Model surfaces engineered with nanoscale roughness and RGD tripeptides promote osteoblast activity.

Cell adhesion to biomaterials is a prerequisite for tissue integration with the implant surface. Herein, we show that we can generate a model silica surface that contains a minimal-length arginine-glycine-aspartic acid (RGD) peptide that maintains its biological activity. In the first part of this study, attachment of MC3T3-E1 osteoblast-like cells was investigated on silicon oxide, amine terminated substrates [i.e., 3-aminopropyl triethoxysilane (APTS)], grafted RGD, and physisorbed RGD control. The APTS layer exhibited nanoscale roughness and presented amine functional groups for grafting a minimal RGD tripeptide devoid of any flanking groups or spacers. Contact angle measurements indicated that the hydrophobicity of the APTS surface was significantly lower than that of the surface with grafted RGD (RGD-APTS). Atomic force microscopy showed that surfaces covered with RGD-APTS were smoother (Ra = 0.71 nm) than those covered with APTS alone (Ra = 1.59 nm). Focusing mainly on cell morphology, experiments showed that the RGD-APTS hybrid provided an optimum surface for cell adhesion, spreading, and cytoskeletal organization. Discrete focal adhesion plaques were also observed consistent with successful cell signaling events. In a second set of experiments, smooth, monolayers of APTS (Ra = 0.1 nm) were used to prepare arginine-glycine-aspartic acid-serine (RGDS)-APTS and arginine-glycine-glutamic acid-serine (RGES)-APTS (control) substrates. Focusing mainly on cell function, integrin and gene expression were all enhanced for rate osteosarcoma cells on surfaces containing grafted RGDS. Both sets of studies demonstrated that grafted molecules of RGD(S) enhance both osteoblast-like cell adhesion and function.

Relative influence of polymer fiber diameter and surface charge on fibrous capsule thickness and vessel density for single-fiber implants.

Single polypropylene microfibers plasma-coated with polymers of different surface charge [N,N-dimethylaminoethyl methacrylate (NN) (positive charge), methacrylic acid (MA) (negative charge), and hexafluoropropylene (HF) (neutral)] were implanted in the subcutaneous dorsum of Sprague-Dawley rats for 5-week intervals. Thee groups of fiber diameters were used: (I) 1.0 to 5.9 microm; (II) 6.0 to 10.9 microm; and (III) 11.0 to 15.9 microm. Fibrous capsule thickness and blood-vessel density (number of vessels within 100 microm of the fiber) were assessed in tissue sections in the planes of microfiber cross-sections. Results from a multifactorial analysis of variance demonstrated statistically significant main effects (p < 0.05) for microfiber diameter but not for surface-charge coating. The mean differences in capsule thickness among the microfiber diameter groups were: between groups II and I: 5.4 microm; between groups III and I: 10.2 microm; and between groups III and II: 4.7 microm. The mean differences in capsule thickness among surface-charge coatings were: between MA and NN: 0.7 microm; between MA and HF: 1.4 microm; and between NN and HF: 0.7 microm. Many of the 1.0 to 5.9 microm-in-diameter fibers had no capsule and no sign of a foreign-body reaction. For the vessel density analysis, neither microfiber diameter nor surface-charge coating had a statistically significant effect. Thus the geometric feature of microfiber diameter was more important than was surface charge relative to fibrous capsule formation but not relative to local vessel density. This ranking of the relative influence of design features in relation to tissue response provides useful information for prioritization in biomaterial design.

Control of in vivo microvessel ingrowth by modulation of biomaterial local architecture and chemistry.

We developed a method for controlling local architecture and chemistry simultaneously in biomaterial implants to control microvessel ingrowth in vivo. Porous polypropylene disks (5 mm in diameter and 40 microm thick) were plasma-coated with a fluoropolymer and then laser-drilled with 50-microm-diameter holes through their thickness. We then oxidized the disks to create hydroxyl functionality on the exposed polypropylene (inside the holes). Acrylamide was grafted to the hydroxyl groups through polymerization in the presence of activating ceric ions. Staining with toluidine blue O demonstrated that grafting occurred only inside the holes. We used the Hoffman degradation reaction to convert the amide groups of acrylamide to amine groups, and then we used ethylene glycol diglycidyl ether to attach biomolecules of interest inside the holes: secreted protein acidic and rich in cysteine (SPARC) peptide Lys-Gly-His-Lys (KGHK; angiogenic), thrombospondin-2 (TSP; antiangiogenic), or albumin (rat; neutral). In vivo testing in a rat subcutaneous dorsum model for a 3-week interval demonstrated a greater vessel surface area (p = 0.032) and a greater number of vessels (p = 0.043) in tissue local to the holes with KGHK-immobilized disks than with TSP-immobilized disks. However, differences between KGHK-immobilized and albumin-immobilized disks were less significant (p = 0.120 and p = 0.289 for the vessel surface area and number of vessels, respectively). The developed methods have potential applications in biomaterial design applications for which selective neovascularization is desired.

Plasma lithography--thin-film patterning of polymers by RF plasma polymerization II: Study of differential binding using adsorption probes.

In this study we present methods to physico-chemically modify micropatterned cell culture substrates that were manufactured using plasma lithography to incorporate affinity structures for specific cell binding. The surfaces consist of a pattern of a fluorocarbon plasma polymer with feature sizes between 5 and 100 microm on a background of a non-fouling tetraglyme (tetraethylene glycol dimethyl ether) plasma polymer. The tetraglyme polymer blocks virtually all non-specific binding of proteins, and it is non-adhesive for a fluorocarbon-polyethylene glycol (FC-PEG) surfactant designed to act as a 'hydrophobic anchor' for peptides. The surfactant shows a strong affinity for the fluorocarbon polymer pattern, thus enabling us to form a pattern of the surfactant-conjugated peptide. To verify this, we have synthesized a conjugate between histamine (as a model for a more complex peptide) and a commercially available FC-PEG surfactant. Disuccinimidyl carbonate was used to activate the terminal -OH group of the polyethylene glycol headgroup for the reaction with the amine-containing molecule. Affinity pattern formation can easily be achieved by immersion of the patterned substrates in a solution of the peptide-surfactant conjugate. Time of flight secondary ion mass spectroscopy in the imaging mode was used to verify that the surfactant localizes on the pattern, while the background remains bare. A model protein, bovine serum albumin, showed the same behavior. This suggests that these surfaces can be used for the formation of patterns of cell-adhesive proteins. These substrates will be used to investigate the influence of the cell size and shape of vascular smooth muscle cells on their physiology.