The mechanical stress–strain properties of single electrospun collagen type I nanofibers

Knowledge of the mechanical properties of electrospun fibers is important for their successful application in tissue engineering, material composites, filtration and drug delivery. In particular, electrospun collagen has great potential for biomedical applications due to its biocompatibility and promotion of cell growth and adhesion. Using a combined atomic force microscopy (AFM)/optical microscopy technique, the single fiber mechanical properties of dry, electrospun collagen type I were determined. The fibers were electrospun from a 80 mg ml^?1 collagen solution in 1,1,1,3,3,3-hexafluro-2-propanol and collected on a striated surface suitable for lateral force manipulation by AFM. The small strain modulus, calculated from three-point bending analysis, was 2.82 GPa. The modulus showed significant softening as the strain increased. The average extensibility of the fibers was 33% of their initial length, and the average maximum stress (rupture stress) was 25 MPa. The fibers displayed significant energy loss and permanent deformations above 2% strain.     

Structure–function relationships and source-to-ground distance in electrospun polycaprolactone

The strength of electrospun scaffolds has direct relevance to their function within tissue engineering. We characterized the effects of source-to-ground distance on the mechanical properties of electrospun poly(ε-caprolactone) (PCL). Source-to-ground distances of 10, 15 and 20 cm, solids concentrations of 12 and 18 wt.% and mandrel rotation surface speeds of 0–12 m s^?1 were utilized. Tensile tests evaluated elastic modulus, tensile strength and elongation at failure. Scanning electron microscopy provided morphology and quantified fiber alignment. Increased source-to-ground distance yielded a microstructure allowing greater fiber rearrangement under load, tripling the observed tensile strength. Increases in rotational speed generally increased fiber alignment and strength at high but not low to moderate speeds. As fiber is quickly pulled out of a comparatively gentle falling process, collision with neighboring fibers moving at different speeds and in different directions can occur. The source-to-ground distance influences these collisions and thus has critical implications for microstructure and biocompatibility. In larger diameter (18 wt.% PCL), heavily point-bonded fibers (produced using a shorter, 10 cm source-to-ground distance), elongation at failure in the aligned direction increases dramatically due to severe localized necking. These specimens show only half of the tensile strength (from 2.6 to 4.5 MPa) and a dramatic increase (from 94% to 503%) in elongation at failure vs. a longer 20 cm source-to-ground distance. Strains of several hundred per cent are accompanied by periodic necking of large-diameter fibers in which microstructural failure appears to occur in a sequential manner involving an equilibrium between localized strain in the tensile direction and anisotropic point bonding that locally resists strain.     

Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration

Aligned, electrospun fibers have shown great promise in facilitating directed neurite outgrowth within cell and animal models. While electrospun fiber diameter does influence cellular behavior, it is not known how aligned, electrospun fiber scaffolds of differing diameter influence neurite outgrowth and Schwann cell (SC) migration. Thus, the goal of this study was to first create highly aligned, electrospun fiber scaffolds of varying diameter and then assess neurite and SC behavior from dorsal root ganglia (DRG) explants. Three groups of highly aligned, electrospun poly-l-lactic acid (PLLA) fibers were created (1325 + 383 nm, large diameter fibers; 759 + 179 nm, intermediate diameter fibers; and 293 + 65 nm, small diameter fibers). Embryonic stage nine (E9) chick DRG were cultured on fiber substrates for 5 days and then the explants were stained against neurofilament and S100. DAPI stain was used to assess SC migration. Neurite length and SC migration distance were determined. In general, the direction of neurite extension and SC migration were guided along the aligned fibers. On the small diameter fiber substrate, the neurite length was 42% and 36% shorter than those on the intermediate and large fiber substrates, respectively. Interestingly, SC migration did not correlate with that of neurite extension in all situations. SCs migrated equivalently with extending neurites in both the small and large diameter scaffolds, but lagged behind neurites on the intermediate diameter scaffolds. Thus, in some situations, topography alone is sufficient to guide neurites without the leading support of SCs. Scanning electron microscopy images show that neurites cover the fibers and do not reside exclusively between fibers. Further, at the interface between fibers and neurites, filopodial extensions grab and attach to nearby fibers as they extend down the fiber substrate. Overall, the results and observations suggest that fiber diameter is an important parameter to consider when constructing aligned, electrospun fibers for nerve regeneration applications.     

Characterization of dielectrophoresis-aligned nanofibrous silk fibroin–chitosan scaffold and its interactions with endothelial cells for tissue engineering applications

Aligned three-dimensional nanofibrous silk fibroin–chitosan (eSFCS) scaffolds were fabricated using dielectrophoresis (DEP) by investigating the effects of alternating current frequency, the presence of ions, the SF:CS ratio and the post-DEP freezing temperature. Scaffolds were characterized with polarized light microscopy to analyze SF polymer chain alignment, atomic force microscopy (AFM) to measure the apparent elastic modulus, and scanning electron microscopy and AFM to analyze scaffold topography. The interaction of human umbilical vein endothelial cells (HUVECs) with eSFCS scaffolds was assessed using immunostaining to assess cell patterning and AFM to measure the apparent elastic modulus of the cells. The eSFCS (50:50) samples prepared at 10 MHz with NaCl had the highest percentage of aligned area as compared to other conditions. As DEP frequency increased from 100 kHz to 10 MHz, fibril sizes decreased significantly. eSFCS (50:50) scaffolds fabricated at 10 MHz in the presence of 5 mM NaCl had a fibril size of 77.96 ± 4.69 nm and an apparent elastic modulus of 39.9 ± 22.4 kPa. HUVECs on eSFCS scaffolds formed aligned and branched capillary-like vascular structures. The elastic modulus of HUVEC cultured on eSFCS was 6.36 ± 2.37 kPa. DEP is a potential tool for fabrication of SFCS scaffolds with aligned nanofibrous structures that can guide vasculature in tissue engineering and repair.     

Poroelastic response of articular cartilage by nanoindentation creep tests at different characteristic lengths

Nanoindentation is an experimental technique which is attracting increasing interests for the mechanical characterization of articular cartilage. In particular, time dependent mechanical responses due to fluid flow through the porous matrix can be quantitatively investigated by nanoindentation experiments at different penetration depths and/or by using different probe sizes. The aim of this paper is to provide a framework for the quantitative interpretation of the poroelastic response of articular cartilage subjected to creep nanoindentation tests. To this purpose, multiload creep tests using spherical indenters have been carried out on saturated samples of mature bovine articular cartilage achieving two main quantitative results. First, the dependence of indentation modulus in the drained state (at equilibrium) on the tip radius: a value of 500 kPa has been found using the large tip (400 μm radius) and of 1.7 MPa using the smaller one (25 μm). Secon, the permeability at microscopic scale was estimated at values ranging from 4.5 × 10⁻¹⁶ m⁴/N s to 0.1 × 10⁻¹⁶ m⁴/N s, from low to high equivalent deformation. Consistently with a poroelastic behavior, the size-dependent response of the indenter displacement disappears when characteristic size and permeability are accounted for. For comparison purposes, the same protocol was applied to intrinsically viscoelastic homogeneous samples of polydimethylsiloxane (PDMS): both indentation modulus and time response have been found size-independent.     

Effect of addition of hyaluronic acids on the osteoconductivity and biodegradability of synthetic octacalcium phosphate

The present study was designed to investigate whether three sodium hyaluronic acid (HyA) medical products, Artz®, Suvenyl® and a chemically modified derivative of sodium HyA Synvisc®, can be used as suitable vehicles for an osteoconductive octacalcium phosphate (OCP). OCP granules (300–500 μm diameter) were mixed with these sodium HyAs with molecular weights of 90 × 10⁴ (Artz®), 190 × 10⁴ (Suvenyl®) and 600 × 10⁴ (Synvisc®) (referred to as HyA90, HyA190 and HyA600, respectively). OCP–HyA composites were injected using a syringe into a polytetrafluoroethylene ring, placed on the subperiosteal region of mouse calvaria for 3 and 6 weeks, and then bone formation was assessed by histomorphometry. The capacity of the HyAs for osteoclast formation from RAW264 cells with RANKL was examined by TRAP staining in vitro. Bone formation was enhanced by the OCP composites with HyA90 and HyA600, compared to OCP alone, through enhanced osteoclastic resorption of OCP. HyA90 and HyA600 facilitated in vitro osteoclast formation. The results suggest that the osteoconductive property of OCP was accelerated by the HyAs-associated osteoclastic resorption of OCP, and therefore that HyA/OCP composites are attractive bone substitutes which are injectable and bioactive materials.     

Cytocompatible cross-linking of electrospun zein fibers for the development of water-stable tissue engineering scaffolds

This paper reports a new method of cross-linking electrospun zein fibers using citric acid as a non-toxic cross-linker to enhance the water stability and cytocompatibility of zein fibers for tissue engineering and other medical applications. The electrospun structure has many advantages over other types of structures and protein-based biomaterials possess unique properties preferred for tissue engineering and other medical applications. However, ultrafine fiber matrices developed from proteins have poor mechanical properties and morphological stability in the aqueous environments required for medical applications. Efforts have been made to improve the water stability of electrospun protein scaffolds using cross-linking and other approaches, but the current methods have major limitations, such as cytotoxicity and low efficiency. In this research electrospun zein fibers were cross-linked with citric acid without using any toxic catalysts. The stability of the cross-linked fibers in phosphate-buffered saline and their ability to support the attachment, spreading and proliferation of mouse fibroblast cells were studied. The cross-linked electrospun fibers retained their ultrafine fibrous structure even after immersion in PBS at 37 °C for up to 15 days. Citric acid cross-linked electrospun zein scaffolds showed better attachment, spreading and proliferation of fibroblast cells than uncross-linked electrospun zein fibers, cross-linked zein films and electrospun polylactide fibers.     

Electrically conductive nanofibers with highly oriented structures and their potential application in skeletal muscle tissue engineering

Recent trends in scaffold design have focused on materials that can provide appropriate guidance cues for particular cell types to modulate cell behavior. In this study highly aligned and electrically conductive nanofibers that can simultaneously provide topographical and electrical cues for cells were developed. Thereafter their potential to serve as functional scaffolds for skeletal muscle tissue engineering was investigated. Well-ordered nanofibers, composed of polyaniline (PANi) and poly(ε-caprolactone) (PCL), were electrospun by introducing an external magnetic field in the collector region. Incorporation of PANi into PCL fibers significantly increased the electrical conductivity from a non-detectable level for the pure PCL fibers to 63.6 ± 6.6 mS cm^?1 for the fibers containing 3 wt.% PANi (PCL/PANi-3). To investigate the synergistic effects of topographical and electrical cues using the electrospun scaffolds on skeletal myoblast differentiation, mouse C2C12 myoblasts were cultured on random PCL (R-PCL), aligned PCL (A-PCL), random PCL/PANi-3 (R-PCL/PANi) and aligned PCL/PANi-3 (A-PCL/PANi) nanofibers. Our results showed that the aligned nanofibers (A-PCL and A-PCL/PANi) could guide myoblast orientation and promote myotube formation (i.e. approximately 40% and 80% increases in myotube numbers) compared with R-PCL scaffolds. In addition, electrically conductive A-PCL/PANi nanofibers further enhanced myotube maturation (i.e. approximately 30% and 23% or 15% and 18% increases in the fusion and maturation indices) compared with non-conductive A-PCL scaffolds or R-PCL/PANi. These results demonstrated that a combined effect of both guidance cues was more effective than an individual cue, suggesting a potential use of A-PCL/PANi nanofibers for skeletal muscle regeneration.     

A small diameter,fibrous vascular conduit generated from a poly(ester urethane)urea and phospholipid polymer blend

The thrombotic and hyperplastic limitations associated with synthetic small diameter vascular grafts have generated sustained interest in finding a tissue engineering solution for autologous vascular segment generation in situ. One approach is to place a biodegradable scaffold at the site that would provide acute mechanical support while vascular tissue develops. To generate a scaffold that possessed both non-thrombogenic character and mechanical properties appropriate for vascular tissue, a biodegradable poly(ester urethane)urea (PEUU) and non-thrombogenic bioinspired phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-methacryloyloxyethyl butylurethane) (PMBU) were blended at PMBU weight fractions of 0–15% and electrospun to create fibrous scaffolds. The composite scaffolds were flexible with breaking strains exceeding 300%, tensile strengths of 7–10 MPa and compliances of 2.9–4.4 × 10^?4 mmHg^?1. In vitro platelet deposition on the scaffold surfaces significantly decreased with increasing PMBU content. Rat smooth muscle cell proliferation was also inhibited on PEUU/PMBU blended scaffolds with greater inhibition at higher PMBU content. Fibrous vascular conduits (1.3 mm inner diameter) implanted in the rat abdominal aorta for 8 weeks showed greater patency for grafts with 15% PMBU blending versus PEUU without PMBU (67% versus 40%). A thin neo-intimal layer with endothelial coverage and good anastomotic tissue integration was seen for the PEUU/PMBU vascular grafts. These results are encouraging for further evaluation of this technique in larger diameter applications for longer implant periods.     

Elastin-like recombinamer catalyst-free click gels: Characterization of poroelastic and intrinsic viscoelastic properties

Elastin-like recombinamer catalyst-free click gels (ELR-CFCGs) have been prepared and characterized by modifying both a structural ELR (VKVx24) and a biofunctionalized ELR-bearing RGD cell-adhesion sequence (HRGD6) to bear the reactive groups needed to form hydrogels via a click reaction. Prior to formation of the ELR-CFCGs, azide-bearing and cyclooctyne-modified ELRs were also synthesized. Subsequent covalent crosslinking was based on the reaction between these azide and cyclooctyne groups, which takes place under physiological conditions and without the need for a catalyst. The correlation among SEM micrographs, porosity, swelling ratio, and rheological measurements have been carried out. The storage and loss moduli at 1 Hz are in the range 1–10 kPa and 100–1000 Pa, respectively. The linear dependence of |G1| on f^½ and the peak value of tan δ were considered to be consistent with a poroelastic mechanism dominating the frequency range 0.3–70 Hz. The discrete relaxation spectrum was obtained from stress relaxation measurements (t > 5 s). The good fit of the relaxation modulus to decrease exponential functions suggests that an intrinsic viscoelastic mechanism dominates the transients. Several recombinamer concentrations and temperatures were tested to obtain gels with fully tuneable properties that could find applications in the biomedical field.     

Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.     

Nanofiber size-dependent sensitivity of fibroblast directionality to the methodology for scaffold alignment

The sensitivity of fibroblast guidance on directional cues provided by aligned nanofibers is studied for scaffolds of successively smaller fiber sizes (740 ± 280, 245 ± 85, 140 ± 40, and 80 ± 10 nm) fabricated using mandrel and electrical alignment methodologies for electrospun nanofibers (～10° angular deviation (AD)), as well as nanoimprint methodologies for perfectly aligned fibers (0° AD). On aligned scaffolds of large fibers (～740 nm) cell directionality closely follows the underlying fibers, irrespective of the alignment method. However, on mandrel aligned scaffolds of successively smaller fibers the cell directionality exhibits greater deviations from the underlying fiber alignment due to the higher likelihood of interaction of cell lamellipodia with multiple, rather than single, nanofibers. Using electrically aligned scaffolds, fibroblast directionality deviations can be maintained in the range of nanofiber alignment deviation for fiber sizes down to ～100 nm. This improvement in cell guidance is attributed to molecular scale directional adhesion cues for cell receptors, which occur within electrically aligned scaffolds due to fiber polarization parallel to the geometric alignment axis of the nanofiber under the modified electric field during electrospinning. While fibroblast directionality is similar on electrically aligned vs. nanoimprinted scaffolds for fiber sizes >100 nm, cell directionality is influenced more strongly by the perfect alignment cues of the latter on ～100 nm fiber scaffolds. The scaffold alignment methodology is hence highly significant, especially for tissue engineering applications requiring sub-100 nm aligned fibers.     

Primary human chondrocyte extracellular matrix formation and phenotype maintenance using RGD-derivatized PEGDM hydrogels possessing a continuous Young’s modulus gradient

Efficient ex vivo methods for expanding primary human chondrocytes while maintaining the phenotype is critical to advancing the sourcing of autologous cells for tissue engineering applications. While there has been significant research reported in the literature, systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using limited cell numbers. Functionalized hydrogels possessing continuous variations in physico-chemical properties are, therefore, an efficient three-dimensional platform for studying several properties simultaneously. Herein we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system with a modulus gradient (～27,000–3800 Pa) containing a uniform concentration of arginine–glycine–aspartic acid (RGD) peptide to enhance cell adhesion in order to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance, and extracellular matrix (ECM) production with hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with a higher storage modulus (>13,100 Pa), while regions with a lower storage modulus maintained their cell number and phenotype. Over 3 weeks culture hydrogel regions possessing a lower Young's modulus experienced an increase in ECM content (～200%) compared with regions with a higher storage modulus. Variations in the amount and organization of the cytoskeletal markers actin and vinculin were observed within the modulus gradient, which are indicative of differences in chondrogenic phenotype maintenance and ECM expression. Thus scaffold mechanical properties have a significant impact in modulating human osteoarthritic chondrocyte behavior and tissue formation.     

Long-term drug release from electrospun fibers for in vivo inflammation prevention in the prevention of peritendinous adhesions

Physical barriers such as electrospun fibrous membranes are potentially useful in preventing peritendinous adhesions after surgery. However, inflammatory responses caused by degradation of barrier materials remain a major challenge. This study aimed to fabricate electrospun composite fibrous membranes based on drug-loaded modified mesoporous silica (MMS) and poly (l-lactic acid) (PLLA). Using a co-solvent-based electrospinning method ibuprofen (IBU)-loaded MMS was successfully and uniformly encapsulated in the PLLA fibers. The electrospun PLLA–MMS–IBU composite fibrous membranes showed significantly lower initial burst release (6% release in the first 12 h) compared with that of electrospun PLLA–IBU fibrous membranes (46% release in the first 12 h) in in vitro release tests. Moreover, the release from PLLA–MMS–IBU was also for significantly longer than that from PLLA–IBU (100 vs. 20 days). In animal studies both PLLA–IBU and PLLA–MMS–IBU showed improved anti-adhesion properties and anti-inflammatory effects compared with PLLA fibrous membrane alone 4 weeks after implantation. Further, animals implanted with PLLA–MMS–IBU for 8 weeks showed the lowest inflammation and best recovery compared with those implanted with PLLA–IBU and PLLA, most likely as a result of its long-term IBU release profile. Therefore, this study provides a platform technique for fabricating fibrous membranes with long-term sustained drug release characteristics which may function as a novel carrier for long-term anti-inflammation and anti-adhesion to prevent peritendinous adhesions.     

Acellular vascular grafts generated from collagen and elastin analogs

Tissue-engineered vascular grafts require long fabrication times, in part due to the requirement of cells from a variety of cell sources to produce a robust, load-bearing extracellular matrix. Herein, we propose a design strategy for the fabrication of tubular conduits comprising collagen fiber networks and elastin-like protein polymers to mimic native tissue structure and function. Dense fibrillar collagen networks exhibited an ultimate tensile strength (UTS) of 0.71 ± 0.06 MPa, strain to failure of 37.1 ± 2.2% and Young’s modulus of 2.09 ± 0.42 MPa, comparing favorably to a UTS and a Young’s modulus for native blood vessels of 1.4–11.1 MPa and 1.5 ± 0.3 MPa, respectively. Resilience, a measure of recovered energy during unloading of matrices, demonstrated that 58.9 ± 4.4% of the energy was recovered during loading–unloading cycles. Rapid fabrication of multilayer tubular conduits with maintenance of native collagen ultrastructure was achieved with internal diameters ranging between 1 and 4 mm. Compliance and burst pressures exceeded 2.7 ± 0.3%/100 mmHg and 830 ± 131 mmHg, respectively, with a significant reduction in observed platelet adherence as compared to expanded polytetrafluoroethylene (ePTFE; 6.8 ± 0.05 × 10⁵ vs. 62 ± 0.05 × 10⁵ platelets mm^–2, p < 0.01). Using a rat aortic interposition model, early in vivo responses were evaluated at 2 weeks via Doppler ultrasound and CT angiography with immunohistochemistry confirming a limited early inflammatory response (n = 8). Engineered collagen–elastin composites represent a promising strategy for fabricating synthetic tissues with defined extracellular matrix content, composition and architecture.     

The role of doxorubicin in non-viral gene transfer in the lung

Proteasome inhibitors have been shown to increase adeno-associated virus (AAV)-mediated transduction in vitro and in vivo. To assess if proteasome inhibitors also increase lipid-mediated gene transfer with relevance to cystic fibrosis (CF), we first assessed the effects of doxorubicin and N-acetyl-l-leucinyl-l-leucinal-l-norleucinal in non-CF (A549) and CF (CFTE29o-) airway epithelial cell lines. CFTE29o- cells did not show a response to Dox or LLnL; however, gene transfer in A549 cells increased in a dose-related fashion (p < 0.05), up to approximately 20-fold respectively at the optimal dose (no treatment: 9.3 × 10⁴ ± 1.5 × 10³, Dox: 1.6 × 10⁶ ± 2.6 × 10⁵, LLnL: 1.9 × 10⁶ ± 3.2 × 10⁵ RLU/mg protein). As Dox is used clinically in cancer chemotherapy we next assessed the effect of this drug on non-viral lung gene transfer in vivo. CF knockout mice were injected intraperitoneally (IP) with Dox (25–100 mg/kg) immediately before nebulisation with plasmid DNA carrying a luciferase reporter gene under the control of a CMV promoter/enhancer (pCIKLux) complexed to the cationic lipid GL67A. Dox also significantly (p < 0.05) increased expression of a plasmid regulated by an elongation factor 1α promoter (hCEFI) approximately 8-fold. Although administration of Dox before lung gene transfer may not be a clinically viable option, understanding how Dox increases lung gene expression may help to shed light on intracellular bottle-necks to gene transfer, and may help to identify other adjuncts that may be more appropriate for use in man.     

A bilayered elastomeric scaffold for tissue engineering of small diameter vascular grafts

A major barrier to the development of a clinically useful small diameter tissue engineered vascular graft (TEVG) is the scaffold component. Scaffold requirements include matching the mechanical and structural properties with those of native vessels and optimizing the microenvironment to foster cell integration, adhesion and growth. We have developed a small diameter, bilayered, biodegradable, elastomeric scaffold based on a synthetic, biodegradable elastomer. The scaffold incorporates a highly porous inner layer, allowing cell integration and growth, and an external, fibrous reinforcing layer deposited by electrospinning. Scaffold morphology and mechanical properties were assessed, quantified and compared with those of native vessels. Scaffolds were then seeded with adult stem cells using a rotational vacuum seeding device to obtain a TEVG, cultured under dynamic conditions for 7 days and evaluated for cellularity. The scaffold showed firm integration of the two polymeric layers with no delamination. Mechanical properties were physiologically consistent, showing anisotropy, an elastic modulus (1.4 ± 0.4 MPa) and an ultimate tensile stress (8.3 ± 1.7 MPa) comparable with native vessels. The compliance and suture retention forces were 4.6 ± 0.5 × 10^?4 mmHg^?1 and 3.4 ± 0.3 N, respectively. Seeding resulted in a rapid, uniform, bulk integration of cells, with a seeding efficiency of 92 ± 1%. The scaffolds maintained a high level of cellular density throughout dynamic culture. This approach, combining artery-like mechanical properties and a rapid and efficient cellularization, might contribute to the future clinical translation of TEVGs.     

Adhesion,migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix

Silk fibroin–chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 ± 4.8% at 15 min, increasing to 92.8 ± 1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47 ± 2.7 μm h^?1 and a mean persistence time of 41.4 ± 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC–ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p < 0.0001) for ASCs seeded on SFCS (69.0 ± 9.0 kPa) than on glass (6.1 ± 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher (p < 0.05) for ASC seeded on SFCS (0.42 ± 0.02 fibers μm^?1) than on glass-seeded controls (0.24 ± 0.03 fibers μm^?1). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell–cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.     

Polyurethane-based leukocyte-inspired biocidal materials

During the neutrophil respiratory burst myeloperoxidase uses hydrogen peroxide and chloride ion to generate hypochlorite which kills pathogens. Synthetic antimicrobial materials based on this chemistry are described herein. The oxidizing enzymes glucose oxidase (GOX) and horseradish peroxidase (HRP) catalyze two reactions in tandem using glucose, hydrogen peroxide and sodium halide (iodide or bromide). The final product of these two consecutive enzymatic reactions is either iodine or bromine. HRP, acting as haloperoxidase, utilizes the H₂O₂ generated by GOX to oxidize halide ions into free halogens. Typically, 15 units/ml HRP and 25 units/ml GOX reacted with 0.8 mm NaI and 5 mm glucose to generate 5–7 ppm free iodine within 30 min. Medical grade polyurethane ChronoFlex AR (CF) was electrospun together with GOX and HRP. The electrospun fibers were collected as a uniform, water-insoluble, flexible elastomeric matrix with an average fiber diameter of 1 ± 0.2 μm. Biocidal activity of CF/enzyme fibers resulted in >6-log unit reduction of both Escherichia coli and Staphylococcus aureus challenges. A time-course of biocidal activity displayed a 3–4 log reduction of E. coli and S. aureus within the first 5 min and complete kill (>6 logs) within 15 min. A dose–response study of fiber weight (0.5–30 mg/ml) exhibited complete kill of E. coli (>6 logs) and at least 99.99% S. aureus kill (>4 logs) with as little as 1 mg fiber. The fibers were reusable with slightly less activity on the second use and significant activity after continuous soaking in buffer for up to 7 days. Electrospun CF/GOX/HRP fibers adhered to a thin film with embedded NaI and glucose caused a complete kill of E. coli (>7-log units) and MRSA (6-log unit reduction) within 1 h at 37 °C.     

T cell receptor diversity in the human thymus

A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCRα and TCRβ gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3 × 10⁶ unique TCRβ and 0.5 × 10⁶ TCRα sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3 × 10⁶ unique TCRβ sequences and 3.7 × 10⁶ TCRα sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70 × 10⁶ unique TCRβ sequences and 60 to 100 × 10⁶ TCRα sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times.