Osteogenic differentiation of human mesenchymal stem cells in the absence of osteogenic supplements: A surface-roughness gradient study.

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (∼0.5-4.7 µm), and mean distance between peaks (RSm) gradually varying from ∼214 µm to 33 µm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra∼1.53 µm/RSm∼79 µm in Dex-deprived OI medium, and Ra∼0.93 µm/RSm∼135 µm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone. STATEMENT OF SIGNIFICANCE: Biodegradable polymers, such as polycaprolactone (PCL), are promising materials in the field of tissue engineering and regenerative medicine, which aims at creating viable options to replace permanent orthopedic implants. The material, cells, and growth-stimulating factors are often referred to as the key components of engineered tissues. In this article, we studied the hypothesis of specific surface modification of PCL being capable of inducing mesenchymal stem cell differentiation in bone cells in the absence of cell-differentiating factors. The systematic investigation of the linearly varying surface-roughness gradient showed that an average PCL roughness of 0.93 µm alone can serve as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone.

Antimicrobial properties of 8-hydroxyserrulat-14-en-19-oic acid for treatment of implant-associated infections.

Treatment options are limited for implant-associated infections (IAI) that are mainly caused by biofilm-forming staphylococci. We report here on the activity of the serrulatane compound 8-hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. EN4 elicited antimicrobial activity toward various Gram-positive bacteria but not to Gram-negative bacteria. It showed a similar bactericidal effect against logarithmic-phase, stationary-phase, and adherent Staphylococcus epidermidis, as well as against methicillin-susceptible and methicillin-resistant S. aureus with MICs of 25 to 50 µg/ml and MBCs of 50 to 100 µg/ml. The bactericidal activity of EN4 was similar against S. epidermidis and its Δica mutant, which is unable to produce polysaccharide intercellular adhesin-mediated biofilm. In time-kill studies, EN4 exhibited a rapid and concentration-dependent killing of staphylococci, reducing bacterial counts by >3 log(10) CFU/ml within 5 min at concentrations of >50 µg/ml. Investigation of the mode of action of EN4 revealed membranolytic properties and a general inhibition of macromolecular biosynthesis, suggesting a multitarget activity. In vitro-tested cytotoxicity on eukaryotic cells was time and concentration dependent in the range of the MBCs. EN4 was then tested in a mouse tissue cage model, where it showed neither bactericidal nor cytotoxic effects, indicating an inhibition of its activity. Inhibition assays revealed that this was caused by interactions with albumin. Overall, these findings suggest that, upon structural changes, EN4 might be a promising pharmacophore for the development of new antimicrobials to treat IAI.

One-step method for generating PEG-like plasma polymer gradients: chemical characterization and analysis of protein interactions.

In this work we report a one-step method for the fabrication of poly(ethylene glycol) PEG-like chemical gradients, which were deposited via continuous wave radio frequency glow discharge plasma polymerization of diethylene glycol dimethyl ether (DG). A knife edge top electrode was used to produce the gradient coatings at plasma load powers of 5 and 30 W. The chemistry across the gradients was analyzed using a number of complementary techniques including spatially resolved synchrotron source grazing incidence FTIR microspectroscopy, X-ray photoelectron spectroscopy (XPS) and synchrotron source near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Gradients deposited at lower load power retained a higher degree of monomer like functionality as did the central region directly underneath the knife edge electrode of each gradient film. Surface derivatization experiments were employed to investigate the concentration of residual ether units in the films. In addition, surface derivatization was used to investigate the reactivity of the gradient films toward primary amine groups in a graft copolymer of poly (L-lysine) and poly(ethylene glycol) (PLL-g-PEG copolymer) which was correlated to residual aldehyde, ketone and carboxylic acid functionalities within the films. The protein adsorption characteristics of the gradients were analyzed using three proteins of varying size and charge. Protein adsorption varied and was dependent on the chemistry and the physical properties (such as size and charge) of the proteins. A correlation between the concentration of ether functionality and the protein fouling characteristics along the gradient films was observed. The gradient coating technique developed in this work allows for the efficient and high-throughput study of biomaterial gradient coating interactions.

Characterization of supported lipid bilayers incorporating the phosphoinositides phosphatidylinositol 4,5-biphosphate and phosphoinositol-3,4,5-triphosphate by complementary techniques.

Phosphoinositides are involved in a large number of processes in cells and it is very demanding to study individual protein-lipid interactions in vivo due to their rapid turnover and involvement in simultaneous events. Supported lipid bilayers (SLBs) containing controlled amounts of phosphoinositides provide a defined model system where important specific recognition events involving phosphoinositides can be systematically investigated using surface sensitive analytical techniques. The authors have demonstrated the formation and characterized the assembly kinetics of SLBs incorporating phosphatidylinositol 4,5-biphosphate (PIP(2); 1, 5, and 10 wt %) and phosphoinositol-3,4,5-triphosphate (1 wt %) using the quartz crystal microbalance with dissipation monitoring and fluorescence recovery after photobleaching. An increased fraction of phosphoinositides led to a higher barrier to liposome fusion, but full fluidity for the phosphatidylcholine lipids in the formed SLB. Significantly, the majority of phosphoinositides were shown to be immobile. X-ray photoelectron spectroscopy was used for the first time to verify that the PIP(2) fraction of lipids in the SLB scales linearly with the amount mixed in from stock solutions.

Covalent immobilization of antibacterial furanones via photochemical activation of perfluorophenylazide.

N-(3-trimethoxysilylpropyl)-4-azido-2,3,5,6-tetrafluorobenzamide (PFPA-silane) was used as a photoactive cross-linker to immobilize antibacterial furanone molecules on silicon oxide surfaces. This immobilization strategy is useful, especially for substrates and molecules that lack reactive functional groups. To this end, cleaned wafers were initially incubated in solutions of different concentrations of PFPA-silane to form a monolayer presenting azido groups on the surface. The functionalized surfaces were then treated with a furanone solution followed by illumination with UV light and extensive rinsing with ethanol to remove noncovalently adhered molecules. In the presented study, we demonstrate the ability to control the surface density of the immobilized furanone molecules by adjusting the concentration of PFPA-silane solution used for surface functionalization using complementary surface analytical techniques. The fluorine in PFPA-silane and the bromine in furanone molecules were convenient markers for the XPS study. The ellipsometric layer thickness of the immobilized furanone molecules on the surface decreased with decreasing PFPA-silane concentration, which correlated with a decline of water contact angle as a sign of film collapse. The intensity of characteristic azide vibration in the MTR IR spectra was monitored as a function of PFPA-silane concentration, and the peak disappeared completely after furanone application followed by UV irradiation. As a complementary technique to XPS, TOF-SIMS provided valuable information on the chemical and molecular structure of the modified surfaces and spatial distribution of the immobilized furanone molecules. Finally, this report presents a convenient, reproducible, and robust strategy to design antibacterial coating based on furanone compounds for applications in human health care.

Bovine serum albumin adsorption onto colloidal Al2O3 particles: a new model based on zeta potential and UV-vis measurements.

We investigated the adsorption of bovine serum albumin (BSA) on colloidal Al2O3 particles in an aqueous environment. Changes in the zeta potential of the Al2O3 particles upon the adsorption of BSA were measured using an electro-acoustic technique. The mass of protein adsorbed was determined by using UV-vis spectroscopy. The change of the isoelectric point of the Al2O3 powder-protein suspension was found to be a function of adsorbed protein mass. It was shown that approximately one monolayer of BSA was needed to fully mask the surface and to compromise the charge of Al2O3. From titration experiments it follows that about 30-36% of the negatively charged groups of the protein form bonds with the protonated and charged Al2O3 surface. On the basis of our observations we introduced a new adsorption model for BSA on Al2O3 particles.