In serum veritas-in serum sanitas? Cell non-autonomous aging compromises differentiation and survival of mesenchymal stromal cells via the oxidative stress pathway.

Even tissues capable of complete regeneration, such as bone, show an age-related reduction in their healing capacity. Here, we hypothesized that this decline is primarily due to cell non-autonomous (extrinsic) aging mediated by the systemic environment. We demonstrate that culture of mesenchymal stromal cells (MSCs) in serum from aged Sprague-Dawley rats negatively affects their survival and differentiation ability. Proteome analysis and further cellular investigations strongly suggest that serum from aged animals not only changes expression of proteins related to mitochondria, unfolded protein binding or involved in stress responses, it also significantly enhances intracellular reactive oxygen species production and leads to the accumulation of oxidatively damaged proteins. Conversely, reduction of oxidative stress levels in vitro markedly improved MSC function. These results were validated in an in vivo model of compromised bone healing, which demonstrated significant increase regeneration in aged animals following oral antioxidant administration. These observations indicate the high impact of extrinsic aging on cellular functions and the process of endogenous (bone) regeneration. Thus, addressing the cell environment by, for example, systemic antioxidant treatment is a promising approach to enhance tissue regeneration and to regain cellular function especially in elderly patients.

MICROPATTERNING OF GOLD SUBSTRATES BASED ON POLY(PROPYLENE SULFIDE-BL-ETHYLENE GLYCOL), (PPS-PEG) BACKGROUND PASSIVATION AND THE MOLECULAR-ASSEMBLY PATTERNING BY LIFT-OFF (MAPL) TECHNIQUE.

Poly(propylene sulfide-bl-ethylene glycol (PPS-PEG) is an amphiphilic block copolymer that spontaneously adsorbs onto gold from solution. This results in the formation of a stable polymeric layer that renders the surface protein resistant when an appropriate architecture is chosen. The established molecular assembly patterning by lift-off (MAPL) technique can convert a prestructured resist film into a pattern of biointeractive chemistry and a noninteractive background. Employing the MAPL technique, we produced a micron-scale PPS-PEG pattern on a gold substrate, and then characterized the patterned structure with Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) and Atomic Force Microscopy (AFM). Subsequent exposure of the PPS-PEG/gold pattern to protein adsorption (full human serum) was monitored in situ; SPR-imaging (i-SPR) shows a selective adsorption of proteins on gold, but not on PPS-PEG areas. Analysis shows a reduction of serum adsorption up to 93% on the PPS-PEG areas as compared to gold, in good agreement with previous analysis of homogenously adsorbed PPS-PEG on gold. MAPL patterning of PPS-PEG block copolymers is straightforward, versatile and reproducible, and may be incorporated into biosensor-based surface analysis methods.

Conformation of poly(L-lysine)-graft-poly(ethylene glycol) molecular brushes in aqueous solution studied by small-angle neutron scattering.

Small-angle neutron scattering (SANS) has been employed for the analysis of conformations of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular bottle brushes in aqueous solutions. The degree of polymerisation of the PEG chains was systematically varied in order to unravel dependence of the conformational properties of the bottle brushes on the molecular weight of the grafted chains. The grafting density was kept constant and high enough to ensure strong overlap of the PEG chains. The scattering spectra were fitted on the basis of the model of an effective worm-like chain with the account of average radial distribution and local fluctuations of the PEG density in the bottle brush. The results of the fits indicate that molecular brushes retain weakly bent configuration on the length scale of the order of (or larger than) the brush thickness. This finding is in agreement with earlier simulation and recent theoretical results.

Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces.

Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polished Ti (P), and rough grit-blasted Ti (B), acid-etched Ti (AE), and grit-blasted and acid-etchedTi (SLA) surfaces and their growth, area, adhesion, and membrane-Ti proximity assessed. Rough surfaces decreased the growth of E cells compared to smooth surfaces in cultures up to 28 days. In general rough surfaces decreased the spreading of E cells as assessed by their area with the most pronounced affect for the SLA surface. On the other hand, the strength of E cells adhesion as inferred by immunofluorescence staining of vinculin in focal adhesions indicated that E cells formed more and larger focal adhesions on the smooth P surface compared to the rougher AE surface. As this finding indicates a stronger adhesion to smooth surfaces, it is likely that E cells on rough surfaces are more susceptible to mechanical removal. An immunogold labeling method was developed to visualize focal adhesions using back-scattered electron imaging with a scanning electron microscope (SEM). On rough surfaces focal adhesions were primarily localized on to the ridges rather than the valleys and the cells tended to bridge over the valleys. Transmission electron microscopy (TEM) measurements of membrane proximity to the Ti surface indicated that average distance of cell to the Ti increased as the Ti surface roughness increased. Therefore, the size and shape of surface features are important determinants of epithelial adhesive behavior and epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.

Locally Addressable Electrochemical Patterning Technique (LAEPT) applied to poly(L-lysine)-graft-poly(ethylene glycol) adlayers on titanium and silicon oxide surfaces.

The protein-resistant polycationic graft polymer, poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), was uniformly adsorbed onto a homogenous titanium surface and subsequently subjected to a direct current (dc) voltage. Under the influence of an ascending cathodic and anodic potential, there was a steady and gradual loss of PLL-g-PEG from the conductive titanium surface while no desorption was observed on the insulating silicon oxide substrates. We have implemented this difference in the electrochemical response of PLL-g-PEG on conductive titanium and insulating silicon oxide regions as a biosensing platform for the controlled surface functionalization of the titanium areas while maintaining a protein-resistant background on the silicon oxide regions. A silicon-based substrate was micropatterned into alternating stripes of conductive titanium and insulating silicon oxide with subsequent PLL-g-PEG adsorption onto its surfaces. The surface modified substrate was then subjected to +1800 mV (referenced to the silver electrode). It was observed that the potentiostatic action removed the PLL-g-PEG from the titanium stripes without inducing any polyelectrolyte loss from the silicon oxide regions. Time-of-flight secondary ions mass spectroscopy and fluorescence microscopy qualitatively confirmed the PLL-g-PEG retention on the silicon oxide stripes and its absence on the titanium region. This method, known as "Locally Addressable Electrochemical Patterning Technique" (LAEPT), offers great prospects for biomedical and biosensing applications. In an attempt to elucidate the desorption mechanism of PLL-g-PEG in the presence of an electric field on titanium surface, we have conducted electrochemical impedance spectroscopy experiments on bare titanium substrates. The results showed that electrochemical transformations occurred within the titanium oxide layer; its impedance and polarization resistance were found to decrease steadily upon both cathodic and anodic polarization resulting in the polyelectrolyte desorption from the titanium surface.

Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-functionalized poly(L-lysine)-grafted-poly(ethylene glycol) copolymers.

Implanted biomaterials are coated immediately with host plasma constituents, including extracellular matrix (ECM); this reaction may be undesirable in some cases. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) has been shown to spontaneously adsorb from aqueous solution onto metal oxide surfaces, effectively reducing the degree of non-specific adsorption of blood and ECM proteins, and decreasing the adhesion of fibroblastic and osteoblastic cells to the coated surfaces. Cell adhesion through specific peptide-integrin receptors could be restored on surfaces coated with PLL-g-PEG functionalized with peptides of the RGD (Arg-Asp-Gly) type. To date, no study has examined the effect of surface modifications by PLL-g-PEG-based polymers on bacterial adhesion. The ability of Staphylococcus aureus to adhere to the ECM and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of medical-device-related infections. This study describes methods for visualizing and quantifying the adhesion of S. aureus to smooth and rough (chemically etched) titanium surfaces without and with monomolecular coatings of PLL-g-PEG, PLL-g-PEG/PEG-RGD and PLL-g-PEG/PEG-RDG. The different surfaces were exposed to S. aureus cultures for 1-24h and bacteria surface density was evaluated using scanning electron microscopy and fluorescence microscopy. Coating titanium surfaces with any of the three types of copolymers significantly decreased the adhesion of S. aureus to the surfaces by 89-93% for PLL-g-PEG, and 69% for PLL-g-PEG/PEG-RGD. Therefore, surfaces coated with PLL-g-PEG/PEG-RGD have the ability to attach cells such as fibroblasts and osteoblasts while showing reduced S. aureus adhesion, resulting in a selective biointeraction pattern that may be useful for applications in the area of osteosynthesis, orthopaedic and dental implantology.

RGD-containing peptide GCRGYGRGDSPG reduces enhancement of osteoblast differentiation by poly(L-lysine)-graft-poly(ethylene glycol)-coated titanium surfaces.

Osteoblasts exhibit a more differentiated morphology on surfaces with rough microtopographies. Surface effects are often mediated through integrins that bind the RGD motif in cell attachment proteins. Here, we tested the hypothesis that modulating access to RGD binding sites can modify the response of osteoblasts to surface microtopography. MG63 immature osteoblast-like cells were cultured on smooth (Ti sputter-coated Si wafers) and rough (grit blasted/acid etched) Ti surfaces that were modified with adsorbed monomolecular layers of a comb-like graft copolymer, poly-(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), to limit nonspecific protein adsorption. PLL-g-PEG coatings were functionalized with varying amounts of an integrin-receptor-binding RGD peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGD) or a nonbinding RDG control sequence GCRGYGRDGSPG (PLL-g-PEG/PEG-RDG). Response to PLL-g-PEG alone was compared with response to surfaces on which 2-18% of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGD dose-response, peptide surface concentration was varied between 0 and 6.4 pmol/cm(2). In addition, cells were cultured on uncoated Ti or Ti coated with PLL-g-PEG or PLL-g-PEG/PEG-RGD at an RGD surface concentration of 0.7 pmol/cm(2), and free RGDS was added to the media to block integrin binding. Analyses were performed 24 h after cultures had achieved confluence on the tissue culture plastic surface. Cell number was reduced on smooth Ti compared to plastic or glass and further decreased on surfaces coated with PLL-g-PEG or PLL-g-PEG/PEG-RDG, but was restored to control levels when PLL-g-PEG/PEG-RGD was present. Alkaline phosphatase specific activity and osteocalcin levels were increased on PLL-g-PEG alone or PLL-g-PEG/PEG-RDG, but PLL-g-PEG/PEG-RGD reduced the parameters to control levels. On rough Ti surfaces, cell number was reduced to a greater extent than on smooth Ti. PLL-g-PEG coatings reduced alkaline phosphatase and increased osteocalcin in a manner that was synergistic with surface roughness. The RDG peptide did not alter the PLL-g-PEG effect but the RGD peptide restored these markers to their control levels. PLL-g-PEG coatings also increased TGF-beta1 and PGE(2) in conditioned media of cells cultured on smooth or rough Ti; there was a 20x increase on rough Ti coated with PLL-g-PEG. PLL-g-PEG effects were inhibited dose dependently by addition of the RGD peptide to the surface. Free RGDS did not decrease the effect elicited by PLL-g-PEG surfaces. These unexpected results suggest that PLL-g-PEG may have osteogenic properties, perhaps correlated with effects that alter cell attachment and spreading, and promote a more differentiated morphology.

Characterization of poly(L-lysine)-graft-poly(ethylene glycol) assembled monolayers on niobium pentoxide substrates using time-of-flight secondary ion mass spectrometry and multivariate analysis.

Control of protein adsorption onto solid surfaces is a critical area of biomaterials and biosensors research. Application of high performance surface analysis techniques to these problems can improve the rational design and understanding of coatings that control protein adsorption. We have used static time-of-flight secondary ion mass spectrometry (TOF-SIMS) to investigate several poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) adlayers adsorbed electrostatically onto negatively charged niobium pentoxide (Nb(2)O(5)) substrates. By varying the PEG graft ratio (i.e., the number of lysine monomers per grafted PEG chain) and the molecular weights of the PLL and PEG polymers, the amount of protein adsorption can be tailored between 1 and 300 ng/cm(2). Detailed multivariate analysis using principal component analysis (PCA) of the positive and negative ion TOF-SIMS spectra showed changes in the outermost surface of the polymer films that were related to the density and molecular weight of the PEG chains on the surface. However, no significant differences were noted due to PLL molecular weight, despite observed differences in the serum adsorption characteristics for adlayers of PLL-g-PEG polymers with different PLL molecular weights. From the PCA results, multivariate peak intensity ratios were developed that correlated with the thickness of the adlayer and the enrichment of the PEG chains and the methoxy terminus of the PEG chains at the outermost surface of the adlayer. Furthermore, partial least squares regression was used to correlate the TOF-SIMS spectra with the amount of protein adsorption, resulting in a predictive model for determining the amount of protein adsorption on the basis of the TOF-SIMS spectra. The accuracy of the prediction of the amount of serum adsorption depended on the molecular weight of the PLL and PEG polymers and the PEG graft ratio. The combination of multivariate analysis and static TOF-SIMS provides detailed information on the surface chemistry and insight into the mechanism for protein resistance of the coatings.

Peptide functionalized poly(L-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma.

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivatives (PLL-g-PEG/PEG-peptide) were assembled from aqueous solutions on titanium (oxide) surfaces. The polymers were characterized by NMR in order to determine quantitatively the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomolecular adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 degrees C or 37 degrees C, whereas clean, uncoated titanium surfaces adsorbed approximately 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clinical performance.

Osteoblast proliferation and differentiation on dentin slices are modulated by pretreatment of the surface with tetracycline or osteoclasts.

BACKGROUND: Implant surface roughness and chemical composition, as well as other factors, affect the ability of osteogenic cells to form bone adjacent to an implant. The same principles may also apply to the tooth root and some reports have shown that surface modification of the root may lead to improved restoration of the periodontal apparatus. The most common of these surface modification techniques involves demineralization with citric acid or treatment with tetracycline to expose collagen fibrils. In addition, during normal bone remodeling, osteoclasts demineralize the extracellular matrix, leaving resorption pits and exposed collagen fibrils. In this study, the effect of different dentin surface-preparation techniques on osteoblasts were compared. METHODS: Slices of sperm whale dentin were mechanically polished and surfaces were treated with tetracycline-HCl (TCN) or were cultured with mouse bone marrow cells to create a surface with osteoclast (OC) resorption pits or left untreated. Profilometry, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to evaluate the 3 different dentin surfaces. MG63 osteoblast-like cells were cultured on the 3 different surfaces and the effect of dentin surface preparation technique on MG63 cell proliferation (cell number), differentiaton (alkaline phosphatase specific activity of isolated cells and cell layer lysates; osteocalcin production), and local factor production (transforming growth factor (TGF)-beta1 and prostaglandin E2 (PGE2) compared. RESULTS: Profilometry showed the polished and TCN surfaces were smooth with comparable Ra values, whereas the OC surfaces were slightly rougher due to resorption pits which covered 3.7% of the surface. XPS measurements showed that TCN treatment reduced the Ca and P content of the surface, indicating that it had dissolved the mineral. Osteoclast-resorption also reduced the Ca and P content, but to a lesser extent. MG63 cell proliferation on polished dentin and tissue culture polystyrene was equivalent. In contrast, cells grown on the TCN- and OC-treated surfaces exhibited increased proliferation. No effect of surface treatment on cell alkaline phosphatase activity was observed, but activity in the cell layer lysates was increased on the TCN- and OC-treated surfaces. Osteocalcin production was reduced on all dentin surfaces, but the greatest reduction was found on the TCN-treated surface. Production of both TGF-beta1 and PGE2 was increased on the treated surfaces. All effects were greatest in cultures grown on the TCN-treated dentin. CONCLUSIONS: These data indicate that demineralization of the dentin surface promotes proliferation of osteoblasts and early differentiation events like production of alkaline phosphatase and autocrine mediators such as PGE2 and TGF-beta1. However, later differentiation events like osteocalcin production are decreased. Osteoclast-mediated bone resorption elicits similar responses; less than 4% of the dentin surface resulted in approximately 75% of the response caused by TCN treatment. These observations suggest that greater attention should be paid to the effects of osteoclastic resorption in designing methods for enhancing bone and cementum formation adjacent to root surfaces.

Immobilization of the cell-adhesive peptide Arg-Gly-Asp-Cys (RGDC) on titanium surfaces by covalent chemical attachment.

Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg-Gly-Asp-Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.