The role of the conformation and bioactivity of adsorbed vitronectin in enhancing osteoblast (the bone-forming cells) adhesion on nanophase (that is, grain sizes less than 100 nm) alumina was investigated in the present in vitro study. Results obtained from surface-enhanced Raman scattering (SERS) provided the first evidence of increased unfolding of vitronectin adsorbed on nanophase alumina. This conclusion was further supported by dose-dependent inhibition of cell adhesion on nanophase alumina pretreated with vitronectin following preincubation of osteoblasts with either Arginine-Glycine-Aspartic Acid-Serine (RGDS) or Lysine-Arginine-Serine-Arginine (KRSR) to block respective cell-membrane receptors. These events, namely enhanced unfolding of vitronectin that leads to exposure of bioactive epitopes (such as RGDS) in adsorbed vitronectin may explain the observed increased osteoblast adhesion on nanophase alumina.

The amino acid glutamate is the major excitatory neurotransmitter used in the nervous system for interneuronal communication. It is used throughout the brain by various neuronal pathways including those involved in learning and memory, locomotion, and sensory perception. Because glutamate is released from neurons on a millisecond time scale into sub-micrometer spaces, the development of a glutamate biosensor with high temporal and spatial resolution is of great interest for the study of neurological function and disease. Here, we demonstrate the feasibility of an optical glutamate sensor based on the sol-gel encapsulation of the enzyme glutamate dehydrogenase (GDH). GDH catalyses the oxidative deamination of glutamate and the reduction of NAD+ to NADH. NADH fluorescence is the basis of the sensor detection. Thermodynamic and kinetic studies show that GDH remains active in the sol-gel matrix and that the reaction rate is correlated to the glutamate concentration.

A dual scaffold structure made of biodegradable polymers and seeded with neural stem cells has been developed to address the issues of spinal cord injury including axonal severance and the loss of neurons and glia. The general design of the scaffold is derived the structure of the spinal cord with an outer section which mimics the white matter with long axial pores to provide axonal guidance and an inner section seeded with neural stem cells to address the issues of cell replacement and mimic the general character of the gray matter. The seeded scaffold leads to improved functional recovery as compared with the lesion control or cells alone following spinal cord injury.

Diffusional dynamics of polymers can be very sensitive to polymer architecture. Polymers with novel (or time-varying) architectures could facilitate the release of therapeutic compounds from gels or concentrated polymer solutions with unusual or novel kinetic profiles. Towards this end, we are studying the behavior of model dendritic polymers, the poly(amidoamine)(PAMAM) dendrimers, in aqueous solutions and in concentrated solutions of a “matrix” polymer, poly(ethylene oxide)(PEO). Fluorescence measurements of the environmental polarity of the dendrimers provide evidence for pH-induced confomational changes in mid-sized (generation 6), but not in small (generation 2) dendrimers. In aqueous solution, dendrimer diffusion measurements reveal the fractal-like growth of these molecules, but measurements in aqueous PEO solutions failed to detect any pH dependence of the diffusion coefficient. Specific chemical interactions between the PEO and the PAMAM molecules may dominate their dynamic behavior.

A methodology for the preparation of porous scaffolds for tissue engineering using co-extrusion is presented. Poly(ε-caprolactone) is blended with poly(ethylene oxide) in a twin-screw extruder to form a two-phase material with micrometer-sized domains. Selective dissolution of the poly(ethylene oxide) with water results in a porous material. This method of polymer extrusion permits the preparation of scaffolds having continuous void space and controlled characteristic length scales without the use of potentially toxic organic solvents. A range of blend volume fractions results in co-continuous networks of polymer and void spaces. Annealing studies demonstrate that the characteristic pore size may be increased to larger than 100 μm. The mechanical properties of the scaffolds are characterized by a compressive modulus on the order of 1 MPa at low strains and approximately 10 MPa at higher strains. The results of osteoblast seeding suggest it is possible to use co-extrusion to prepare polymer scaffolds without the introduction of toxic contaminants.

A cylindrically-configured plasma treatment system in Radio Frequency Glow Discharges fed with ammonia was used with the aim of modifying the internal surface of ePTFE arterial prostheses to improve their biocompatibility. In order to understand the effects of this treatment on the PTFE polymer surface, we have first realized RF-plasma treatment experiments on PTFE films. Preliminary XPS analyses have shown that about 15% of the surface atoms were substituted by nitrogen (N/C ratio of 0.3) whereas the F/C ratio decreased from 2 to 0.6, therefore leading to the conclusion that several chemical species are created onto the surface upon an ammonia plasma treatment. As X-Ray Photoelectron Spectroscopy (XPS) analysis does not allow the direct determination of the nature of the N-species grafted on the surface (chemical shifts are not different enough), vapor phase chemical derivatization was carried out on PTFE films to quantify the concentration of these new surface moities grafted on the polymer surface.

We have studied the polarized hydroxyapatite (HAp) whose surface was negatively or positively charged. In this study, we assessed the interfaces in vitro and in vivo periodically. As in vitro experiment, samples were immersed in simulated body fluid for 7 days and the surface was examined by scanning electron microscope (SEM). As in vivo experiments, cortical bone defects were created on the femoral trochanters and the condyles of the six Japanese white rabbits and the samples were implanted. The rabbits were sacrificed at 1, 2 and 4 W after the operation to analyze the surfaces by the SEM and the optical microscopy. In this study, a new thick apatite layer was formed on the negatively charged surface (N-surface) after 1week immersion in SBF in vitro. Besides, significant new bone formation was found at 2 weeks after the operation on N-surface in vivo, which was earlier than positively charged or non-polarized HAp surface. From this study negatively charged HAp surface by polarization accelerated the HAp crystal growth or the new bone formation. Thus, this N-surface will be promising for earlier fixation of the prosthesis or better recovery of the bone defect.

The objective of this research is to fabricate injectable, polymeric composites that will act as scaffolds for bone ingrowth as well as carriers for the controlled release of bone growth factors. To that end, the injectable polyester poly(propylene fumarate) (PPF) was loaded with poly(DLlactic-co-glycolic acid) (PLGA) microparticles carrying the model drug FITC-dextran. This preparation was then crosslinked with N-vinyl pyrrolidinone in the presence of benzoyl peroxide as initiator and sodium chloride (NaCl) as leachable porogen. The encapsulation of growth factors in microparticles is necessary to minimize their denaturation during scaffold crosslinking. PLGA microparticles (0.04 g microparticles/g PPF) were incorporated into PPF composites having variable NaCl weight percents (50 and 70 wt% NaCl) and the effect on FITC-dextran release kinetics was determined in vitro for cylinders of diameter 6.5 mm and height 13.0 mm. The FITC-dextran loaded microparticles alone exhibited a large initial burst effect, while the composite materials displayed a smaller burst effect and a longer linear region of release. At day 3, 54.6±2.1%, 5.1±0.9%, and 12.5±0.3% of loaded FITC-dextran was released into pH 7.4 phosphate buffered saline from the microparticles, the 50 wt% NaCl, and the 70 wt% NaCl composites, respectively. By day 28, 90.9±6.9%, 12.7±1.7%, and 34.4±0.4% of loaded FITC-dextran was released. Our results demonstrate that PLGA microparticles can be incorporated into PPF composites and that the release kinetics of FITC-dextran can be systematically manipulated through alteration of the composite initial salt content.

This paper describes the results of bioactivity responses to different ultra high molecular weight polyethylene (UHMWPE) particles. Particles were produced by a wear tester using two different textures of steel counters, one with cross-hatched and the other with uni-hatched grooves. These two textured surfaces produced two distinct populations of wear particles. One is larger and more elongated (fibril shapes) than the other. The mean sizes and aspect ratios of the particles are in the ranges of 5 μm to 25 μm and about 1.5 to 3, respectively. These two distinct UHMWPE particles were examined through in-vitro and in-vivo tests. Macrophages RAW 264.7 and the murine air-pouch model of inflammation were employed to characterize the effect of the particle size and shape. Preliminary in-vivo tests results showed that more elongated and larger particles enhanced bio-reactions.

A novel, water-soluble AB-block copolymer of diethylaminoethyl methacrylate (DEAEM) and poly(ethylene glycol) (PEG) was synthesized by anionic polymerization. Poly(ethylene glycol) methyl ether (PEGME) was converted into the corresponding potassium salt by reacting with potassium metal. The PEG salt was used as a macroinitiator for the polymerization of DEAEM to yield a PEG-b-PDEAEM block copolymer. Carbon dioxide was used to terminate DEAEM polymerization with a carboxylic acid group. This polymer, loaded with dye, was tested for pH sensitivity by release studies into solutions of various pH.

We report the synthesis of environmentally responsive hydrogels as nano-sized particles with core-shell morphologies. Composed of co-polymers of N-isopropylacrylamide with various co-monomers, these materials can be designed to render the core and shell responsive to different stimuli or to different magnitudes of the same stimulus. The measured phase transitions reflect the degree to which the two materials interact and thereby modulate the responsivity of the particle as a whole. Characterization of these materials is accomplished via dynamic light scattering and electron microscopy.

The development and implementation of successful transdermal devices for drug delivery requires an understanding of the adhesion occurring between the device and the soft dermal layer. This study utilizes a mechanics approach to quantify the adhesive properties of representative pressure sensitive adhesives (PSAs) used as the adhesive layer in these systems. Debonding of PSAs is accompanied by cavitation in the PSA and the formation of an extensive cohesive zone behind the debond tip. The presence of such large-scale bridging provides significant energy dissipation and increased resistance to delamination. The strain energy release rate (G) during debonding of a cantilever-beam sample, containing at its midline a thin layer of PSA, was utilized to quantify the adhesion of the PSA. The analysis accounts for both the work of adhesion as well as the viscoelastic constitutive behavior of the soft adhesive layer. Effects of strain rate, physiological environment, and permeation-enhancement additions are considered. The resistance of human stratum corneum to debonding between corneocyte layers is also presented, as knowledge of this parameter is essential for developing techniques to test the fracture resistance of the PSA-stratum corneum interface present in the clinical use of these transdermal devices.

This research examines the microstructure of polyanhydride blends for use in drug delivery devices. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) studies were performed on the homopolymers and blends of the polyanhydrides poly(1,6-carboxyphenoxy hexane) (CPH) and poly(sebacic anhydride) (SA). AFM of the CPH/SA blends 20:80, 50:50, and 80:20 showed distinct patterns indicating spinodal decomposition and phase separation on the micron-scale. Because it has been shown that incorporated drugs will thermodynamically partition into phase-separated domains depending on their hydrophobicity, polyanhydride blends will be able to encapsulate larger bioactive compounds including nucleotides, proteins, and vaccines. Preliminary SAXS studies of the CPH/SA blend systems provide information on the crystalline morphology of the polymer. A peak shift to a lower q from poly(SA) to the blends indicates that the poly(CPH) is incorporated into and causes swelling of the interlamellar amorphous regions of poly(SA).

The effect of lower critical solution temperature (LCST) phase separation on the crystallization of poly(ε-caprolactone) PCL in PCL/poly(D,L-lactide) (PDLA) blends is studied by simultaneous small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS). Phase separation is induced by controlled temperature jumps into the LCST (two-phase) region, which is above the melting temperature (60 °C) of PCL and the glass transition temperature (50 °C) of PDLA. We have followed the nanoscale structural changes (< 100 nm) during subsequent crystallization at 45 °C of critical (0.36 PCL) and off-critical (0.50 PCL) blend compositions in both one-phase and two-phase melts. The spherulite morphology (1-100 μm) is examined with optical microscopy. When crystallization follows LCST phase separation, the shape, size and distribution of the spherulites depends on the extent of melt phase separation. In our x-ray measurements, the WAXS crystallinity of PCL is less than 40 % for the temperature range of interest. We perform a correlation function and intensity model analysis of our SAXS data to obtain morphological variables that characterize the intraspherulitic morphology. These morphological variables are relatively constant during crystallization and are also independent of melt phase separation. On the other hand, the ultimate crystallinity and the crystallization rate depend on the extent of melt phase separation.

Tissue engineering aims at creating new tissues as alternatives to organ transplants. Our approach is to incorporate cells into biodegradable polymer scaffolds designed to temporarily support new tissue formation. An important requirement of scaffolds is homogeneity. Homogeneity ensures structural integrity, uniform distribution of cells, and uniform porosity throughout the scaffold. Controlling pore sizes is necessary to regulate exchange of nutrients and waste products for cells. Pores too large can provide entryway to immune cells that can harm allogenic cells and developing tissue. We have fabricated three-dimensionally defined, homogeneous, ionically crosslinked alginate gels with a controlled slow-gelation system involving CaCO3 and D-glucono-δ-lactone. We varied the structural parameters and alginate types of these gels to control the diffusion of glucose, vitamin B12 and FITC-dextran (molecular weights of 180, 1355 and 9500, respectively) through the gels. Experiments were performed with gel discs placed between side-by-side donor and receptor chambers in a humidified incubator at 37°C. Samples were taken periodically and measured on a UV-Vis spectrophotometer. Generally, diffusion coefficient (D) increased with decreasing solute size. Varying structural parameters of the gels did not have a significant effect on diffusivity of vitamin B12. In contrast, for gels made with a Ca2+ to carboxyl molar ratio of 0.36, D of FITCdextran increased from (2.88 ± 0.52)×10-7 to (4.66 ± 0.48)×10-7 cm2/sec as alginate concentration decreased from 3.2% to 1.5%, respectively. D of FITC-dextran also increased from (3.17 ± 0.30)×10-7 to (4.66 ± 0.48)×10-7 cm2/sec as crosslinking density decreased for 1.5% alginate gels from a Ca2+ to carboxyl molar ratio of 0.72 to 0.36, respectively. D was highest for alginate gels with the highest guluronic acid content. Controlling diffusivity allows alginate gels with specific properties to be fabricated for tissue engineering scaffolding and other biomedical applications.

Calcium phosphate in the form of apatite has been successfully precipitated on the surface of liposomes. Liposome vesicles were prepared by sonication of phosphatidylcholine and this was introduced into an aqueous solution of calcium and phosphate ions supersaturated with respect to hydroxyapatite. Calcium phosphate was shown to precipitate solely on the outer layer surface of the liposome vesicles. These composite assemblies were then deposited onto a stainless steel cathode substrate using an electrophoretic method at physiological temperatures.

Scanning electron microscopy (SEM), transmission electron microscopy (TEM), powder xray diffraction (PXRD) and Fourier transform infrared spectroscopy (FTIR) were used to analyse the morphology, structure and chemical composition of the composite coating. The results from PXRD and FTIR show a mixture of amorphous and poor crystalline hydroxyapatite (HAp). This was verified by electron diffraction. Dark field images confirmed that the precipitated HAp deposited solely at the outer surface of the liposomes. SEM micrographs demonstrated a thin uniform coating at the microstructure level. These results suggest that these calcium phosphateliposome composites can be formed. They have tremendous potential for use as drug delivery devices in aiding the treatments of bone disorders.

Two alternatives to standard tensile testing of arteries are discussed. The first involves inflation of arteries and simultaneous measurement of radial displacement with intra-vascular ultrasound (IVUS). The second involves the measurement of load versus displacement during micro-indentation of the intimal surface. The IVUS technique is used to study the nonlinear stiffening of porcine coronaries during inflation and, ultimately, it may provide a method to determine mechanical properties in vivo. Processing of the IVUS data relies on accurate determination of the luminal/intimal and medial/advential boundaries during inflation. The microindentation technique is used to study the effect of loading rate on tissue stiffness, recovery, and internal dissipation. Ultimately, this technique may provide a method to measure local mechanical properties in the vicinity of an atherosclerotic plaque, for example. Accurate determination of the initial contact point between the indenter and intima is required, however. The techniques appear to successfully capture significant, nonlinear, time-dependent properties of arterial tissue.

We have developed a novel bilayer matrix composed of a porous type I collagen layer that transitions into a hyaluronate gel layer. This study evaluates the potential of the bilayer matrix to support the in vitro and in vivo formation of both bone and cartilage tissue. In the presence of recombinant human growth and differentiation factor-5, fetal rat calvarial cells cultured in the HA layer grew in a round, aggregated, chondrocyte-like morphology, while those in the collagen layer grew flattened and spread. Biochemical analysis demonstrated that cells in the collagen layer expressed higher levels of alkaline phosphatase activity, and lower levels of sulfated glycosaminoglycans and type II collagen when compared to cells in the HA layer. Intramuscular implants of the bilayer matrix with growth factor retrieved at 28 days revealed the presence of bone and cartilage tissue in the collagen and hyaluronate layers, respectively. These results demonstrate that the differentiation of cells in response to a single growth factor can be guided by specific compositional changes of the extracellular matrix.

An experimental study was conducted to determine the mechanisms of transport for delivery of cardiovascular agents using a pH-sensitive hydrogel as the carrier. Copolymer gels based on hydrophilic (2-hydroxyethyl methacrylate) and polybasic (N,N-diethylaminoethyl methacrylate) monomers were formed as membranes and analyzed for their potential to control the diffusion of model solutes as well as heparin and streptokinase. The polybasic materials were selected because they would allow drug delivery to be triggered by microenvironmental pH fluctuations around the site of a blood clot. In slightly basic solutions, the polymers remained in a thermodynamic state of phase-separation, while the polymer absorbed more solution and the mesh size increased once the pH was less than the pKb of the polybasic moiety. The hydrogels' equilibrium swelling ratios were determined as a function of pH, and the mesh sizes were determined by rubber elasticity measurements. Diffusion of model solutes, as well as heparin, was studied using side-by-side diffusion cells to determine the influence of gel morphology and mesh size on the screening of the solutes. Streptokinase release from these gels was modulated by environmental pH.

This research involves synthesis and characterization of biodegradable 3-dimensional polymer/calcium phosphate hybrid matrices as scaffolds for bone tissue regeneration. The scaffolds are comprised of chitosan and calcium phosphates [β-tricalcium phosphate (β-TCP), invert glass] and have combined optimum mechanical and biological properties of the two materials. Chitosan is a biocompatible and biodegradable polymer, and has rich hydroxyl groups for surface modification. Calcium phosphate crystals and invert glass are used as powder fillers to reinforce the scaffolds and increase bioactivity of the scaffolds. The scaffolds are fabricated using a thermally induced phase separation technique. The principal advantages of this technique are its low cost, low shrinkage levels, low sintering temperatures, and its ability to produce a variety of microstructures of various shapes and sizes. The osteoblast-like MG63 cells are seeded on the scaffolds to study the attachment and proliferation of the cells. The hydroxyl groups on the scaffolds are used to graft arginine-glycine-aspartate (RGD) peptides for promoting bone cell attachment and new tissue formation.