D. Bruce Chase

Evaluation of Cross-Linking Methods for Electrospun Gelatin on Cell Growth and Viability

The creation of a tissue engineering scaffold via electrospinning that has minimal toxicity and uses a solvent system composed of solvents with low toxicity and different cross-linking agents was investigated. First, a solvent system of acetic acid/ethyl acetate/water (50:30:20) with gelatin as a solute was evaluated. The optimum system for electrospinning a scaffold with the desired properties resulted from a gelatin concentration of 10 wt %. Several different methods were used to cross-link the electrospun gelatin fibers, including vapor-phase glutaraldehyde, aqueous phase genipin, and glyceraldehyde, as well as reactive oxygen species from a plasma cleaner. Because glutaraldehyde at high concentrations has been shown to be toxic, we explored other cross-linking methods. Using reactive oxygen species from a plasma cleaner is an easy alternative; however, the degradation reaction dominated the cross-linking reaction and the scaffolds degraded after only a few hours in aqueous medium at 37 °C. Glyceraldehyde and genipin were established as good options for cross-linking agents because of the low toxicity of these cross-linkers and the resistance to dissolution of the cross-linked fibers in cell culture medium at 37 °C. MG63 osteoblastic cells were grown on each of the cross-linked scaffolds. A proliferation assay showed that the cells proliferated as well or better on the cross-linked scaffolds than on traditional two-dimensional polystyrene culture plates.

AFM-IR: Combining Atomic Force Microscopy and Infrared Spectroscopy for Nanoscale Chemical Characterization

Polymer and life science applications of a technique that combines atomic force microscopy (AFM) and infrared (IR) spectroscopy to obtain nanoscale IR spectra and images are reviewed. The AFM–IR spectra generated from this technique contain the same information with respect to molecular structure as conventional IR spectroscopy measurements, allowing significant leverage of existing expertise in IR spectroscopy. The AFM–IR technique can be used to acquire IR absorption spectra and absorption images with spatial resolution on the 50 to 100 nm scale, versus the scale of many micrometers or more for conventional IR spectroscopy. In the life sciences, experiments have demonstrated the capacity to perform chemical spectroscopy at the sub-cellular level. Specifically, the AFM–IR technique provides a label-free method for mapping IR-absorbing species in biological materials. On the polymer side, AFM–IR was used to map the IR absorption properties of polymer blends, multilayer films, thin films for active devices such as organic photovoltaics, microdomains in a semicrystalline polyhydroxyalkanoate copolymer, as well as model pharmaceutical blend systems. The ability to obtain spatially resolved IR spectra as well as high-resolution chemical images collected at specific IR wavenumbers was demonstrated. Complementary measurements mapping variations in sample stiffness were also obtained by tracking changes in the cantilever contact resonance frequency. Finally, it was shown that by taking advantage of the ability to arbitrarily control the polarization direction of the IR excitation laser, it is possible to obtain important information regarding molecular orientation in electrospun nanofibers.

Fiber diameters control osteoblastic cell migration and differentiation in electrospun gelatin

Defined electrospinning conditions were used to create scaffolds with different fiber diameters to investigate their interactions with osteoblastic MG63 cells. Nonwoven gelatin scaffolds were electrospun with varied fiber diameters to investigate the effect of fiber size and resultant porosity on cell proliferation, viability, migration, and differentiation. The low toxicity solvent acetic acid:ethyl acetate:water ratio and gelatin concentrations were optimized to create small and large diameter fibers. The fiber diameters obtained by this procedure were 110 +/- 40 nm for the small and 600 +/- 110 nm for the large fibers. Cell viability assays showed that MG63 cells grew similarly on both fibers at the early time point (day 3) but preferred the scaffold with large diameter fibers by the later time points (day 5 and day 7). Confocal microscopic imaging showed that MG63 cells migrated poorly (maximum depth of 18 microm) into the scaffold of small diameter fibers, but readily penetrated (maximum depth of 50 microm) into the scaffold of large diameter fibers. Alkaline phosphatase (ALP) assays showed that MG63 cells differentiated on scaffolds made from both diameter fibers. In longer term experiments, MG63 cells differentiated to a greater extent on scaffolds made from small diameter fibers compared to large diameter fibers at days 3 and 7, but the ALP levels were the same for both diameter fibers by day 14. These results indicate that cells can perceive differences in the diameter and resultant pore size of electrospun gelatin fibers and that they process this information to alter their behavior.

Characterization of biodegradable polyurethane microfibers for tissue engineering

A polyurethane designed to be biodegradable via hydrolysis and enzyme-mediated chain cleavage, has been investigated for its use as a temporary scaffold in tissue-engineering applications. The phase-segregated nature of the polyurethane imparts elastomeric properties that are attractive for soft tissue engineering. This polyurethane has been electrospun in order to create scaffolds that incorporate several biomimetic features including small fiber diameter, large void volume, and an interconnected porous network. Material properties were evaluated via gel-permeation chromatography, differential scanning calorimetry and Raman spectroscopy before and after processing. Analysis by gel-permeation chromatography showed that the molecular weights were similar, indicating that the bulk of the polymer chains were not degraded during processing. Thermal analysis revealed that the glass transition temperature did not shift and Raman spectra of the bulk polyurethane film compared to the electrospun mat were identical, confirming that the conformation of the polymer was unaffected by the shear and electric field used in the electrospinning process. In addition, field emission scanning electron microscopy revealed that the morphology of the electrospun mats had a broad fiber diameter distribution, and mechanical analysis showed that the mats had an ultimate tensile stress of 1.33 MPa and ultimate tensile strain of 78.6%. The degradation profile was investigated in the presence of chymotrypsin. These results were compared to a previous study of thin films of this polyurethane, and it was found that the increase of surface area aided the surface-mediated erosion of the material. It is believed that an electrospun matrix of this biodegradable polyurethane shows promise for use in soft tissue engineering and regenerative medicine applications.

FT-Raman Microscopy: Discussion and Preliminary Results:

This paper reports on the first spectra obtained using a microscope apparatus to perform FT-Raman spectroscopy. It is considered desirable to use a standard FT-IR microscope for these measurements, since this approach may lead to the attainment of both FT-IR and FT-Raman microscope data from a single instrument. The relative performance of the microscope apparatus compared to that of a previously designed macro-apparatus is studied. The described micro-apparatus is then used to measure the FT-Raman spectrum of a single crystal of bis-methyl-styry I benzene and of a single fiber of Kevlar®. The results indicate an acceptable sensitivity, for these small samples.

Immobilization of Gold Nanorods onto Electrospun Polycaprolactone Fibers Via Polyelectrolyte Decoration—A 3D SERS Substrate

We report the fabrication of a homogeneous and highly dense gold nanorod (AuNR) assembly on electrospun polycaprolactone (PCL) fibers using electrostatic interaction as the driving force. Specifically, decoration of a poly(sodium 4-styrenesulfonate) (PSS) layer onto the AuNRs imposed negative charges on the nanorod surface, and the interactions between PSS and the AuNRs were investigated using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Positive charges on the PCL fibrous substrate were established via polyelectrolyte layer-by-layer deposition, which was investigated using multiple characterization techniques. Driven by the attractive electrostatic interaction, immobilization of AuNRs on the PCL fibers was initiated upon substrate immersion, and the kinetics of the immobilization process were studied using UV-vis spectroscopy. Electron microscopy characterization of the AuNR/PCL nanocomposite fibers reveals a uniform AuNR coating on the fiber surface with the immobilized AuNR density being high enough to provide full surface coverage. By using both 4-mercaptopyridine and Rhodamine 6G as probe molecules, the performance of the AuNR/PCL fibrous mesh as a three-dimensional (3D) surface-enhanced Raman scattering (SERS) substrate was investigated. The nanocomposite fibers allowed detection at concentrations as low as 10(-7) M of the probe molecule in solution and exhibited excellent reproducibility in the SERS measurements. In addition, a comparison between the 3D AuNR/PCL fibrous mesh and a 2D AuNR/PCL film reveals that the enhanced surface area in the 3D substrate effectively improved the SERS performance with a 6-fold increase in the Raman intensity.

Performance and Application of a New Planar Array Infrared Spectrograph Operating in the Mid-Infrared (2000–975 cm -1 ) Fingerprint Region

A no-moving-part planar array infrared spectrograph (PA-IR) equipped with a 256 × 256 mercury cadmium telluride (MCT) focal plane array has been designed and constructed. The performance of the instrument, whose frequency range extends from 2000–975 cm−1, has been assessed in terms of resolution, bandwidth, and signal-to-noise ratio. The PA-IR spectrograph is able to record spectra with an 8.7 ms time resolution and has peak-to-peak noise levels as low as 2.4 × 10−4 A.U. As a demonstration of the potential of PA-IR, the dynamics of reorientation of a liquid crystalline sample exposed to a single electric field pulse has been studied. It was shown that PA-IR can be used for the simultaneous acquisition of two orthogonally polarized spectra. The advantages and limitations of PA-IR, step-scan Fourier transform infrared (FT-IR), and ultra-rapid-scanning FT-IR for real-time studies of reversible and irreversible phenomena are thoroughly discussed.

Design and Performance of a Planar Array Infrared Spectrograph that Operates in the 3400 to 2000 cm -1 Region

A prototype, no-moving-parts, plane array infrared spectrograph (PA-IR) capable of routine spectral acquisition in the 3400 to 2000 cm−1 region has been constructed. The instrument includes a continuous source, Czerney–Turner type monochromator system and an infrared camera that incorporates a 320 × 256 pixel InSb focal plane array detector cooled with liquid nitrogen. PA-IR spectra (∼3400 to 2550 cm−1) of polystyrene (PS) and poly(ethylene naphthalate) (PEN) films have been obtained at a resolution of ∼8 cm−1 with excellent signal-to-noise ratios (SNR). Peak-to-peak noise levels of ∼1 × 10−3 absorbance units are observed for single acquisition spectra with 1.5 ms integration times and 17 ms total acquisition times. Integration times as low as 10 μs are possible (with good SNR); however, data acquisition is limited by the frame rate (60 frames/s) of the software acquisition package currently used. In this work, an apertured image of the source is displayed over ∼20 rows of the array and the expected square root improvement in SNR is observed when multiple rows and/or frames are averaged. We have also shown that a square root improvement in SNR continues to occur with further signal averaging, providing noise levels as low as 1.5 × 10−5 absorbance units. Several additional advantages and options associated with the PA-IR method are discussed, including time-resolved spectroscopy, real-time monitoring, and spectroscopic mapping.

“Real Time” Raman Studies of Electrospun Fibers

Raman spectra of as-spun fibers produced through electrospinning have shown that high S/N data can be obtained on 50-μm diameter fibers in relatively short collection times (25 s). Using this same instrumental approach, “real time” Raman spectra of the electrospinning liquid fiber jet at the origin of the jet and 1 cm downstream have been obtained. The results show that “on-line” analysis of the solvent/polymer ratio and spectroscopic measurements of polymer orientation are possible and will lead to a more quantitative understanding of the development of the polymer microstructure during the electrospinning process.

New developments in planar array infrared spectroscopy

A planar array infrared (PA-IR) spectrograph offers several advantages over other infrared approaches, including high acquisition rate and sensitivity. However, it suffers from some important drawbacks, such as a limited spectral range and a significant curvature of the recorded spectral images, which still need to be addressed. In this article, we present new developments in PA-IR spectroscopy that overcome these drawbacks. First, a data processing method for the correction of the curvature observed in the spectral images has been developed and refined. In addition, a dual-beam instrument that allows the simultaneous recording of two independent spectral images has been developed. These two improvements have been combined to demonstrate the real-time background correction capability of PA-IR instruments. Finally, the accessible spectral range of the PA-IR spectrograph has been extended to cover simultaneously the methylene stretching (3200–2800 cm−1) and the fingerprint (2000–1000 cm−1) spectral regions.