increased as the cross-ply angle increased. At 180°, the strength and strain values of the fibers depended on tensile loading directions. At an alignment of 90°, the PHBV scaffolds had a stress value of 3.5?MPa, which was more than two times higher than the random structure. The cell morphology and proliferation of L-929 cells was strongly dependant on the fiber alignment and the best results were observed when the angle alignment was high. The results of this study showed that the cross-ply structure of the PHBV scaffold affected not only the cell adhesion and spreading properties but also dictated the mechanical properties, which were dependent on the angles of alignment.     

Electrospun bio-composite P(LLA-CL)/collagen I/collagen III scaffolds for nerve tissue engineering

One of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that could mimic the extracellular matrix (ECM) and assist in nerve regeneration. Bio-composite nanofibrous scaffolds made from synthetic and natural polymeric blends provide suitable substrate for tissue engineering and it can be used as nerve guides eliminating the need of autologous nerve grafts. Nanotopography or orientation of the fibers within the scaffolds greatly influences the nerve cell morphology and outgrowth, and the alignment of the fibers ensures better contact guidance of the cells. In this study, poly (L-lactic acid)-co-poly(ε-caprolactone) or P(LLA-CL), collagen I and collagen III are utilized for the fabrication of nanofibers of different compositions and orientations (random and aligned) by electrospinning. The morphology, mechanical, physical, and chemical properties of the electrospun scaffolds along with their biocompatibility using C17.2 nerve stem cells are studied to identify the suitable material compositions and topography of the electrospun scaffolds required for peripheral nerve regeneration. Aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds with average diameter of 253 ± 102 nm were fabricated and characterized with a tensile strength of 11.59 ± 1.68 MPa. Cell proliferation studies showed 22% increase in cell proliferation on aligned P(LLA-CL)/collagen I/collagen III scaffolds compared with aligned pure P(LLA-CL) scaffolds. Results of our in vitro cell proliferation, cell-scaffold interaction, and neurofilament protein expression studies demonstrated that the electrospun aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds mimic more closely towards the ECM of nerve and have great potential as a substrate for accelerated regeneration of the nerve.     

Hydrophilic nanofibrous structure of polylactide; fabrication and cell affinity

Microstructure and architecture of the scaffolds along with the surface chemistry exert profound effect on biological activity (cell distribution, proliferation, and differentiation). For the biological activity, scaffolds in tissue engineering have been widely designed. The objective of this study was to develop hydrophilic nanofibrous structure of polylactides (PLLA) polymer in the form of nonwoven mat by electrospinning technique, and further evaluate the fibroblast NIH3T3 cell proliferation, morphology, and cell-matrix interaction. Hydrophilicity of the PLLA fibers was improved by adding small fraction of low molecular weight polyethylene glycol (PEG) into the electrospinning solution. Four different ratio types (100/0, 80/20, 70/30, and 50/50) of PLLA/PEG electrospun matrices were fabricated, and the pore characteristics, tensile properties, contact angle, and hydrolytic degradation were observed. Furthermore, scanning electron microscope (SEM) and fluorescence actin staining images were used for micro-observation of cell-matrix interaction and cell morphology. It was found that the electrospun mat of PLLA/PEG (80/20), composed of fibers with diameters in the range 540-850 nm, majority of pore diameter less than 100 microm, tensile strength 8 MPa, elongation 150%, porosity more than 90%, and improved hydrophilicity with slow hydrolytic degradation, is favorable for biological activity of NIH3T3 fibroblast cell. Based on these results, the correct composition of PLLA and PEG in the porous electrospun matrix (i.e., PLLA/PEG (80/20)) will be a better candidate rather than other compositions of PLLA/PEG as well as hydrophobic PLLA for application in tissue engineering.     

Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold

Scaffolds made of chitosan nanofibers are often too mechanically weak for their application and often their manufacturing processes involve the use of harmful and flammable organic solvents. In the attempt to improve the mechanical properties of nanofibrous scaffolds made of chitosan without the use of harmful chemicals, pectin, an anionic polymer was blended with chitosan, a cationic polymer, to form a polyelectrolyte complex and electrospun into nanofibers for the first time. The electrospun chitosan-pectin scaffolds, when compared to electrospun chitosan scaffolds, had a 58% larger diameter, a 21% higher Young’s modulus, a 162% larger strain at break, and a 104% higher ultimate tensile strength. Compared to the chitosan scaffolds, the chitosan-pectin scaffolds’ swelling ratios decreased by 55% after 60?min in a saline solution and more quickly released the preloaded tetracycline HCl. The L929 fibroblast cells proliferated slightly slower on the chitosan-pectin scaffolds than on the chitosan scaffolds. Nonetheless, cells on both materials deposited similar levels of extracellular type I collagen on a per DNA basis. In conclusion, a novel chitosan-pectin nanofibrous scaffold with superior mechanical properties than a chitosan nanofibrous scaffold was successfully made without the use of harmful solvents.     

Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications

In the engineering of soft tissues, scaffolds with high elastance and strength coupled with controllable biodegradable properties are necessary. To fulfill such design criteria we have previously synthesized two kinds of biodegradable polyurethaneureas, namely poly(ester urethane)urea (PEUU) and poly(ether ester urethane)urea (PEEUU) from polycaprolactone, polycaprolactone-b-polyethylene glycol-b-polycaprolactone, 1,4-diisocyanatobutane and putrescine. PEUU and PEEUU were further fabricated into scaffolds by thermally induced phase separation using dimethyl sulfoxide (DMSO) as a solvent. The effect of polymer solution concentration, quenching temperature and polymer type on pore morphology and porosity was investigated. Scaffolds were obtained with open and interconnected pores having sizes ranging from several mum to more than 150 microm and porosities of 80-97%. By changing the polymer solution concentration or quenching temperature, scaffolds with random or oriented tubular pores could be obtained. The PEUU scaffolds were flexible with breaking strains of 214% and higher, and tensile strengths of approximately 1.0 MPa, whereas the PEEUU scaffolds generally had lower strengths and breaking strains. Scaffold degradation in aqueous buffer was related to the porosity and polymer hydrophilicity. Smooth muscle cells were filtration seeded in the scaffolds and it was shown that both scaffolds supported cell adhesion and growth, with smooth muscle cells growing more extensively in the PEEUU scaffold. These biodegradable and flexible scaffolds demonstrate potential for future application as cell scaffolds in cardiovascular tissue engineering or other soft tissue applications.     

Microbial degradation,cytotoxicity and antibacterial activity of polyurethanes based on modified castor oil and polycaprolactone

The objective of this study was to assess the effects of type of polyol and concentration of polycaprolactone (PCL) in polyurethanes (PUs) on microbial degradability, cytotoxicity, biological properties and antibacterial activity to establish whether these materials may have biomedical applications. Chemically modified and unmodified castor oil, PCL and isophorone diisocyanate in a 1:1 ratio of NCO/OH were used. PUs were characterized by stress/strain fracture tests and hardness (ASTM D 676-59). Hydrophilic character was determined by contact angle trials and morphology was evaluated by scanning electron microscopy. Degradability with Escherichia coli and Pseudomonas aeruginosa was evaluated by measuring variations in the weight of the polymers. Cytotoxicity was evaluated using the ISO 10993-5 (MTT) method with mouse embryonic fibroblasts L-929 (ATCC® CCL-1) in direct contact with the PUs and with NIH/3T3 cells (ATCC® CRL-1658) in indirect contact with the PUs. Antimicrobial activity against E. coli and P. aeruginosa was determined. PUs derived from castor oil modified (P0 and P1) have higher mechanical properties than PUs obtained from castor oil unmodified (CO). The viability of L-929 mouse fibroblasts in contact with polymers was greater than 70%. An assessment of NIH/3T3 cells in indirect contact with PUs revealed no-toxic degradation products. Finally, the antibacterial effect of the PUs decreased by 77% for E. coli and 56% for P. aeruginosa after 24 h. These results indicate that PUs synthesized with PCL have biocidal activity against Gram-negative bacteria and do not induce cytotoxic responses, indicating the potential use of these materials in the biomedical field.     

Degradation-induced changes of mechanical properties of an electro-spun polyester-urethane scaffold for soft tissue regeneration

The aim of this study was the in vitro investigation of the change in mechanical properties of a fast-degrading electro-spun polymeric scaffold for the use in soft tissue regenerative implants. Tubular scaffolds were electro-spun from a DegraPol? D30 polyesther-urethane solution (target outer diameter: 5.0 mm; scaffold wall thickness: 0.99 ± 0.18 mm). Scaffold samples were subjected to hydrolytic in vitro degradation for up to 34 days. The fiber network structure and fiber surfaces were inspected on scanning electron micrographs. Following vacuum drying and determination of mass, flat samples (9.69 ± 0.21 × 18.47 ± 2.62 mm, n = 5) underwent uni-axial tensile testing (5 load cycles, strain ε = 0 to 20%; final extension to failure) in circumferential scaffold direction after 5, 10, 14, 18, 22, 26, 30, and 34 days of degradation. Scaffold mass did not change with degradation. Maximum elastic modulus, maximum stress and associated strain were E(max) = 1.14 ± 0.23 MPa, σ(max) = 0.52 ± 0.12 MPa and ε(max) = 176.8 ± 21.9% before degradation and E(max) = 0.43 ± 0.26 MPa, σ(max) = 0.033 ± 0.028 MPa and ε(max) = 24.6 ± 3.0% after 34 days of degradation. The deterioration of mechanical properties was not reflected in the ultrastructural surface morphology of the fibers. The current exploratory study provides a basis for the development of constitutive computational models of biodegradable scaffolds with future extension of the investigation most importantly to capture mechanical effects of regenerating tissue. Future studies will include degradation in biological fluids and assessment of molecular weight for an advanced understanding of the material changes during degradation.     

Synthesis,characterization, and cytocompatibility of elastomeric,biodegradable poly(ester-urethane)ureas based on poly(caprolactone) and putrescine

The engineering of tissue for mechanically demanding applications in the cardiovascular system is likely to require mechanical conditioning of cell-scaffold constructs prior to their implantation. Scaffold properties amenable to such an application include high elasticity and strength coupled with controllable biodegradative and cell-adhesive properties. To fulfill such design criteria, we have synthesized a family of poly(ester-urethane)ureas (PEUUs) from polycaprolactone and 1,4-diisocyanatobutane. Lysine ethyl ester (Lys) or putrescine was used as chain extenders. To encourage cell adhesion, PEUUs were surface modified with radio-frequency glow discharge followed by coupling of Arg-Gly-Asp-Ser (RGDS). The synthesized PEUUs were highly flexible, with breaking strains of 660-895% and tensile strengths from 9.2-29 MPa. Incubation in aqueous buffer for 8 weeks resulted in mass loss, from >50% (Lys chain extender) to 10% (putrescine chain extender). Human endothelial cells cultured for 4 days with medium containing the degradation products from PEUUs with either the Lys or putrescine chain extender showed no toxic effects. Cell adhesion was 85% of that measured on tissue-culture polystyrene for unmodified PEUU surfaces (p < 0.01) and >160% (p < 0.001) of polystyrene on RGDS-modified PEUUs. These biodegradable PEUUs demonstrate potential for future application as cell scaffolds in cardiovascular tissue-engineering or other soft-tissue applications.     

Influences of tensile load on in vitro degradation of an electrospun poly(l-lactide-co-glycolide) scaffold

Scaffolds for tissue engineering and regenerative medicine are usually subjected to different mechanical loads during in vitro and in vivo degradation. In this study, the in vitro degradation process of electrospun poly(l-lactide-co-glycolide) (PLGA) scaffolds was examined under continuous tensile load and compared with that under no load. As PLGA degraded in phosphate-buffered saline solution (pH 7.4) at 37 °C over a 7-week period, the tensile elastic modulus and ultimate strength of the loaded specimen increased dramatically, followed by a decrease, which was much faster than that of the unloaded specimen, whereas break elongation of the loaded samples declined more quickly over the whole degradation period. Moreover, molecular weight, thermal properties and lactic acid release showed greater degradation under load. Also, a ruptured morphology was more obvious after degradation under tensile load. The results demonstrate that tensile load increased the degradation rate of electrospun PLGA and it may be necessary to consider the effects of mechanical load when designing or applying biodegradable scaffolds. Finally, some possible explanation for the faster degradation under load is given.     

The use of thermal treatments to enhance the mechanical properties of electrospun poly(epsilon-caprolactone) scaffolds

Nonwoven nanofiber scaffolds fabricated by electrospinning technology have been widely used for tissue engineering applications. Although electrospun nanofiber scaffolds fulfill many requirements for tissue engineering applications, they sometimes lack the necessary biomechanical properties. To attempt to improve the biomechanical properties of electrospun poly(epsilon-caprolactone) (PCL) scaffolds, fibers were bonded by thermal treatment. The thermal fiber bonding was performed in Pluronic F127 solution at a range of temperatures from 54 degrees C to 60 degrees C. Thermally bonded electrospun PCL scaffolds were characterized by analyzing the changes in morphology, fiber diameter, pore area, tensile properties, suture retention strength, burst pressure strength, and compliance. The biomechanical properties of the thermally bonded electrospun PCL scaffolds were significantly increased without any gross observable and ultrastructural changes when compared to untreated PCL scaffolds. This study suggests that the introduction of thermal fiber bonding to electrospun PCL scaffolds improved the biomechanical properties of these scaffolds, making them more suitable for tissue engineering applications.     

Synthesis and characterization of biodegradable elastomeric polyurethane scaffolds fabricated by the inkjet technique

Biodegradable polyurethanes (PUs) were synthesized from methylene di-p-phenyl-diisocyanate (MDI), polycaprolactone diol (PCL-diol) and N,N-bis (2-hydorxyethyl)-2-aminoethane-sulfonic acid (BES), serving as a hard segment, soft segment and chain extender, respectively. MDI was chosen due to its reactivity and wide application in synthesis of biomedical polyurethanes due to its reactivity; PCL-diol was chosen because of its biodegradability; and BES was chosen because it allowed the introduction sulfonic acid groups onto the polymer chains. We evaluated the polyurethanes' degradation rate, mechanical properties, hydrophilicity, antithrombogenecity, and ability to support fibroblast cell attachment and growth by comparing with polymers having a 2,2-(methylimino)diethanol (MIDE) chain extender. Mechanical testing demonstrated that the PU containing BES has tensile strengths of about 17 MPa and elongations up to 400%, about three times the strength and four times the elongation than the MIDE based PUs. The polymers showed decreased in vitro degradation rates, lower glass transition temperature (T(g)) and hydrophilicity possibly due to enhanced microphase separation. Preliminary cytocompatibility studies showed that all the PUs are non-toxic, but PU containing BES exhibited much lower cell attachment and proliferation than the MIDE chain extended polymers. An in vitro platelet adhesion assay showed lower platelet attachment on BES containing PU. Additionally, due to the existence of sulfonic acid groups, the BES extended PU became water soluble in basic condition and insoluble in acidic condition, a phenomenon that is reversible at pH value of 8.7, making this a pH sensitive polymer attractive for bioprinting applications. By adding acetic acid into an inkjet cartridge and printing it onto a PU solution with pH above 8.7, precision fabricated scaffolds can be obtained, suggesting that BES based PUs are promising candidates as synthetic inks used for customizable fabrication of tissue engineering scaffolds.     

Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering

Fibrous materials have morphological similarities to natural cartilage extracellular matrix and have been considered as candidate for bone tissue engineering scaffolds. In this study, we have evaluated a novel electrospun chitosan mat composed of oriented sub-micron fibers for its tensile property and biocompatibility with chondrocytes (cell attachment, proliferation and viability). Scanning electronic microscope images showed the fibers in the electrospun chitosan mats were indeed aligned and there was a slight cross-linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on the electrospun mat was 69% of the cells on tissue-culture polystyrene (TCP control) after three days in culture, which was slightly higher than that on the cast films (63% of the TCP control). Cells on the electrospun mat grew slowly the first week but the growth rate increased after that. By day 10, cell number on the electrospun mat was almost 82% that of TCP control, which was higher than that of cast films (56% of TCP). The electrospun chitosan mats have a higher Young's modulus (P < 0.01) than cast films and provide good chondrocyte biocompatibility. The electrospun chitosan mats, thus, have the potential to be further processed into three-dimensional scaffolds for cartilage tissue repair.     

Polypyrrole-contained electrospun conductive nanofibrous membranes for cardiac tissue engineering

Cardiac tissue engineering (TE) is one of the most promising strategies to reconstruct infarct myocardium and the major challenge is to generate a bioactive substrate with suitable chemical, biological, and conductive properties, thus mimicking the extracellular matrix (ECM) both structurally and functionally. In this study, polypyrrole/poly(ε-caprolactone)/gelatin nanofibrous scaffolds were electrospun by incorporating different concentrations of polypyrrole (PPy) to PCL/gelatin (PG) solution. Morphological, chemical, mechanical, and biodegradation properties of the electrospun nanofibers were evaluated. Our data indicated that by increasing the concentration of PPy (0-30%) in the composite, the average fiber diameters reduced from 239 ± 37 nm to 191 ± 45 nm, and the tensile modulus increased from 7.9 ± 1.6 MPa to 50.3 ± 3.3 MPa. Conductive nanofibers containing 15% PPy (PPG15) exhibited the most balanced properties of conductivity, mechanical properties, and biodegradability, matching the requirements for regeneration of cardiac tissue. The cell proliferation assay, SEM, and immunostaining analysis showed that the PPG15 scaffold promote cell attachment, proliferation, interaction, and expression of cardiac-specific proteins better than PPG30. Electrospun PPG15 conductive nanofibrous scaffold could be desirable and promising substrates suitable for the regeneration of infarct myocardium and cardiac defects.     

Biodegradable fibrous scaffolds with diverse properties by electrospinning candidates from a combinatorial macromer library

The properties of electrospun fibrous scaffolds, including degradation, mechanics and cellular interactions, are important for their use in tissue engineering applications. Although some diversity has been obtained previously in fibrous scaffolds, optimization of scaffold properties relies on iterative techniques in both polymer synthesis and processing. Here, we electrospun candidates from a combinatorial library of biodegradable and photopolymerizable poly(β-amino ester)s (PBAEs) to show that the diversity in properties found in this library is retained when processed into fibrous scaffolds. Specifically, three PBAE macromers were electrospun into scaffolds and possessed similar initial mechanical properties, but exhibited mass loss ranging from rapid (complete degradation within ～2 weeks) to moderate (complete degradation within ～3 months) to slow (only partial degradation after 3 months). These trends in mechanics and degradation mimicked what was previously observed in the bulk polymers. Although cellular adhesion was dependent on the polymer composition in films, adhesion to scaffolds that were electrospun with gelatin was similar on all formulations and controls. To further illustrate the diverse properties that are attainable in these systems, the fastest and slowest degrading polymers were electrospun together into one scaffold, but as distinct fiber populations. This dual-polymer scaffold exhibited behavior in mass loss and mechanics with time that fell between the single-polymer scaffolds. In general, this work indicates that combinatorial libraries may be an important source of information and specific polymer compositions for the fabrication of electrospun fibrous scaffolds with tunable properties.     

Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional,two-dimensional,and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering

This article reports on the experimental determination and finite element modeling of tensile and compressive mechanical properties of solid polycaprolactone (PCL) and of porous PCL scaffolds with one-dimensional, two-dimensional and three-dimensional orthogonal, periodic porous architectures produced by selective laser sintering (SLS). PCL scaffolds were built using optimum processing parameters, ensuring scaffolds with nearly full density (>95%) in the designed solid regions and with excellent geometric and dimensional control (within 3–8% of design). The tensile strength of bulk PCL ranged from 10.5 to 16.1 MPa, its modulus ranged from 343.9 to 364.3 MPa, and the tensile yield strength ranged from 8.2 to 10.1 MPa. These values are consistent with reported literature values for PCL processed through various manufacturing methods. Across porosity ranged from 56.87% to 83.3%, the tensile strength ranged from 4.5 to 1.1 MPa, the tensile modulus ranged from 140.5 to 35.5 MPa, and the yield strength ranged from 3.2 to 0.76 MPa. The compressive strength of bulk PCL was 38.7 MPa, the compressive modulus ranged from 297.8 to 317.1 MPa, and the compressive yield strength ranged from 10.3 to 12.5 MPa. Across porosity ranged from 51.1% to 80.9%, the compressive strength ranged from 10.0 to 0.6 MPa, the compressive modulus ranged from 14.9 to 12.1 MPa, and the compressive yield strength ranged from 4.25 to 0.42 MPa. These values, while being in the lower range of reported values for trabecular bone, are the highest reported for PCL scaffolds produced by SLS and are among the highest reported for similar PCL scaffolds produced through other layered manufacturing techniques. Finite element analysis showed good agreement between experimental and computed effective tensile and compressive moduli. Thus, the construction of bone tissue engineering scaffolds endowed with oriented porous architectures and with predictable mechanical properties through SLS is demonstrated.     

Collagen fibers constructed by gravity filament forming process

Fibers comprised of reconstituted type I collagen were prepared by a gravity filament forming process and crosslinked with 0.1% glutaraldehyde. These fibers have a crosslinking index of about 90% (89.89 ± 1.82%) with higher denature temperature (74.43 ± 0.08°C) as compared to that without glutaraldehyde treatment (52.1 ± 0.17°C). The ultimate tensile strength of the collagen fibers increases from 99.4 ± 12.9 to 174.4 ± 9.0 MPa after glutaraldehyde-crosslinking. L929 fibroblast cells were seeded and cultured using these newly developed collagen fibers. The fibroblast cells proliferated well and covered all surface areas of the collagen fiber. These collagen fibers have a great potential for application in 3-D tissue engineering.     

Structural characterization and cell response evaluation of electrospun PCL membranes: micrometric versus submicrometric fibers

Electrospinning is a valuable technique to fabricate fibrous scaffolds for tissue engineering. The typical nonwoven architecture allows cell adhesion and proliferation, and supports diffusion of nutrients and waste products. Poly(epsilon-caprolactone) (PCL) electrospun membranes were produced starting from 14% w/v solutions in (a) mixture 1:1 tetrahydrofuran and N,N-dimethylformamide and (b) chloroform. Matrices made up of randomly arranged uniform fibers free of beads were obtained. The average fiber diameters were (a) 0.8 +/- 0.2 microm and (b) 3.6 +/- 0.8 microm. PCL matrices showed the following tensile mechanical properties: tensile modulus (a) 5.0 +/- 0.7 MPa (b) 6.4 +/- 0.2 MPa, yield stress (a) 0.55 +/- 0.06 MPa (b) 0.43 +/- 0.02 MPa, and ultimate tensile stress (a) 1.7 +/- 0.2 MPa and (b) 0.8 +/- 0.1 MPa. The ultimate strain ranged between 300% and 400%. Cytotoxicity of electrospun membranes was continuously evaluated by means of electric cell-substrate impedance sensing technique using human umbilical vein endothelial cells (HUVEC). PCL matrices resulted free of toxic amounts of contaminants and/or process by-products. In vitro studies performed by culturing HUVEC on micrometric and submicrometric fibrous mats showed that both structures supported cell adhesion and spreading. However, cells cultured on the micrometric network showed higher vitality and improved interaction with the polymeric fibers, suggesting an increased ability to promote cell colonization.     

Synthesis and inhibition study on tripeptide inhibitor modified poly(L-lysine) dendrimers

Peptide dendrimers are attractive nonviral gene vectors. But a biological barrier for their application in gene delivery is the fast degradation catalyzed by proteasomes. Proteasome inhibitors are efficient at prohibiting the degradation of peptide nonviral vectors, thus enhancing gene transfection efficiency. In this study, N(α)-Boc-protected leucine vinyl ester proteasome inhibitor Boc-Leu-Leu-Leu-ve was synthesized by the liquid-phase method and was then immobilized onto poly(L-lysine) dendrimers. Suc-Leu-Leu-Val-Tyr-AMC was used as fluorimetric substrate and the inhibition capacity of Boc-Leu-Leu-Leu-ve immobilized onto G(3) and G(6) poly(L-lysine) dendrimers for the chymotrypsin-like activity of ACHN cell proteasome was tested. The results indicated that both Boc-Leu-Leu-Leu-ve peptide and the peptide immobilized on G(3) dendrimer showed low inhibition capacity when the concentration was below 0.2 μM. When the inhibitor concentrations were increased to 5.0 μM, however, the percentage inhibition of Boc-Leu-Leu-Leu-ve peptide and the peptide immobilized on G(3) dendrimer became about 50% and 25%, and that of peptide immobilized on the G(6) dendrimer was 7.5% only. These results indicated that dendritic structure and linker length could be the main factors affecting proteasome inhibition capacity. The cytotoxicity of the dendritic inhibitors was found to be low. Thus, whilst the synthetic production of poly(L-lysine) dendrimers immobilized with peptide inhibitors was successful and these modified dendrimers could work to inhibit proteasome activity, further studies will need to be carried out to improve inhibition capacity.     

Low-temperature electrospun silk scaffold for in vitro mucosal modeling

Electrospinning is often used to create scaffolding as a biomimetic of the extracellular matrix of tissues. A frequent limitation of this technique for three-dimensional tissue modeling is poor cell infiltration throughout the void volume of scaffolds. Here, we generated low-temperature electrospun silk scaffolds and compared these with conventional electrospun silk scaffolds in terms of mechanical properties, void volume, cell infiltration, cell viability, and potential to support mucosal models under three-dimensional culture conditions. Low-temperature electrospun silk scaffolds supported fibroblast attachment and infiltration throughout the volume of the scaffolds, while conventional electrospun scaffolds exhibited limited cell infiltration with fibroblasts attaching exclusively to the seeding surface of the scaffolds. The porosity of low-temperature electrospun scaffolds was 93% compared with 88% of conventional electrospun silk scaffolds. Uniaxial tensile testing showed a 3.5-fold reduction in strength of low-temperature electrospun silk compared with the conventional in terms of peak stress and modulus but no significant change in strain at break. Mucosal modeling with fibroblast-keratinocyte or fibroblast-carcinoma cocultures showed similar results, with cell infiltration occurring only in low-temperature electrospun scaffolds. Cell viability was confirmed using live/dead staining after 21 days in culture. Furthermore, low-temperature electrospun silk scaffolds were able to support keratinocyte differentiation, as judged by involucrin immunoreactivity. The low-temperature electrospun silk scaffold that we have developed eliminates the limitation of electrospun silk scaffolds in terms of cell infiltration and, therefore, can potentially be used for a wide range of tissue engineering purposes ranging from in vitro tissue modeling to in vivo tissue regeneration purposes.