Uni-Directionally Oriented Fibro-Porous PLLA/Fibrin Bio-Hybrid Scaffold: Mechano-Morphological and Cell Studies.

In this study, we report a biohybrid oriented fibrous scaffold based on nanofibers of poly(l-lactic acid) (PLLA)/fibrin produced by electrospinning and subsequent post-treatment. Induced hydrolytic degradation of the fibers in 0.25 M NaOH solution for various time periods followed by the immobilization of fibrin on the hydrolyzed fiber surfaces was shown to significantly affect the mechanical properties, with the tensile strength (40.6 MPa ± 1.3) and strain at failure (38% ± 4.5) attaining a value within the range of human ligaments and ligament-replacement grafts. Unidirectional electrospinning with a mandrel rotational velocity of 26.4 m/s produced highly aligned fibers with an average diameter of 760 ± 96 nm. After a 20-min hydrolysis treatment in NaOH solution, this was further reduced to an average of 457 ± 89 nm, which is within the range of collagen bundles found in ligament tissue. Based on the results presented herein, the authors hypothesize that a combination of fiber orientation/alignment and immobilization of fibrin can result in the mechanical and morphological modification of PLLA tissue scaffolds for ligament-replacement grafts. Further, it was found that treatment with NaOH enhanced the osteogenic differentiation of hMSCs and the additional inclusion of fibrin further enhanced osteogenic differentiation, as demonstrated by decreased proliferative rates and increased ALP activity.

A novel device to quantify the mechanical properties of electrospun nanofibers.

Mechanical deformation of cell-seeded electrospun matrices plays an important role in cell signaling. However, electrospun biomaterials have inherently complex geometries due to the random deposition of fibers during the electrospinning process. This confounds attempts at quantifying strains exerted on adherent cells during electrospun matrix deformation. We have developed a novel mechanical test platform that allows deposition and tensile testing of electrospun fibers in a highly parallel arrangement to simplify mechanical analysis of the fibers alone and with adherent cells. The device is capable of optically recording fiber strain in a cell culture environment. Here we report on the mechanical and viscoelastic properties of highly parallel electrospun poly(ε-caprolactone) fibers. Force-strain data derived from this device will drive the development of cellular mechanotransduction studies as well as the customization of electrospun matrices for specific engineered tissue applications.

Electrospinning of Biosyn(®)-based tubular conduits: structural, morphological, and mechanical characterizations.

Electrospinning has garnered special attention recently due to its flexibility in producing extracellular matrix-like non-woven fibers on the nano-/microscale and its ability to easily fabricate seamless three-dimensional tubular conduits. Biosyn(®), a bioabsorbable co-polymer of glycolide, dioxanone, and trimethylene carbonate, was successfully electrospun into tubular conduits for the first time for soft tissue applications. At an electric field strength of 1 kV cm(-1) over a distance of 22 cm (between the Taylor cone and the collector) and at a flow rate of 1.5 ml h(-1) different concentrations of Biosyn/HFP solutions (5-20%) were spun into nanofibers and collected on a rotating mandrel (diameter 4 mm) at 300 and 3125 r.p.m. Scaffolds were characterized for structural and morphological properties by differential scanning calorimetry and scanning electron microscopy and for mechanical properties by uniaxial tensile testing (in both the circumferential and longitudinal directions). Biosyn(®) tubular scaffolds (internal diameter 4 mm) have been shown to exhibit a highly porous structure (60-70%) with a randomly oriented nanofibrous morphology. The polymer solution concentration directly affects spinnability and fiber diameter. At very low concentrations (≤5%) droplets were formed due to electrospraying. However, as the concentration increased the solution viscosity increased and a "bead-on-string" morphology was observed at 10%. A further increase in concentration to 13% resulted in "bead-free" nanofibers with diameters in the range 500-700 nm. Higher concentrations (≥20%) resulted in the formation of microfibers (1-1.4 µm diameter) due to increased solution viscosity. It has also been noted that increasing the mandrel speed from 300 to 3125 r.p.m. produced a reduction in the fiber size. Uniaxial tensile testing of the scaffolds revealed the mechanical properties to be attractive for soft tissue applications. As the fiber diameters of the scaffold decrease the tensile strength and modulus increase. There is no drastic change in tensile properties of the scaffolds tested under hydrated and dry conditions. However, a detailed study on the biodegradation and biomechanics of electrospun Biosyn conduits under physiological pressure conditions is required to ensure potential application as a vascular graft.

A hybrid biomimetic nanomatrix composed of electrospun polycaprolactone and bioactive peptide amphiphiles for cardiovascular implants.

Current cardiovascular therapies are limited by the loss of endothelium, restenosis and thrombosis. The goal of this study was to develop a biomimetic hybrid nanomatrix that combined the unique properties of electrospun polycaprolactone (ePCL) nanofibers with self-assembled peptide amphiphiles (PAs). ePCL nanofibers have interconnected nanoporous structures, but are hampered by a lack of surface bioactivity to control cellular behavior. It has been hypothesized that PAs could self-assemble onto the surface of ePCL nanofibers and endow them with the characteristic properties of native endothelium. The PAs, which comprised hydrophobic alkyl tails attached to functional hydrophilic peptide sequences, contained enzyme-mediated degradable sites coupled to either endothelial cell-adhesive ligands (YIGSR) or polylysine (KKKKK) nitric oxide (NO) donors. Two different PAs (PA-YIGSR and PA-KKKKK) were successfully synthesized and mixed in a 90:10 (YK) ratio to obtain PA-YK. PA-YK was reacted with pure NO to develop PA-YK-NO, which was then self-assembled onto ePCL nanofibers to generate a hybrid nanomatrix, ePCL-PA-YK-NO. Uniform coating of self-assembled PA nanofibers on ePCL was confirmed by transmission electron microscopy. Successful NO release from ePCL-PA-YK-NO was observed. ePCL-YK and ePCL-PA-YK-NO showed significantly increased adhesion of human umbilical vein endothelial cells (HUVECs). ePCL-PA-YK-NO also showed significantly increased proliferation of HUVECs and reduced smooth muscle cell proliferation. ePCL-PA-YK-NO also displayed significantly reduced platelet adhesion compared with ePCL, ePCL-PA-YK and a collagen control. These results indicate that this hybrid nanomatrix has great potential application in cardiovascular implants.

Cell infiltration and growth in a low density, uncompressed three-dimensional electrospun nanofibrous scaffold.

A limiting factor of traditional electrospinning is that the electrospun scaffolds consist entirely of tightly packed nanofiber layers that only provide a superficial porous structure due to the sheet-like assembly process. This unavoidable characteristic hinders cell infiltration and growth throughout the nanofibrous scaffolds. Numerous strategies have been tried to overcome this challenge, including the incorporation of nanoparticles, using larger microfibers, or removing embedded salt or water-soluble fibers to increase porosity. However, these methods still produce sheet-like nanofibrous scaffolds, failing to create a porous three-dimensional scaffold with good structural integrity. Thus, we have developed a three-dimensional cotton ball-like electrospun scaffold that consists of an accumulation of nanofibers in a low density and uncompressed manner. Instead of a traditional flat-plate collector, a grounded spherical dish and an array of needle-like probes were used to create a Focused, Low density, Uncompressed nanoFiber (FLUF) mesh scaffold. Scanning electron microscopy showed that the cotton ball-like scaffold consisted of electrospun nanofibers with a similar diameter but larger pores and less-dense structure compared to the traditional electrospun scaffolds. In addition, laser confocal microscopy demonstrated an open porosity and loosely packed structure throughout the depth of the cotton ball-like scaffold, contrasting the superficially porous and tightly packed structure of the traditional electrospun scaffold. Cells seeded on the cotton ball-like scaffold infiltrated into the scaffold after 7 days of growth, compared to no penetrating growth for the traditional electrospun scaffold. Quantitative analysis showed approximately a 40% higher growth rate for cells on the cotton ball-like scaffold over a 7 day period, possibly due to the increased space for in-growth within the three-dimensional scaffolds. Overall, this method assembles a nanofibrous scaffold that is more advantageous for highly porous interconnectivity and demonstrates great potential for tackling current challenges of electrospun scaffolds.

Acellular dermal matrix graft: synergistic effect of rehydration and natural crosslinking on mechanical properties.

This investigation studied how the incorporation of a natural crosslinking agent, genipin (Gp), into the AlloDerm® (AD) rehydration protocol affects the biomechanical properties and the stability of the collagenous matrix. AD is a minimally processed, noncrosslinked, freeze-dried collagen-based graft. Samples were immersed in a saline solution for 5 min and then randomly assigned for further rehydration (30 min) into three groups, according to the crosslinking agent: G1-control (saline), G2-1 wt % genipin, and G3-1 wt % glutaraldehyde. Gp crosslinking for a prolonged time of 6 h (G4) was also investigated. After washing (5 min), samples were mechanically tested wet in tension. G2 demonstrated a significantly higher ultimate tensile strength (UTS) and E relative to G1. However, G3 did not show a noteworthy increase in these properties. A significant enhancement in UTS was found when Gp exposure time was increased from 30 min to 6 h. FT-IR revealed a protein backbone with no significant peak shifting for all samples due to crosslinking. However, a considerable decrease in -NH(2) peak intensity occurred due to crosslinking reactions. Additionally, DSC analyses indicated an important shift in the denaturation temperature for crosslinked samples. SEM micrographs revealed no alterations in the native fibrous morphology after crosslinking. Simultaneous genipin incorporation during the rehydration protocol of AlloDerm significantly enhances its biomechanical properties.

Freeze-dried acellular dermal matrix graft: effects of rehydration on physical, chemical, and mechanical properties.

OBJECTIVES: To test the effect of rehydration time over the range prescribed in the manufacturer's protocol on (1) the biomechanical properties and on (2) the recovery and stabilization of the collagenous matrix of AlloDerm. METHODS: A sterile dish containing warm saline solution was prepared, and samples rehydrated for 5 min. Subsequently, three other dishes with the solution were prepared and samples assigned into three groups according to the total rehydration time: 10 min (G1), 20 min (G2), and 40 min (G3). Uni-axial tensile testing was used to assess the biomechanical properties of the different groups and the control (dry condition). Physico-chemical properties were examined by Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC) as a function of rehydration time. RESULTS: ANOVA revealed a significant change in tensile strength (p=0.0269) and in elastic modulus (p=0.0306) for AlloDerm following different rehydration times. The lowest tensile strength was in the dry condition, whereas the highest was achieved after a 40 min rehydration. The shortest rehydration periods did not result in a statistically significant (p>0.05) change in elastic modulus. However, after 40 min the elastic modulus increased significantly when compared to the shortest periods. FT-IR confirmed the protein backbone recovery of the graft matrix after rehydration. DSC scans of rehydrated samples showed visible shifts in the denaturation temperature to higher values compared to as-received sample (dry) suggesting stronger polymer-water bridge formation, supporting the increase in the biomechanical properties. SIGNIFICANCE: The current study suggests that there are major changes on the biomechanical properties of the collagenous graft as rehydration time increases, which were also structurally confirmed by the physico-chemical analyses. Clinicians must be aware that the rehydration times of the manufacturer's protocol result in a significant range in mechanical and physico-chemical properties. Therefore, a rehydration time of at least 20 min guarantees not only better handling and mechanical properties but, most importantly, supplies a material that closely resembles the natural tissue.

Aligned PLGA/HA nanofibrous nanocomposite scaffolds for bone tissue engineering.

Aligned nanofibrous scaffolds based on poly(d,l-lactide-co-glycolide) (PLGA) and nano-hydroxyapatite (nano-HA) were synthesized by electrospinning for bone tissue engineering. Morphological characterization using scanning electron microscopy showed that the addition of different amounts of nano-HA (1, 5, 10 and 20wt.%) increased the average fiber diameter from 300nm (neat PLGA) to 700nm (20% nano-HA). At higher concentrations (>or=10%), agglomeration of HA was observed and this had a marked effect at 20% concentration whereby the presence of nano-HA resulted in fiber breaking. Thermal characterization showed that the fast processing of electrospinning locked in the amorphous character of PLGA; this resulted in a decrease in the glass transition temperature of the scaffolds. Furthermore, an increase in the glass transition temperature was observed with increasing nano-HA concentration. The dynamic mechanical behavior of the scaffolds reflected the morphological observation, whereby nano-HA acted as reinforcements at lower concentrations (1% and 5%) but acted as defects at higher concentrations (10% and 20%). The storage modulus value of the scaffolds increased from 441MPa for neat PLGA to 724MPa for 5% nano-HA; however, further increasing the concentration leads to a decrease in storage modulus, to 371MPa for 20% nano-HA. Degradation characteristics showed that hydrophilic nano-HA influenced phosphate-buffered saline uptake and mass loss. The mechanical behavior showed a sinusoidal trend with a slight decrease in modulus by week 1 due to the plasticizing effect of the medium followed by an increase due to shrinkage, and a subsequent drop by week 6 due to degradation.

Nanostructured biocomposite scaffolds based on collagen coelectrospun with nanohydroxyapatite.

Nanofibrous biocomposite scaffolds of type I collagen and nanohydroxyapatite (nanoHA) of varying compositions (wt %) were prepared by electrostatic cospinning. The scaffolds were characterized for structure and morphology by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD) techniques. The scaffolds have a porous nanofibrous morphology with random fibers in the range of 500-700 nm diameters, depending on the composition. FT-IR and XRD showed the presence of nanoHA in the fibers. The surface roughness and diameter of the fibers increased with the presence of nanoHA in biocomposite fiber as evident from AFM images. Tensile testing and nanoindendation were used for the mechanical characterization. The pure collagen fibrous matrix (without nanoHA) showed a tensile strength of 1.68 +/- 0.10 MPa and a modulus of 6.21 +/- 0.8 MPa with a strain to failure value of 55 +/- 10%. As the nanoHA content in the randomly oriented collagen nanofibers increased to 10%, the ultimate strength increased to 5 +/- 0.5 MPa and the modulus increased to 230 +/- 30 MPa. The increase in tensile modulus may be attributed to an increase in rigidity over the pure polymer when the hydroxyapatite is added and/or the resulting strong adhesion between the two materials. The vapor phase chemical crosslinking of collagens using glutaraldehyde further increased the mechanical properties as evident from nanoindentation results. A combination of nanofibrous collagen and nanohydroxyapatite that mimics the nanoscale features of the extra cellular matrix could be promising for application as scaffolds for hard tissue regeneration, especially in low or nonload bearing areas.

Mechano-morphological studies of aligned nanofibrous scaffolds of polycaprolactone fabricated by electrospinning.

Mechanical and morphological studies of aligned nanofibrous meshes of poly(epsilon-caprolactone) (PCL) fabricated by electrospinning at different collector rotation speeds (0, 3000 and 6000 rpm) for application as bone tissue scaffolds are reported. SEM, XRD and DSC analyses were used for the morphological characterization of the nanofibers. Scaffolds have a nanofibrous morphology with fibers (majority) having a diameter in the range of 550-350 nm (depending on fiber uptake rates) and an interconnected pore structure. With the increase of collector rotation speed, the nanofibers become more aligned and oriented perpendicular to the axis of rotation. Deposition of fibers at higher fiber collection speeds has a profound effect on the morphology and mechanical properties of individual fibers and also the bulk fibrous meshes. Nanoindentation was used for the measurement of nanoscopic mechanical properties of individual fibers of the scaffolds. The hardness and Young's modulus of aligned fibers measured by nanoindentation decreased with collector rotation speeds. This reveals the difference in the local microscopic structure of the fibers deposited at higher speeds. The sequence of nanoscopic mechanical properties (hardness and modulus) of three fibers is PCL at 0 rpm > PCL at 3000 rpm > PCL at 6000 rpm. This may be explained due to the decrease in crystallinity of fibers at higher uptake rates. However, uni-axial tensile properties of (bulk) scaffolds (tensile strength and modulus) increased with increasing collector rotation speed. The average ultimate tensile strength of scaffolds (along the fiber alignment) increased from 2.21 +/- 0.23 MPa for PCL at uptake rate of zero rpm, to a value of 4.21 +/- 0.35 MPa for PCL at uptake rate of 3000 rpm and finally to 9.58 +/- 0.71 MPa for PCL at 6000 rpm. Similarly, the tensile modulus increased gradually from 6.12 +/- 0.8 MPa for PCL at uptake rate of zero rpm, to 11.93 +/- 1.22 MPa for PCL at uptake rate of 3000 rpm and to 33.20 +/- 1.98 MPa for PCL at 6000 rpm. The sequence of macroscopic mechanical properties (tensile strength and modulus) of three fibers, from highest to lowest, is PCL at 0 rpm < PCL at 3000 rpm < PCL at 6000 rpm. This is attributed to the increased fiber alignment and packing and decrease in inter-fiber pore size at higher uptake rates.

Electrospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapatite for bone tissue engineering.

Nanocomposite scaffolds based on nanofibrous poly(epsilon-caprolactone) (PCL) and nanohydroxyapatite (nanoHA) with different compositions (wt%) were prepared by electrostatic co-spinning to mimic the nano-features of the natural extracellular matrix (ECM). NanoHA was found to be well dispersed in polymers up to the addition of 20 wt%, after ultrasonication. The composite scaffolds were characterized for structure and morphology using XRD, EDX, SEM, and DSC. The scaffolds have a porous nanofibrous morphology with fibers (majority) having diameters in the range of 450-650 nm, depending on composition, and interconnected pore structures. SEM, EDX, and XRD analyses have confirmed the presence of nanoHA in the fibers. As the nanoHA content in the fibers increases, the surface of fibers becomes rougher. The mechanical (tensile) property measurement of the electrospun composites reveals that as the nanoHA content increases, the ultimate strength increases from 1.68 MPa for pure PCL to 2.17, 2.65, 3.91, and 5.49 MPa for PCL/nanoHA composites with the addition of 5, 10, 15, and 20 wt% nanoHA, respectively. Similarly the tensile modulus also increases gradually from 6.12 MPa to 21.05 MPa with the increase of nanoHA content in the PCL/nanoHA fibers, revealing an increase in stiffness of the fibers due to the presence of HA. DSC analysis reveals that as nanoHA in the composite scaffolds increases, the melting point slightly increases due to the good dispersion and interface bonding between PCL and nanoHA.