Comparison of the response of cultured osteoblasts and osteoblasts outgrown from rat calvarial bone chips to nonfouling KRSR and FHRRIKA-peptide modified rough titanium surfaces.

Mimicking proteins found in the extracellular matrix (ECM) using specific peptide sequences is a well-known strategy for the design of biomimetic surfaces, but has not yet been widely exploited in the field of biomedical implants. This study investigated osteoblast and, as a control, fibroblast proliferation to novel consensus heparin-binding peptides sequences KRSR and FHRIKKA that were immobilized onto rough (particle-blasted and chemically etched) commercially pure titanium surfaces using a poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system. This platform enabled a detailed study of specific cell-peptide interactions even in the presence of serum in the culture medium; thanks to the excellent nonfouling properties of the PLL-g-PEG surface. Cell-binding peptide sequence RGD in combination with KRSR or FHRRIKA was used to examine a potentially-enhanced or synergistic effect on osteoblast proliferation. Bare titanium and bioinactive surfaces (i.e., unfunctionalized PLL-g-PEG and scrambled KSSR, RFHARIK, and RDG) were used as control substrates. Additionally, in a newly developed experimental setup, freshly harvested bone chips from newborn rat calvariae were placed onto the same type of surfaces investigating size and pattern of osteoblast outgrowths. The findings of the current study demonstrated that the difference in osteoblast and fibroblast proliferation was influenced by surface topography more so than by the presence of surface-bound KRSR and FHRRIKA. On the other hand, in comparison with the control surfaces, osteoblast outgrowths from rat calvarial bone chips covered a significantly larger area on RGD, KRSR, and FHRRIKA surfaces after 8 days and also migrated in an isotropic way unlike cells on the bioinactive substrates. Furthermore, the stimulatory effect of 0.75 pmol cm(-2) RGD on osteoblast migration pattern could be enhanced when applied in combination with 2.25 pmol cm(-2) KRSR.

Fabrication of TiO2-coated epoxy replicas with identical dual-type surface topographies used in cell culture assays.

The goal of this study was to reproducibly generate samples with complex surface topographies and chemistries identical to a "master surface" and to test their response in cell culture using rat calvarial cells. Negative replicas of dual-type topography were fabricated using dental impression material with half of the surface exhibiting smooth and rough topography, respectively. Positive epoxy resin replicas were cast from the same negative replica eight times consecutively and coated with a 60-nm thin film of titanium dioxide using a vapor deposition technique. Atomic force microscopy, scanning electron microscopy, confocal white light microscopy, and X-ray photoelectron spectroscopy indicated that TiO(2)-coated epoxy replicas had surface topographical features and surface compositions nearly indistinguishable from the original titanium master surfaces. The described technique showed high reproducibility over at least eight generations of replication using the same negative replica. Rat calvarial osteoblasts proliferated just as well on dual topography surfaces as on single topography surfaces. The advantage of the dual-type substrates is that they facilitate comparison within a single culture dish, thus eliminating dish-to-dish variation as well as saving material, time and costs compared to the usual method of evaluating surfaces in separate dishes.

Systematic study of osteoblast response to nanotopography by means of nanoparticle-density gradients.

Features over a wide range of length scales affect the biological response to a surface. While the influence of micro-features has been extensively studied, the effect of nano-features has only rarely been systematically investigated. We have developed a simple method to produce nano-featured gradients by kinetically controlled adsorption of negatively charged silica nanoparticles onto positively charged, poly(ethylene imine) (PEI)-coated silicon wafers. Subsequent sintering of the particles allowed a tuning of the particle morphology and resulted in a firm anchoring of the particles to the surface. Particle-density gradients were characterized by atomic force microscopy (AFM). Cell experiments with rat calvarial osteoblasts (RCO) on nano-featured gradients exhibited a significant decrease in proliferation at locations with higher particle coverage. Seven days post seeding, the number of osteoblasts was eight times higher at positions without particles compared to positions with maximum particle coverage. While cells spread well and developed a well-organized actin network in the absence of particles, spreading and formation of a strong actin network was considerably hindered at locations with maximum particle density.

Functionalizable Nanomorphology Gradients via Colloidal Self-Assembly.

We present a novel approach for the fabrication of tailored nanomorphology gradients on metal oxide surfaces. We first show the direct formation of a nanocolloidal density gradient by a dip-coating process. The obtained silica nanoparticle gradients are then subjected to a heat treatment. Control of this sintering step allows the precise tailoring of the particle morphology on the surface. Both these processes together provide a new tool to form precise, tunable, and material-independent nanomorphology gradients.

Systematic study of osteoblast and fibroblast response to roughness by means of surface-morphology gradients.

The surface roughness of a medical implant is of great importance since the surface is in direct contact with the host tissue (e.g. bone, fibrous tissue). The response of cells to roughness is different depending on the cell type. However, the influence of roughness on cell behavior has only rarely been systematically studied. We have developed a surface-modification process to produce roughness gradients that cover a wide range of roughness values on one substratum. Such gradients allow for systematic investigations of roughness on cell behavior. Gradients were fabricated using a two-step roughening and smoothening process, involving sandblasting and a subsequent chemical polishing step. In order to produce a set of identical surfaces we applied a replica technique. Cell experiments were carried out with rat calvarial osteoblasts (RCO) and human gingival fibroblasts (HGF). RCOs showed a significantly increased proliferation rate with increasing surface roughness. The footprint of osteoblasts varied in size at different positions on the gradient, remaining small on the rough end of the gradient and increasing considerably as the roughness decreased. HGF showed the opposite proliferation behavior, proliferation decreasing with increasing roughness. The fibroblast morphology was found to be similar to that seen for osteoblasts.

Material properties of and tissue reaction to the Slocum TPLO plate.

OBJECTIVE: To determine the material properties of Slocum TPLO plates and assess the soft tissue reaction adjacent to these plates in dogs that had undergone tibial plateau leveling osteotomy (TPLO). SAMPLE POPULATION: 3 new TPLO plates, 8 retrieved TPLO plates, and 1 new Synthes dynamic compression plate. PROCEDURES: Metallurgic analyses were performed. Tissue samples were obtained from areas adjacent to retrieved plates and submitted for histologic examination. RESULTS: All of the TPLO plates had a 2-phase microstructure consisting of austenite and ferrite in various amounts. Residua, inclusions, and cavities were seen during microscopic examination of the plate surface. The major differences between new and retrieved TPLO plates were the presence of small gaps separating many inclusions from the surrounding matrix and the presence of various-sized pits on the surface of the retrieved plates. The dynamic compression plate had a nearly pure austenitic structure and was largely free from residua, inclusions, and cavities. Histologic examination of tissue samples obtained from areas adjacent to retrieved TPLO plates revealed intra- and extracellular particulate debris. Two types of particles (one consisting of chromium, nickel, molybdenum, and iron and the other consisting of aluminum and silicon) were seen. CONCLUSIONS AND CLINICAL RELEVANCE: Results determined that new and retrieved TPLO plates were manufactured from 316L stainless steel and produced by a casting process, but not all plates met specifications for chemical composition of cast surgical implants (American Society for Testing Materials standard F745); tissues surrounding retrieved plates had evidence of adverse reactions, probably as a result of plate corrosion.