DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells.

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes/cm(2) for 6 or 24 h (1 dyne = 10 microN), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF/GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine/spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells.

Analytical and mechanical testing of high velocity oxy-fuel thermal sprayed and plasma sprayed calcium phosphate coatings.

Plasma spraying (PS) is the most frequently used coating technique for implants; however, in other industries a cheaper, more efficient process, high-velocity oxy-fuel thermal spraying (HVOF), is in use. This process provides higher purity, denser, more adherent coatings than plasma spraying. The primary objective of this work was to determine if the use of HVOF could improve the mechanical properties of calcium phosphate coatings. Previous studies have shown that HVOF calcium phosphate coatings are more crystalline than plasma sprayed coatings. In addition, because the coatings are exposed to more complex loading profiles in vivo than standard ASTM tensile tests provide, a secondary objective of this study was to determine the applicability of four-point bend testing for these coatings. Coatings produced by HVOF and PS were analyzed by profilometry, diffuse reflectance Fourier transform infrared spectroscopy, X-ray diffraction, four-point bend, and ASTM C633 tensile testing. HVOF coatings were found to have lower amorphous calcium phosphate content, higher roughness values, and lower ASTM C633 bond strengths than PS coatings; however, both coatings had similar crystal unit cell sizes, phases present (including hydroxyapatite, beta-tricalcium phosphate, and tetracalcium phosphate), and four-point bend bond strengths. Thus, the chemical, structural, and mechanical results of this study, in general, indicate that the use of HVOF to produce calcium phosphate coatings is equivalent to those produced by plasma spraying.

Dissolution/reprecipitation of calcium phosphate thin films produced by ion beam sputter deposition technique.

The dissolution, reprecipitation and protein adsorption properties of amorphous CaP bioceramic thin films produced with an ion beam sputter deposition technique using hydroxyapatite (HA) and fluorapatite (FA) as starting materials were studied using Fourier transform infrared spectroscopy (FTIR) with attenuated total internal reflectance (ATR). Our studies showed that these amorphous CaP coatings dissolved to a greater extent when exposed to bovine serum albumin (BSA) in saline solution when compared to a protein free saline solution. Analysis of changes in infrared spectra revealed that coatings exposed to BSA solution exhibited a higher degree of crystalline structure after dissolution/reprecipitation than those exposed to saline alone. There was the indication that the association of inorganic and organic contents was achieved on the coating surface in BSA solution. We could detect no significant difference between the coatings produced from HA and FA targets.

Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces.

The protein adsorption behavior of thin films of calcium phosphate (CaP) bioceramic and titanium (Ti) was studied in this research. The thin films were produced with an ion beam sputter deposition technique using targets of hydroxyapatite (HA), fluorapatite (FA) and titanium (Ti). Fourier transform infrared spectroscopy (FTIR) with attenuated total internal reflectance (ATR) was used to evaluate protein adsorption on these surfaces. This study showed that surface composition and structure influenced the kinetics of protein adsorption and the structure of adsorbed protein. CaP surfaces adsorbed greater amount of protein than the Ti surface, and caused more alteration of the structure of adsorbed BSA than did the Ti surface. The differences in protein adsorption behavior could result in very different initial cellular behavior on CaP and Ti implant surfaces.

FTIR/ATR for protein adsorption to biomaterial surfaces.

It is now well accepted that the initial rapid adsorption of blood proteins to biomaterial surfaces is important in the long-term performance of the implant. Cells that interact with the implant will be reacting to a layer (single or multiple) of adsorbed protein. The parameters of importance in a study of protein adsorption to surfaces of biomaterial interest include total amounts of different adsorbed proteins and the conformation and orientation of these adsorbed proteins. Researchers have developed a number of techniques with which we can now address all these questions. In this paper, we have discussed how Fourier transform infrared (FTIR) attenuated total internal reflection (ATR) techniques can be used for the study of biomaterial surfaces and events at biomaterial surfaces such as protein adsorption. FTIR spectroscopy offers higher signal-to-noise and speeds than spectrometers that use gratings and hence offers the capability of observing the critical early events when proteins interact with surfaces. Perhaps the biggest advantage of the FTIR technique over dispersive spectrometers is wavelength precision. This allows the subtraction of water, a strong infrared absorber, from the spectra of proteins in aqueous solutions. This review starts with an introduction of how ATR can be used to provide information about proteins on surfaces. Equations to calculate the amount of proteins adsorbed to surfaces from analysis of ATR spectra are presented. A discussion of the kinds of surfaces that can be analyzed by FTIR/ATR and difficulties with the subtraction of H2O is given. The rest of the review deals with how information of interest to biomaterials researchers such as kinetics of protein adsorption, changes in protein secondary structure and orientation upon adsorption to surfaces can be obtained by FTIR/ATR.

Dissolution/reprecipitation and protein adsorption studies of calcium phosphate coatings by FT-IR/ATR techniques.

The surfaces of bioactive Ca-P ceramics immediately change when exposed to proteinaceous solutions. The dissolution behavior and protein interactions of these bioactive materials at the bone/implant interface need to be investigated to understand their material-cellular interactions fully. In this study, FT-IR/ATR techniques were used to study the in situ phosphate release kinetics of Ca-P coatings. The net loss of phosphate molecules from coatings was slower in saline solutions compared with alpha-MEM solutions. Coatings exposed to alpha-MEM solutions containing fibronectin released phosphate molecules slower than coatings exposed to alpha-MEM solutions containing albumin. Conformational changes in fibronectin and albumin adsorbed onto Ca-P and uncoated germanium surfaces were also investigated using FT-IR/ATR spectroscopy. Analysis of changes in the amide I bands indicated that there was a greater loss of beta-sheet structure in adsorbed fibronectin on Ca-P coatings when compared with bare germanium surfaces. Although albumin did change its structure upon adsorption on both Ca-P and germanium, unlike fibronectin, adsorbed albumin structure was similar on Ca-P coatings and germanium. Furthermore, with time the conformation of adsorbed fibronectin and albumin appeared to be very stable on Ca-P coatings, whereas albumin adsorbed to germanium exhibited an increase in ratio of alpha-helix to beta-turn.

Evaluation of infrared spectroscopy as a bacterial identification method.

A study motivated by the recent revival of interest in the use of IR spectroscopy to identify bacteria is reported. A library of FT-IR spectra of dried bacterial films was complied using 16 different strains. A test set was compiled from spectra of the same strains grown several months later. The test set was quantitatively compared with the library on the basis of spectral similarity in the region 980-1190 cm-1. Six of the strains in the test set were not matched with the correct strain in the library despite efforts to reproduce the conditions under which cells were grown and prepared. The results suggest that reproducibility of the bacterial spectra is a potential difficulty that must be addressed by any attempts to develop FT-IR spectroscopy as a bacterial identification method.

IR spectral changes of bovine serum albumin upon surface adsorption.

The adsorption of bovine serum albumin from flowing solutions onto germanium and three polyetherurethanes varying in soft segment content was studied by a Fourier transform infrared/attenuated total reflectance technique. Spectral differences observed in the amide I, II, and III regions upon adsorption to all four surfaces were consistent with a loss of helix and gain of beta-structure. There appeared to be a slight difference between BSA adsorbed to germanium and the PEUs, but no distinction could be made between BSA adsorbed to the different PEUs.

Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins.

Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by 125I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml.