Shane A. Catledge

An electrospun triphasic nanofibrous scaffold for bone tissue engineering

A nanofibrous triphasic scaffold was electrospun from a mixture of polycaprolactone (PCL), type-I collagen and hydroxyapatite nanoparticles (nano-HA) with a mixture dry weight ratio of 50/30/20, respectively. Scaffolds were characterized by evaluating fiber morphology and chemical composition, dispersion of HA particles and nanoindentation. Scanning electron microscopy revealed fibers with an average diameter of 180 +/- 50 nm, which coincides well with the collagen fiber bundle diameter characteristic of the native extracellular matrix of bone. The triphasic fibers, stained with calcein and imaged with confocal microscopy, show a uniform dispersion of apatite particles throughout their length with minor agglomeration. Scaffold fibers of triphasic (50/30/20), collagen/nano-HA (80/20), PCL/nano-HA (80/20), pure PCL and pure collagen were each pressure consolidated into non-porous pellets for evaluation by transmission electron microscopy and nanoindentation. While the majority of apatite particles are uniformly dispersed having an average size of 30 nm, agglomerated particles as large as a few microns are sparsely distributed. Nanoindentation of the pressure-consolidated scaffolds showed a range of Youngs modulus (0.50-3.9 GPa), with increasing average modulus in the order of (PCL < PCL/nano-HA < collagen < triphasic < collagen/nano-HA). The modulus data emphasize the importance of collagen and its interaction with other components in affecting mechanical properties of osteoconductive scaffolds.

Rapid dissolution of shells of weakly calcified Antarctic benthic macroorganisms indicates high vulnerability to ocean acidification.

Abstract Antarctic calcified macroorganisms are particularly vulnerable to ocean acidification because many are weakly calcified, the dissolution rates of calcium carbonate are inversely related to temperature, and high latitude seas are predicted to become undersaturated in aragonite by the year 2100. We examined the post-mortem dissolution rates of aragonitic and calcitic shells from four species of Antarctic benthic marine invertebrates (two bivalves, one limpet, one brachiopod) and the thallus of a limpet shell-encrusting coralline alga exposed to acidified pH (7.4) or non-acidified pH (8.2) seawater at a constant temperature of 4°C. Within a period of only 14–35 days, shells of all four species held in pH 7.4 seawater had suffered significant dissolution. Despite calcite being 35% less soluble in seawater than aragonite, there was surprisingly, no consistent pattern of calcitic shells having slower dissolution rates than aragonitic shells. Outer surfaces of shells held in pH 7.4 seawater exhibited deterioration by day 35, and by day 56 there was exposure of aragonitic or calcitic prisms within the shell architecture of three of the macroinvertebrate species. Dissolution of coralline algae was confirmed by differences in weight loss in limpet shells with and without coralline algae. By day 56, thalli of the coralline alga held in pH 7.4 displayed a loss of definition of the conceptacle pores and cracking was evident at the zone of interface with limpet shells. Experimental studies are needed to evaluate whether there are adequate compensatory mechanisms in these and other calcified Antarctic benthic macroorganisms to cope with anticipated ocean acidification. In their absence, these organisms, and the communities they comprise, are likely to be among the first to experience the cascading impacts of ocean acidification.

Functionally graded electrospun scaffolds with tunable mechanical properties for vascular tissue regeneration

Electrospun tubular scaffolds (4 mm inner diameter) based on bio-artificial blends of polyglyconate (Maxon) and proteins such as gelatin and elastin having a spatially designed multilayer structure were prepared for use as vascular tissue scaffolds. Scanning electron microscopy analysis of scaffolds showed a random nanofibrous morphology with fiber diameter in the range of 200-400 nm for protein-blended Maxon, which mimics the nanoscale dimensions of collagen (50-500 nm). The scaffolds have a well interconnected pore structure and porosity up to 82%, with protein blending and multi-layering in contrast to electrospun Maxon scaffolds (67%). Fourier-transform infrared spectroscopy, x-ray diffraction and differential scanning calorimetry results confirmed the blended composition and crystallinity of fibers. Uniaxial tensile testing revealed a strength of 14.46 +/- 0.42 MPa and a modulus of 15.44 +/- 2.53 MPa with a failure strain of 322.5 +/- 10% for a pure Maxon scaffold. The blending of polyglyconate with biopolymers decreased the tensile properties in general, with an exception of the tensile modulus (48.38 +/- 2 MPa) of gelatin/Maxon mesh, which was higher than that of the pure Maxon scaffold. Trilayered tubular scaffolds of gelatin/elastin, gelatin/elastin/Maxon and gelatin/Maxon (GE-GEM-GM) that mimic the complex trilayer matrix structure of natural artery have been prepared by sequential electrospinning. Tensile testing under dry conditions revealed a tensile strength of 2.71 +/- 0.2 MPa and a modulus of 20.4 +/- 3 MPa with a failure strain of 140 +/- 10%. However, GE-GEM-GM scaffolds tested under wet conditions after soaking in a phosphate buffered saline medium at 37 degrees C for 24 h exhibited mechanical properties (2.5 MPa tensile strength and 9 MPa tensile modulus) comparable to those of native femoral artery.

Epitaxial diamond encapsulation of metal microprobes for high pressure experiments

Diamond anvils with diamond encapsulated thin-film microcircuits have been fabricated for ultrahigh pressure electrical conductivity experiments. The diamond films were homoepitaxially deposited onto the diamond anvil substrates with microwave plasma chemical vapor deposition using a 2% methane in hydrogen gas mixture and a diamond substrate temperature of 1300 °C. The diamond embedded thin-film microprobes remain functional to megabar pressures. We have applied this technology to the study of the pressure-induced metallization of KI under pressures up to 1.8 Mbar. This technology has the potential of greatly advancing the pressure range of a number of existing high-pressure diagnostic techniques, and for expanding the capabilities of diamond anvil cells into new directions.

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite.

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.

Mesenchymal stem cell interaction with ultra smooth nanostructured diamond for wear resistant orthopaedic implants

Ultra-smooth nanostructured diamond (USND) can be applied to greatly increase the wear resistance of orthopaedic implants over conventional designs. Herein we describe surface modification techniques and cytocompatibility studies performed on this new material. We report that hydrogen (H)-terminated USND surfaces supported robust mesenchymal stem cell (MSC) adhesion and survival, while oxygen- (O) and fluorine (F)-terminated surfaces resisted cell adhesion, indicating that USND can be modified to either promote or prevent cell/biomaterial interactions. Given the favorable cell response to H-terminated USND, this material was further compared with two commonly used biocompatible metals, titanium alloy (Ti-6Al-4V) and cobalt chrome (CoCrMo). MSC adhesion and proliferation were significantly improved on USND compared with CoCrMo, although cell adhesion was greatest on Ti-6Al-4V. Comparable amounts of the pro-adhesive protein, fibronectin, were deposited from serum on the three substrates. Finally, MSCs were induced to undergo osteoblastic differentiation on the three materials, and deposition of a mineralized matrix was quantified. Similar amounts of mineral were deposited onto USND and CoCrMo, whereas mineral deposition was slightly higher on Ti-6Al-4V. When coupled with recently published wear studies, these in vitro results suggest that USND has the potential to reduce debris particle release from orthopaedic implants without compromising osseointegration.

Surface crystalline phases and nanoindentation hardness of explanted zirconia femoral heads

One new and nine explanted zirconia femoral heads were studied using glancing angle X-ray diffraction, scanning electron microscopy, and nanoindentation hardness techniques. All starting zirconia implants consisted only of tetragonal zirconia polycrystals (TZP). For comparison, one explanted alumina femoral head was also studied. Evidence for a surface tetragonal-to-monoclinic zirconia phase transformation was observed in some implants, the extent of which was varied for different in-service conditions. A strong correlation was found between increasing transformation to the monoclinic phase and decreasing surface hardness. Microscopic investigations of some of the explanted femoral heads revealed ultra high molecular weight polyethylene and metallic transfer wear debris.

EFFECT OF NITROGEN ADDITION ON THE MICROSTRUCTURE AND MECHANICAL PROPERTIES OF DIAMOND FILMS GROWN USING HIGH-METHANE CONCENTRATIONS

We report on the microstructure and mechanical properties of diamond films grown using varying nitrogen additions to a plasma with a high-CH4 fraction of 15% (in hydrogen) and an operating pressure of 125 Torr. Films were grown at N2/CH4 ratios ranging from 0 to 0.30 by fixing the CH4 flow rate and changing only the N2 flow rate. With increasing nitrogen addition, we observe an increase in intensity and a decrease in the full width at half maximum (FWHM) of the Raman band at 1550 cm−1, while the crystalline diamond peak at 1332 cm−1 decreases in intensity and increases in the FWHM. X-ray diffraction confirms that the film crystallinity and diamond grain size decrease rapidly with increasing nitrogen additions up to a N2/CH4 ratio of 0.10, but then do not change significantly above this ratio. A similar trend is observed for film surface roughness. In addition, we find from indentation testing that all films exhibit high hardness values ranging from 70 to 90 GPa and that the toughness of the films improves...

In vitro studies on the effect of particle size on macrophage responses to nanodiamond wear debris.

Nanostructured diamond coatings improve the smoothness and wear characteristics of the metallic component of total hip replacements and increase the longevity of these implants, but the effect of nanodiamond wear debris on macrophages needs to be determined to estimate the long-term inflammatory effects of wear debris. The objective was to investigate the effect of the size of synthetic nanodiamond particles on macrophage proliferation (BrdU incorporation), apoptosis (Annexin-V flow cytometry), metabolic activity (WST-1 assay) and inflammatory cytokine production (qPCR). RAW 264.7 macrophages were exposed to varying sizes (6, 60, 100, 250 and 500 nm) and concentrations (0, 10, 50, 100 and 200 μg ml(-1)) of synthetic nanodiamonds. We observed that cell proliferation but not metabolic activity was decreased with nanoparticle sizes of 6-100 nm at lower concentrations (50 μg ml(-1)), and both cell proliferation and metabolic activity were significantly reduced with nanodiamond concentrations of 200 μg ml(-1). Flow cytometry indicated a significant reduction in cell viability due to necrosis irrespective of particle size. Nanodiamond exposure significantly reduced gene expression of tumor necrosis factor-α, interleukin-1β, chemokine Ccl2 and platelet-derived growth factor compared to serum-only controls or titanium oxide (anatase 8 nm) nanoparticles, with variable effects on chemokine Cxcl2 and vascular endothelial growth factor. In general, our study demonstrates a size and concentration dependence of macrophage responses in vitro to nanodiamond particles as possible wear debris from diamond-coated orthopedic joint implants.

Nanoindentation hardness and adhesion investigations of vapor deposited nanostructured diamond films

The effect of changing the N2/CH4 feedgas ratio on the structure and mechanical properties of microwave plasma chemical vapor deposited diamond films grown on Ti–6Al–4V alloy substrates was investigated. The relative concentration of CH4 and N2 (in a balance of H2) was shown to strongly influence film structure, hardness, and adhesion. For high CH4 concentration (15% by volume), nanostructured diamond films with roughness magnitudes of 15–30 nm, good adhesion and a high hardness value of 90 GPa was obtained. A distinct correlation was found between the nanoindentation hardness of the deposited film and the N2/CH4 ratio in the plasma, as well as a correlation of hardness to the ratio of the Raman peak intensities (1332 and 1555 cm−1). Scratch adhesion testing of nanostructured diamond films showed delamination at a critical force of 33 N using acoustic emission techniques. These results demonstrate that nanostructured diamond films can be tailored on metallic surfaces with hardness ranging from 10 GPa (med...