Surface properties of electrically conducting polymers, including charge-density and wettability, can be altered reversibly by oxidation or reduction. Thus, these materials are interesting systems on which to study the interactions of proteins and cells with surfaces. The properties of polypyrrole were studied in environments suitable for protein adsorption and cell culture. Optically transparent films of polypyrrole were synthesized, and the stability of the oxidized and reduced states in aqueous media were studied. UV/VIS spectroscopy, cyclic voltammetry, and spectroelectrochemistry measurements confirmed the oxidation state of the polymer films. The positively-charged form of polypyrrole could be changed to its neutral form by applying a constant potential (-0.5 V vs. Ag/AgCl reference). It was found that the shape of bovine aortic endothelial cells on fibronectin-coated polypyrrole films can be controlled by varying the oxidation state of the polypyrrole.

Metastasis formation is dependent on the arrest and stabilization of adhesive interactions to prevent detachment from secondary sites. Primary receptor-ligand interactions are not sufficient to maintain prolonged adhesive contacts without secondary events that lead to stabilization. Tumor cell arrest and stabilization were studied under physiologically relevant shear conditions. We used a parallel-plate flow chamber with surfaces coated with human plasma fibronectin or vitronectin. Our previous work suggested that stabilization of cells to immobilized proteins is in part attributed to transglutaminase covalently cross-linking cytoskeletal-integrin-fibronectin multiprotein complexes via lysine-glutamine linkages. To study the role of integrins in mediating arrest and initiating stabilization we used a human melanoma line (70w) and polyclonal antibodies that inhibit the function of the fibronectin (α5β1) and vitronectin (αvβ3/β5) integrin receptors. To confirm the role of integrins in initiating stabilization we used CHO (Chinese hamster ovary) cells selected for low levels of α5β1 integrin expression and integrin transfected CHO cells selected for α5βl overexpression. The level of fibronectin receptor surface expression was inversely related to the adhesion stabilization lag time. These studies confirmed that integrins are essential for mediating arrest and initiating stabilization. They also confirm that secondary events are necessary for complete stabilization to occur. Finally, it is important to note that the arrest and stabilization methods we have developed are capable of detecting biologic effects at far greater sensitivity than static adhesion assays. Some examples of pharmacologic agents or biomaterials effects that can be detected using stabilization assays include: 1) very low drug doses, 2) very low levels of peptide, carbohydrate, and antibody inhibitors, 3) slight modification of endogenous protein expression by antisense oligonucleotides or transfected genetic expression constructs.

Cell-substratum adhesion is important in wound healing [4], embryogenic development [11], tissue architecture [6], and metastasis [7]. Integrins constitute a major class of heterodimeric cell-surface glycoproteins involved in receptor-mediated adhesion to the extracellular matrix (ECM). Focal contacts are regions of the cell-substratum adhesion in which clusters of integrin receptors connect the cytoskeleton to extracellular matrix molecules such as fibronectin. Focal contacts strengthen cell-substrate adhesion, and are sites of biochemical activity. Since cell adhesion strength in part depends on the cell's ability to cluster receptors and cytoskeleton into focal contacts, the integrity of the focal contact, and hence a cell's adhesive strength, will depend both on integrin-cytoskeletal binding as well as integrin-ligand binding.

Using a centrifugation assay, we have quantified cell-substratum adhesion strength of mouse 3T3 cells transfected with the avian β1 integrin receptor (wild type), including various deletion mutants of its cytoplasmic domain, to surfaces containing varying concentrations of CSAT, a monoclonal antibody against the extracellular domain of the avian β1 subunit. For all the transfectants, adhesion strength decreases with decreasing CSAT concentration and increasing centrifugal strength. Different truncations of the cytoplasmic domain lead to different levels of adhesion. There is no simple correlation between the length of the cytoplasmic domain and the strength of adhesion.

Polymers derived from lactic and glycolic acid have achieved success in many medical applications and have been investigated as possible materials for degradable scaffolds required in tissue transplantation. Previous investigators have found significant differences in the behavior of cells cultured on chemically similar polyesters of this family but the mechanisms for these differences have remained unclear.

In this study, we fabricated polylactic acid, polylactic-co-glycolic acid, and blend films by melt-processing. The bulk and surface properties of these films were characterized by GPC, DSC, WAXS, SEM, and contact angle. All films were smooth and amorphous and differed only in chemical composition. The films were assessed for their ability to sustain or promote hepatocyte attachment, growth, and function. Cells adhered to and exhibited similar morphology on all substrates. Cells decreased in number to 50–60% of the initial cell number but exhibited high retention of differentiated function on all of the polymer substrates over five days. Cell response was not statistically different on the different substrates indicating that chemical composition, in our range of polymer formulations, does not affect cell behavior.

Protein-permeable dense (non-porous) urethane membranes have been evaluated for in vitro cell culture, and in vivo cell encapsulation. Polyurethane membranes were designed to exhibit permeability to proteins, gases, and nutrients without the existence of pores. The membranes are non-cytotoxic, angiogenic, and permeable to gases, nutrients, secretagogues and cell products via purely concentration-driven transport. Non-anchorage and anchorage dependent cells were grown encapsulated within the membrane and with the membrane as a growth substrate. Several non-anchorage dependent cell types proliferated within the membrane both in-vitro and in-vivo. Anchorage-dependent cells were grown on the membranes as a substrate. Encapsulated cells have been maintained in culture for up to six months with nutrients supplied only by the external media. Immuno-isolation has been demonstrated with cells implanted into murine hosts. Explants of membrane encapsulated cells exhibited a high degree of vascularization, with little or no fibrous tissue. The ability to support cell growth and function, and the ability to protect xenogenic cells from immunologic rejection suggest that the membranes would be useful in the construction of hybrid artificial organs, devices for cell transplantation, and substrates for cell and tissue culture.

Encapsulated cells are likely to encounter transport limitations of trace nutrients carried by macromolecules due to a high diffusion resistance in the encapsulating membrane. We modeled the transport of transferrin to encapsulated cells and found that the transport limitation of transferrin is potentially significant in comparison to oxygen, which is limiting in unencapsulated tissue. Since transferrin is the primary biological iron carrier, deprivation of transferrin to encapsulated cells may limit long term function and viability in vivo.

The series of cyclized RGD peptides in this study demonstrated a very low partition into octanol as judged by HPLC. Thus, these molecules are likely to move predominantly through the paracellular pathway. Permeability across Caco-2 monolayers was determined using reversed phase HPLC and found to be restricted by molecular weight and possibly charge-charge interactions between the solute and charged moieties within the paracellular shunt. When normalized for molecular weight, molecules with a net charge between -1 and -2 demonstrated the highest permeabilities, which suggests an optimal net charge with respect to permeability.

There is a need for an in situ assay to quantify tissue reactivity to sustained release of bFGF to better understand and control growth factor-induced angiogenesis. To this end we have adapted the alginate/heparin-sepharose release system for use in the mouse dorsal skinfold chamber. A mathematical model was used to predict the time dependence of bFGF release as a function of bFGF loading, heparin concentration, and device geometry. The model predictions agreed well with previously reported in vitro data. In vivo studies to correlate blood vessel growth as a function of release rate are in progress.

The glucose responsive hydrogels developed by Horbett et al. [1, 2] are potentially useful as materials for constructing glucose sensors. The construction of microsensors presents challenges in developing processes for preparing these gels that are compatible with semiconductor fabrication technology.

We have lithographically patterned a pH-sensitive copolymer (poly-(2-hydroxyethyl methacrylate-co-dimethyl aminoethyl methacrylate) 90:10 (mole ratio) (HEMA/DMA) from a prepolymer solution of monomers, crosslinker, solvent, and a viscosity agent (p(HEMA)). Varying the spin speed and p(HEMA) concentration resulted in reproducible thicknesses between 5 and 25 μm. pH sensors produced by this method showed sensitivity ranging from 20 to 50%/pH and a response time (10%–90%) of 6.8 minutes. Sensitivity dropped by a factor of two over a 24 day testing period. The limited lifetime is thought to be due to loss of adhesion of the polymer to the substrate.

Derivatizing glucose oxidase with a photoactivatible crosslinker allowed it to be covalently immobilized onto the surface of the gel layer in a spatially specific manner. After immobilization, GOX specific staining showed that enzyme activity was preserved, however the amount of enzyme immobilized was insufficient to create a working glucose sensor.

Novel graft copolymeric, pH responsive hydrogels have been synthesized by solution polymerization. Poly(methacrylic acid-g-ethylene glycol) hydrogels exhibit reversible complexation as a function of pH because of hydrogen bonding between the PEG grafts and the PMAA main chains. These gels are being developed as pH responsive membranes for use in biomedical applications. ATR-FTIR results indicate that there is hydrogen bonding occurring in membranes swollen under conditions favorable for complexation. Equilibrium swelling studies have shown that the pH of the surrounding environment has a significant effect on the swelling behavior of these membranes. The swelling behavior has also been shown to respond rapidly to changing pH conditions. P(MAA-g-EG) membranes also show pH sensitive permeability toward solutes as a result of this environmentally sensitive swelling.

The temperature response of hydrogels made from hydrophobically modified water soluble polymers is complicated by the fact that while the hydrophobilic backbone becomes dehydrated and thus less compatible with aqueous solvents as the temperature is increased, the hydrophobic side chains become more compatible. These opposing effects taken together will govern gel properties such as volume, storage modulus, etc. We will discuss the effects of temperature on the composition, volume and reheological properties of hydrogels made from hydrophobically modified hydroxyethyl cellulose (HMHEC) in aqueous solutions containing the surfactant sodium dodecyl sulfate (SDS).

Polymeric hydrogels capable of reversible swelling with changes in environmental temperature and pH were synthesized and studied as matrix systems capable of controlled release of antithrombotic drugs to the site of a blood clot. Monomers N-isopropylacrylamide (NIPAAm) and methacrylic acid (MAA) were chosen for the temperature- and pHdependent swelling characteristics of their polymers, respectively. Equilibrium swelling studies were performed as functions of pH and of temperature, and kinetic swelling studies were performed on the thermally responsive gels as a function of time after the gel was subjected to a step-change in temperature. Experimental results indicate that P(NIPAAmco- MAA) hydrogels can be synthesized so that they are either pH- or temperaturesensitive, depending upon the composition of the network. The pure PNIPAAm gels showed a lower critical solubility temperature (LCST) of around 32°C, below which the gel was in its swollen state, and above which the gel collapsed. As the amount of methacrylic acid in the gels was increased, the degree of swelling in deionized water decreased and the temperature sensitivity was lost. The hydrogels containing MAA displayed a transition in swelling behavior between pH 4 and 6. The mesh sizes of the hydrogel networks were calculated from the results of the swelling studies by Flory-Rehner theory. A kinetic experiment on the temperature-sensitive pure PNIPAAm hydrogel showed that the polymer network collapsed rapidly upon temperature changes across the LCST.

Release studies from a lipid-based matrix, known as the bicontinuous cubic phase, are presented. This matrix consists of nano-sized pores within which various proteins and drugs can be dispersed and subsequently released to the exterior. To control the release rate, the aqueous pores of the cubic phase were gelled using sodium alginate, a water soluble polysaccharide. Studies show that the release rate is significantly lowered upon gelation and the first order release profile exhibited by the ungelled cubic phase is converted to a zeroorder linear profile. Further, it has been shown that the release trends can be reversed by degelation. This opens up the possibility of releasing large quantities of the protein when required (drugs on demand concept) by degelling the gelled samples.

Poly(vinyl alcohol) microparticles were prepared by a novel freezing-thawing process in the absence of a crosslinking agent. An aqueous PVA solution to which 1.25 wt% sodium lauryl sulfate was added was dispersed in corn oil. The system was agitated and the ensuing suspended droplets of PVA solution were solidified by a cyclic freezing-thawing process. Key parameters of the process were the PVA to corn oil ratio, the amount of surfactant added, agitation speed, number of freeze cycles, temperatures of freezing and thawing, and presence of additional components.

Crystallization was observed during the freezing-thawing process. The degree of crystallinity was measured with differential scanning calorimetry. Bovine serum albumin was incorporated into the particles by an absorption technique. Studies of BSA release from the microparticles in vitro showed that the release could be prolonged for up to 7 days. BSA diffusion coefficients were calculated from these data and the release mechanism was identified.

The interactions between a therapeutic protein and the surfaces of a delivery device may play a pivotal role in the efficacy of a recombinant protein-based biologic therapy program. Protein adsorption may induce conformational changes and aggregation, resulting in the inactivation of the protein of interest and even malfunction of the delivery device itself. We studied the interactions of formulated interleukin-2 (IL-2) with the surfaces of a commonly used pump-based delivery system at 37°C over a 24-hour period. Delivered protein concentration and bioactivity assays were performed at intervals in order to quantitate the impact of exposure time. Adsorption to the catheter tubing walls resulted in 20 to 30% reduction in the concentration of delivered IL-2 relative to the initial concentration. After 2 hours of operation, delivered IL-2 bioactivity levels fell to -10% of the initial activity levels. These activity losses were accompanied by a significant decrease in the protein's helical content, as monitored by circular dichroism spectroscopy. Attenuated total reflectance Fourier transform infrared spectroscopy indicated that adsorbed IL-2 had non native secondary structure contents. We hypothesize that surface-mediated IL-2 denaturation is responsible for the majority of the observed activity losses

Using Fourier-transform infrared (FTIR) spectroscopy we have characterized a polyanhydride copolymer series composed of various ratios of the diacids 1,3-bis(p -carboxyphenoxy)propane (CPP) and sebacic acid (SA). Typical peaks corresponding to the aliphatic-aliphatic (SA-SA), aromatic-aliphatic (CPP-SA), and aromatic-aromatic (CPP-CPP) diads were found in the 1820- 1710 cm−1 wavenumber range. Further peaks corresponding to the SA-SA diads were identified in the fingerprint region at 1382, 1360, 1307, and 1286 cm−1. These peak characterizations facilitate identification of bond distribution in the CPP-SA copolymer as well as other polyanhydride copolymers, and correlate well with previously presented information obtained with nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction.

This paper reports a preliminary spectroscopic characterization of the surface elemental and phase compositions of Ti49Ni51 alloy treated using various sterilization procedures (autoclaving in water, steam, sealed envelopes; boiling in water and chemical etching). The surface of TiNi is found to consist of a thin oxide covered by a carbon-dominated contamination layer. The surface oxide of autoclaved samples is (TiO2)xNiy where y varies in the range 0-6 at.% depending on the surface preparation procedure. Minor amounts of suboxides as well as metallic Ni are also detected. Preliminary estimations of the oxide film thickness showed that it varies in the range 7- 26 nm depending on the employed method of sterilization. Mechanisms of surface state formation are briefly discussed.

A new poly(ethylene oxide) based biomaterial has been synthesized by co-crosslinking linear poly(ethylene oxide) (PEO) with tetraethylene glycol (TEG) in aqueous solution using electron beam radiation. This process results in hydrogels with variable concentrations of hydroxyl groups, tunable by adjusting the concentration of TEG in solution. FTIR analysis of these gels shows no changes in chemical composition on crosslinking.

Our laboratory has been developing a particulate composite material as a temporary replacement for trabecular bone. The material is degradable in physiologic fluids, and uses starting materials that the body can metabolize and excrete. This study investigates the influence of the molecular weight of one component, a linear polyester, on the mechanical strength of the composite material. The compressive strength and modulus increase from “low” to “medium” molecular weight, but do not increase further from “medium” to “high” molecular weight. Low, medium, and high are relative to the highest molecular weight attainable with the current reaction scheme, which is a number average molecular weight (Mn) of 2,038 and a weight average molecular weight (Mw) of 11,916.

Porous glass templates of pore size 75–200 14m and 20–30% porosity were synthesized. The glass disks were conditioned in a modified tris buffer (SBF) for 48 hrs and then treated with tissue culture medium (TCM) for 1 hr at 37°C. Other porous glass templates were treated with either SBF or TCM. The conditioned glass disks were seeded with 106 neonatal rat calvaria osteoblasts and maintained in culture for 2, 5 or 7 days. It was found that the bioactive glass was rapidly invaded by cells which colonized the porous template. Morphological and biochemical analyses suggested that a bone-like tissue was formed inside templates conditioned with both SBF and TCM. By 7 days, the cells exhibited high alkaline phosphatase activity and synthesized osteocalcin. SDS-PAGE indicated the presence of type I collagen. SEM-EDAX analysis showed the formation of a bone-like tissue throughout the entire sample, while inspection of the Ca:P ratio indicated that the cells synthesized a mineral phase. Indeed, the FTIR spectra of the mineral confirmed that it was a biological hydroxyapatite. The rapid formation of the bone-like material within the template in vitro suggests that this template may be valuable clinically for the repair of bone lesions.