Robert P. Apkarian

Engineered collagen–PEO nanofibers and fabrics

Type I collagen-PEO fibers and non-woven fiber networks were produced by the electrospinning of a weak acid solution of purified collagen at ambient temperature and pressure. As determined by high-resolution SEM and TEM, fiber morphology was influenced by solution viscosity, conductivity, and flow rate. Uniform fibers with a diameter range of 100-150 nm were produced from a 2-wt% solution of collagen-PEO at a flow rate of 100 μl min-1. Ultimate tensile strength and elastic modulus of the resulting non-woven fabrics was dependent upon the chosen weight ratio of the collagen-PEO blend. 1H NMR dipolar magnetization transfer analysis suggested that the superior mechanical properties, observed for collagen-PEO blends of weight ratio 1 : 1, were due to the maximization of intermolecular interactions between the PEO and collagen components. The process outlined herein provides a convenient, non-toxic, non-denaturing approach for the generation collagen-containing nanofibers and non-woven fabrics that have potential application in wound healing, tissue engineering, and as hemostatic agents.

TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone

Transmission electron microscopy (TEM) was used to investigate the crystal-collagen interactions in normal and osteoporotic human trabecular bone at the nanostructural level. More specifically, two-dimensional TEM observations were used to infer the three-dimensional information on the shape, the size, the orientation, and the alignment of apatite crystals in collagen fibrils in normal and osteoporotic bone. We found that crystals were of platelet shape with irregular edges and that there was no substantial difference in crystal length or crystal thickness between normal and osteoporotic trabecular bone. The crystal arrangement in cross-sectioned fibrils did not neatly conform to the parallel arrangement of crystals seen in longitudinally-sectioned fibrils. Instead, the crystal arrangement in both normal and osteoporotic trabecular bone took on more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns. The TEM imaging was done using bright fields only. Thus, the results presented are within the limitations of this approach.

Thermoplastic Elastomer Hydrogels via Self‐Assembly of an Elastin‐Mimetic Triblock Polypeptide

Synthetic polymers consisting of well-defined blocks of compositionally dissimilar monomers undergo microscopic phase separation in the solid state and in selective solvents to afford ordered microstructures that display unique, technologically significant properties in comparison to blends of the respective homopolymers. [1] However, the synthetic repertoire of these materials has been limited to tapered blocks of uniform sequence, which potentially restricts the functional complexity of the resulting microstructures. Genetic engineering of synthetic polypeptides enables preparation of block copolymers composed of complex sequences in which the individual blocks may have different mechanical, chemical, or biological properties. [2‐6] The segregation of the protein blocks into compositionally, structurally, and spatially distinct domains should occur in analogy with synthetic block copolymers, affording ordered structures on the nanometer to micrometer size range. The utility of these protein materials depends on the ability to functionally emulate or enhance the materials properties of conventional polymer systems, while retaining the benefits of greater control over the sequence and microstructure that protein engineering affords for the construction of materials. We report herein the genetically directed synthesis and characterization of a triblock copolymer 1 that is derived from an elastin-mimetic polypeptide sequence in which the respective blocks exhibit different mechanical properties (Scheme 1). Moreover, the sequences of the respective blocks were chosen such that the polypeptide undergoes reversible microscopic phase separation from aqueous solution to form a thermoplastic elastomer hydrogel above a lower critical solution temperature Tt.

Changes in cell morphology due to plasma membrane wounding by acoustic cavitation.

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphologic changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced nonlytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure.

Fibrillogenesis in Continuously Spun Synthetic Collagen Fiber

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.

Outer membrane-associated serine protease involved in adhesion of Shewanella oneidensis to Fe(III) oxides.

The facultative anaerobe Shewanella oneidensis MR-1 respires a variety of anaerobic electron acceptors, including insoluble Fe(III) oxides. S. oneidensis employs a number of novel strategies for respiration of insoluble Fe(III) oxides, including localization of respiratory proteins to the cell outer membrane (OM). The molecular mechanism by which S. oneidensis adheres to and respires Fe(III) oxides, however, remains poorly understood. In the present study, whole cell fractionation and MALDI-TOF-MS/MS techniques were combined to identify a serine protease (SO3800) associated with the S. oneidensis OM. SO3800 contained predicted structural motifs similar to cell surface-associated serine proteases that function as bacterial adhesins in other gram-negative bacteria. The gene encoding SO3800 was deleted from the S. oneidensis genome, and the resulting mutant strain (DeltaSO3800) was tested for its ability to adhere to and respire Fe(III) oxides. DeltaSO3800 was severely impaired in its ability to adhere to Fe(III) oxides, yet retained wild-type Fe(III) respiratory capability. Laser Doppler velocimetry and cryoetch high-resolution SEM experiments indicated that DeltaSO3800 displayed a lower cell surface charge and higher amount of surface-associated exopolysaccharides. Results of this study indicate that S. oneidensis may respire insoluble Fe(III) oxides at a distance, negating the requirement for attachment prior to electron transfer.

In-Lens Cryo-High Resolution Scanning Electron Microscopy: Methodologies for Molecular Imaging of Self-Assembled Organic Hydrogels

The micro- and nanoarchitectures of water-swollen hydrogels were routinely analyzed in three dimensions at very high resolution by two cryopreparation methods that provide stable low-temperature specimens for in-lens high magnification recordings. Gemini surfactants (gS), poly-N-isopropylacrylamides (p-NIP Am), and elastin-mimetic di- (db-E) and triblock (tb-E) copolymer proteins that form hydrogels have been routinely analyzed to the sub-10-nm level in a single day. After they were quench or high pressure frozen, samples in bulk planchets were subsequently chromium coated and observed at low temperature in an in-lens field emission SEM. Pre-equilibrated planchets (4-40 degrees C) that hold 5-10 microl of hydrogel facilitate dynamic morphological studies above and below their transition temperatures. Rapidly frozen samples were fractured under liquid nitrogen, low-temperature metal coated, and observed in-lens to assess the dispersion characteristics of micelles and fragile colloidal assemblies within bulk frozen water. Utilizing the same planchet freezing system, the cryoetch-HRSEM technique removed bulk frozen water from the hydrogel matrix by low-temperature, high-vacuum sublimation. The remaining frozen solid-state sample faithfully represented the hydrogel matrix. Cryo- and cryoetch-HRSEM provided vast vistas of hydrogels at low and intermediate magnifications whereas high magnification recordings and anaglyphs (stereo images) provided a three-dimensional prospective and measurements on a molecular level.

Morphological Characterization of Elastin-Mimetic Block Copolymers Utilizing Cryo- and Cryoetch-HRSEM

Elastin-mimetic block copolymers were produced by genetic engineering. Genetically driven synthesis permitted control of the final physiochemical characteristics of the block copolymers. We designed BB and BAB block copolymers in which the A-block was hydrophilic and the B-block was hydrophobic. By designing the copolymers in this manner, it was proposed that they would self-assemble into micellar aggregates that, at high concentration, would form thermoreversible hydrogels. To analyze the three-dimensional fine surface morphology of the copolymers, to the resolution level of a few nanometers, we employed cryo-HRSEM. This method provided vast expanses of the specimen in its frozen hydrated state for survey. In our initial cryo-HRSEM studies, we observed the protein filaments and micelles surrounded by lakes of vitreous ice. Upon examination at low and intermediate magnifications, there was an extensive honeycomb-like filamentous network. To delineate the fine morphology of the hydrogel network at high magnification and to greater depths, we cryoetched away unbound water from the sample surface, in high vacuum, prior to chromium deposition. By using this technique, we were able to visualize for characterization purposes the fine fibril networks formed from the micellar aggregates over the surface of the hydrogel.

The Role of Bacterial Exopolymers in Metal Sorption and Reduction

The dissimilatory metal reducing bacterium, Shewanella oneidensis strain MR-1 has been extensively studied for the past decade for its significant capacity to utilize a wide range of organic compounds and metals during anaerobic respiration. The primary site of electron transport in gramnegative bacteria is usually the inner membrane and the periplasm, the region between inner and outer membranes with very high enzymatic activity [1]. Previous reports have suggested that multiheme cytochromes localized in the outer membrane by the type II protein secretion pathway (T2S) are involved in extracellular metal reduction [2]. In addition to its ability to reduce metals, MR-1 produces extracellular polymers that become heavily mineralized with nanocrystalline uraninite (UO2) following reduction of U(VI). However, the exact ultrastructure, composition, and mechanisms of cellular and extracellular components involved in metal reduction and precipitation are not completely understood. To evaluate the role of the T2S in reduction and localization of U by Shewanella MR-1 as well as several mutants within key genes associated with the T2S were investigated. Resting cell suspensions were incubated with U(VI) for 24h, and then processed anaerobically for TEM [3]. Morphological studies of these cultures showed differences in the sites of U(IV) deposition in the MR-1 compared to the mutants. Additionally, cells were often surrounded by thin (nm) filamentous structures that were heavily covered with nanocrystalline uraninite [Fig. 1]. Previous studies report metal-binding properties of Pseudomona putida with indications that its polysaccharides (PS) are capable of binding divalent metal cations such Cd and Pb [4]. Here we report this capacity in Shewanella, and investigate the hypothesis that extracellular polymeric substances are able to bind and reduce metals in association with specific lipoproteins that include ctype cytochromes.

A Permanent Change In Protein Mechanical Responses Can Be Produced By Thermally Induced Microdomain Mixing

Electrospinning was employed to fabricate 3-D fiber networks from a recombinant amphiphilic elastin-mimetic tri-block protein polymer and the effects of moderate thermal conditioning (60°C, 4 h) on network mechanical responses investigated. Significantly, while cryo-high resolution scanning electron microscopy (cryo-HRSEM) revealed that the macroscopic and microscopic morphology of the network structure was unchanged, solid-state 1H-NMR spectroscopy demonstrated enhanced interphase mixing of hydrophobic and hydrophilic blocks. Significantly, thermal annealing triggered permanent changes in network swelling behavior (28.75 ± 2.80 non-annealed vs. 13.55 ± 1.39 annealed; P < 0.05) and uniaxial mechanical responses, including Youngs modulus (0.170 ± 0.010 MPa non-annealed vs. 0.366 ± 0.05 MPa annealed; P < 0.05) and ultimate tensile strength (0.079 ± 0.008 MPa vs. 0.119 ± 0.015 MPa; P < 0.05). To our knowledge, these investigations are the first to note that mechanical responses of protein polymers can be permanently altered through a temperature-induced change in microphase mixing.