Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

Biomaterials and interface with bone.

In this paper, some examples from the literature or from my own experience will be given to illustrate the influence of surface topography and surface chemistry at the nano- and micro-scale on the cell and tissue response.

Relative influence of surface topography and surface chemistry on cell response to bone implant materials. Part 1: physico-chemical effects.

Knowledge of the complexity of cell-material interactions is essential for the future of biomaterials and tissue engineering, but we are still far from achieving a clear understanding, as illustrated in this review. Many factors of the cellular or the material aspect influence these interactions and must be controlled systematically during experiments. On the material side, it is essential to illustrate surface topography by parameters describing the roughness amplitude as well as the roughness organization, and at the scales pertinent for the cell response, i.e., from the nano-scale to the micro-scale. Authors interested in this field must be careful to develop surfaces or methods systematically, allowing perfect control of the relative influences of surface topography and surface chemistry.

Relative influence of surface topography and surface chemistry on cell response to bone implant materials. Part 2: biological aspects.

A current medical challenge is the replacement of tissue which can be thought of in terms of bone tissue engineering approaches. The key problem in bone tissue engineering lies in associating bone stem cells with material supports or scaffolds that can be implanted in a patient. Beside bone tissue engineering approaches, these types of materials are used daily in orthopaedics and dental practice as permanent or transitory implants such as ceramic bone filling materials or metallic prostheses. Consequently, it is essential to better understand how bone cells interact with materials. For several years, the current authors and others have developed in vitro studies in order to elucidate the mechanisms underlying the response of human bone cells to implant surfaces. This paper reviews the current state of knowledge and proposes future directions for research in this domain.

Antibacterial properties and compressive strength of new one-step preparation silver nanoparticles in glass ionomer cements (NanoAg-GIC).

OBJECTIVES: This work aimed (1) to develop polyacid formulations by the one-step photoreduction of silver nanoparticles (AgNP) in a polyacrylate solution of conventional glass ionomer cement (GIC), imparting antibacterial activity; and (2) to evaluate handling and mechanical properties of experimental ionomers in comparison to a commercially available conventional GIC. METHODS: Formulations with increasing sub-stoichiometric amounts of AgNO3 were monitored during continuous UV light exposure by UV-vis spectroscopy and analyzed by transmission electron microscopy. The resulted synthesis of formulations containing small and disperse spherical nanoparticles (∼6 nm) were used to design the experimental nano-silver glass ionomer cements (NanoAg-GIC). The cements were characterized as to net setting time and compressive strength according to ISO 9917-1:2007 specifications. The antibacterial activity of these cements was assessed by Ag+ diffusion tests on nutritive agar plates (E. coli) and by MTT assay (S. mutans). RESULTS: The higher concentration of silver (0.50% by mass) in the matrix of NanoAg-GIC allowed viable net setting time and increased in 32% compressive strength of the experimental cement. All groups containing AgNP induced statistically significant E. coli growth inhibition zones (p-value <.05), indicating diffusion of Ag+ ions on the material surroundings. Metabolic activity of S. mutans grown on NanoAg-GIG with higher concentration of silver was significantly affected compared to control (p-value <.01). CONCLUSIONS: Silver nanoparticles one-step preparation in polyacrylate solution allowed the production of highly bioactive water-based cements within suitable parameters for clinical use and with large potential of dental and biomedical application.

Synthesis and characterization of Ti-27.5Nb alloy made by CLAD® additive manufacturing process for biomedical applications.

Biocompatible beta-titanium alloys such as Ti-27.5(at.%)Nb are good candidates for implantology and arthroplasty applications as their particular mechanical properties, including low Young's modulus, could significantly reduce the stress-shielding phenomenon usually occurring after surgery. The CLAD® process is a powder blown additive manufacturing process that allows the manufacture of patient specific (i.e. custom) implants. Thus, the use of Ti-27.5(at.%)Nb alloy formed by CLAD® process for biomedical applications as a mean to increase cytocompatibility and mechanical biocompatibility was investigated in this study. The microstructural properties of the CLAD-deposited alloy were studied with optical microscopy and electron back-scattered diffraction (EBSD) analysis. The conservation of the mechanical properties of the Ti-27.5Nb material after the transformation steps (ingot-powder atomisation-CLAD) were verified with tensile tests and appear to remain close to those of reference material. Cytocompatibility of the material and subsequent cell viability tests showed that no cytotoxic elements are released in the medium and that viable cells proliferated well.

Physico-chemical characteristics and protein adsorption potential of hydroxyapatite particles: influence on in vitro biocompatibility of ceramics after sintering.

Through the example of two HA ceramics prepared from two HA powders (HAD and HAL), we explored the relation between the physico-chemical qualities of the initial HA powder and the final HA ceramic and their influence on the protein adsorption and cell response to the final HA ceramics. The powders were characterized by XRD, FT-IR, zeta potential, and specific surface area (SSA). Their protein adsorption potential was tested after immersion in culture medium +15% of fetal calf serum. These results were correlated with the protein adsorption potential of the two ceramics (cHAD and cHAL) prepared from the HAD and HAL powders respectively and to the cell attachment after 4, 24 and 72 h on the ceramics. From our results, it appears that a relation can be established between the physico-chemical characteristics of the initial HA powders and the final biological response to the sintered ceramics prepared from these powders. An inverse relation exists between the SSA and the protein adsorption capacity of HA powders and the protein adsorption and cell attachment on HA ceramics. This inverse relation is related to phenomenon occurring during the sintering phase and the formation of inter-granular micro-porosity.

Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

A kinetic approach to osteoblast adhesion on biomaterial surface.

An incompletely understood question in the field of biomaterials is how eucaryotic cells adhere on material surfaces. The adhesion of cells on materials is generally studied after some hours. Because this evaluation after some hours cannot let us presume about the future of the cells on the material, we have developed a culture model that does allow study in the long term of an elaborate cell/material interface closer to the in vivo situation. For that, we used a progressive trypsin-based detachment method. Here we report on the mathematical modeling of long-term human primary osteoblastic cell adhesion on metallic substrates, which allows us to quantify the real adhesion simultaneously by taking into account the effect of cell proliferation. A time-dependent adhesion index t(d) is proposed, which varies with culture time t according to the power law: t(d)(t) = at(b), a being independent of b. The exponent b is equal to 0.5 +/- 0.03 and is independent of the substrate's characteristics, meaning that the long-term adhesion increases proportionally to the square root of culture time. On the contrary, the parameter a significantly depends on the material's nature, the surface's topography, and the surface chemistry of the substrate and is sufficient to characterize cell adhesion. From this relationship, we suggest that a diffusion-based process related to the kinetic of formation of extracellular matrix should be involved in long-term adhesion on materials.

Statistical correlation between cell adhesion and proliferation on biocompatible metallic materials.

Our ambition for several years is to appreciate and quantify the long-term adhesion of cells on materials at times where the interface between cells and substrate becomes more complex, more closed to the cell/matrix/substrate interface existing in vivo. With this objective, we quantified the long-term adhesion and proliferation of human osteoblasts cultured from 24 h to 21 days on pure titanium, titanium alloy, and stainless-steel substrates presenting six different surface morphologies and two different roughness amplitude. Hence, we did proceed to the statistical correlation of cell adhesion and cell proliferation on 30 different substrates. Additionally, we described surface topography not only by the roughness amplitude but also by the roughness morphology using new specific parameters. By multiple analysis of variance, we demonstrated that nor material composition nor surface roughness amplitude did influence cell proliferation, whereas a very significant influence of the process used to produce the surface was observed meaning that the main influent factor on cell proliferation was the surface morphology. The long-term adhesion and proliferation capacity of cells were positively correlated on 23 types of substrates on 30, this positive correlation being statistically asserted on 13 types of substrates on 23. This study is the first demonstration of the existence of a statistical correlation between long-term adhesion and proliferation capacity of human bone cells on substrates with various chemical composition, surface chemistry, and surface topography.

Topography effects of pure titanium substrates on human osteoblast long-term adhesion.

Classically various treatments are applied to increase the roughness of titanium implants and improve their integration in the tissues. Many in vitro studies have been performed to better understand the mechanisms underlying the adhesion of cells on materials. Frequently, the adhesion is related to the attachment of cells during the first hours of contact with the substrate. For several years, our objective has been to develop experimental methods to evaluate the long-term adhesion of human osteoblasts from some hours to several weeks in order to model in vitro a tissue-like interface. This culture model allows for the formation over 21 days of a complex osteoblast/extracellular matrix/material interface. We recently developed a new parameter called adhesion power (AP) to evaluate this long-term adhesion. In this study, our objective is to check its efficiency in discriminating the long-term adhesion of human osteoblasts on pure titanium substrates with seven different surface morphologies obtained by electro-erosion, sandblasting, polishing, acid-etching and machine-tooling. By scanning electron microscopy, we observed that the human osteoblasts did spread more intimately on surface with low roughness amplitude than on rough ones. However, the AP was higher on rough isotropic surfaces obtained by electro-erosion, sandblasting or acid-etching and lower on smoother surfaces obtained by polishing and machine-tooling. We demonstrated that the AP was pertinent for evaluating human osteoblast's long-term adhesion on pure titanium surfaces with various roughness parameters. Its correlation with the order parameter, which describes the organization of the roughness, confirmed once more that human osteoblasts are more sensitive to the organization and morphology of the roughness than to its amplitude.

Bootstrap analysis of the relation between initial adhesive events and long-term cellular functions of human osteoblasts cultured on biocompatible metallic substrates.

Classically, the evaluation of cellular adhesion of cells on substrates is limited to the evaluation of cell attachment after some hours. We have claimed for several years that this evaluation is incomplete concerning the evaluation of cell adhesion and more precisely of the quality of the in vitro cell/biomaterial interface. With a view to demonstrating this assertion, we develop in this paper statistical correlations between short-term adhesion (STA) evaluating the attachment after 24 h (IA: initial attachment) and long-term adhesion (LTA) evaluating the strength of the cell/matrix substrate interface over 21 days of culture (AP: adhesion power). Additionally, as the adhesion phase is known to influence further growth of cells we proceed to the correlation of STA with the number of cells after 21 days. We demonstrate statistically the expected positive relation existing between STA and cell growth and we show that this relation is totally independent of the substrate's surface topography or chemistry. More surprisingly, we demonstrate the absence of correlation between IA and AP. This illustrates the fact that different mechanisms underlie STA and LTA. Moreover, this study demonstrates that the evaluation of the number of attached cells after some hours cannot let us presume either that cells will survive or that they will adhere at later times by forming a complex cell/substrate interface by synthesis of extracellular matrix proteins. Finally, the originality of this work lies in the extensive statistical correlation analysis performed between biological parameters describing the cell behaviour on a substrate.

Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value to the covalent grafting immobilization methods. However no differences in exposure of the cell binding domains were observed (ELISA results) before SDS rinsing, suggesting that pFN protein grafting to the surface is initially kinetically driven be a stochastic random adsorption phenomenon. Covalent grafting acts in the final stage as a process that simply tethers and stabilizes (or freezes) the initial conformation/orientation of the adsorbed protein on the surface. In addition covalent linkage via the SSMPB approach is likely favored by surface-induce exposure of one of the normally hidden free thiol group pair, thus optimizing covalent linkage to the surface. However after SDS rinsing, this "tethering"/"freezing" effect was significantly more prominent for the GA grafting approach (due to greater number of potential covalent links between the protein and the surface) compared to that for the SSMPB approach. This hypothesis was buttressed by the improved resistance to denaturation (smaller conformational lability) for the GA compared to the SMPB approach and improved exposure of the cell binding domain for the former (>50%) even after SDS rinsing. These results are promising in that they suggest covalent tethering of fibronectin to PS substrate in a monolayer range, with significantly improved irreversible protein surface bonding via both approaches (compared to that for mere adsorption). The latter are likely applicable to a wide range of proteins.

Association of porous hydroxyapatite and bone marrow cells for bone regeneration.

The preparation of hybrid material with osteoinductive capacity may be achieved by association of cultured autologous bone cells with a porous ceramic vehicle. We optimized culture conditions for rabbit marrow stromal stem cells (MSCs), notably by selection from batches of fetal calf serum. Rabbit MSCs formed colony-forming unit-ribroblastic (CFU-Fs) in vitro. Their alkaline phosphatase (ALP) activity was doubled in the presence of dexamethasone. Autologous rabbit serum allowed the formation of ALP-positive CFU-Fs, but results were highly variable depending on the rabbit. We tested the osteogenic potential of autologous cultured (with or without dexamethasone addition in the culture medium) and noncultured rabbit MSCs associated with a porous hydroxyapatite ceramic after a dorsal intramuscular implantation. Nucleated cells (10(7) or 10(8)/mL) were used for the preparation of autologous hybrid material. A significantly higher number of implants containing bone was obtained with a suspension of 10(7) cells/mL cultured in the presence of 10(-8) M dexamethasone. Some positive implants were also obtained with a suspension of 10(8) noncultured cells/mL. We demonstrated the feasibility of preparing rabbit autologous hybrid materials following a process for controlling culture conditions, cell characterization and cell/material association.

Osteoblast adhesion on biomaterials.

The development of tissue engineering in the field of orthopaedic surgery is now booming. Two fields of research in particular are emerging: the association of osteo-inductive factors with implantable materials; and the association of osteogenic stem cells with these materials (hybrid materials). In both cases, an understanding of the phenomena of cell adhesion and, in particular, understanding of the proteins involved in osteoblast adhesion on contact with the materials is of crucial importance. The proteins involved in osteoblast adhesion are described in this review (extracellular matrix proteins, cytoskeletal proteins, integrins, cadherins, etc.). During osteoblast/material interactions, their expression is modified according to the surface characteristics of materials. Their involvement in osteoblastic response to mechanical stimulation highlights the significance of taking them into consideration during development of future biomaterials. Finally, an understanding of the proteins involved in osteoblast adhesion opens up new possibilities for the grafting of these proteins (or synthesized peptide) onto vector materials, to increase their in vivo bioactivity or to promote cell integration within the vector material during the development of hybrid materials.

Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography.

The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 microm in diameter, whereas they did not recognize the 10 microm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 microm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 microm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.

Fate of bioresorbable poly(lactic acid) microbeads implanted in artificial bone defects for cortical bone augmentation in dog mandible.

The fate was examined of poly(lactic acid) microbeads implanted in large artificial defects created in cortical bone of dog mandibles. Two poly(lactic acid) polymers--poly(L-lactic acid) (PLA 100) and poly(DL-lactic acid) (PLA 50)--were used to make microbeads by solvent evaporation with poly(vinyl alcohol) as surfactant. Histological observation of non-decalcified mandibular bone showed that no real bone regeneration existed in the experimental bone defects 18 months after PLA 100 microbeads implantation. The same observation was made 6 months after implantation of PLA 50 microbeads. PLA 100 and PLA 50 microbeads appeared unable to induce regeneration of cortical bone defects of dog mandible, in contrast to previous observations in man for PLA 50 large implants. The failure is tentatively assigned to the presence of poly(vinyl alcohol) at the surface of microbeads.

Improvement in the morphology of Ti-based surfaces: a new process to increase in vitro human osteoblast response.

Surface roughness has been shown to be an influencing parameter for cell response. In this experience we attempted to compare the effect of roughness organization of Ti6A14V or pure titanium substrates on human osteoblast (hOB) response (proliferation, adhesion). Surface roughness was extensively analyzed at scales above the cell size (macro-roughness) or below the cell size (micro-roughness) by calculation of relevant classic amplitude parameters (Ra, Rt) and original frequency parameters (Order, Delta). We developed a new process to prepare isotropic surfaces (electro-erosion), which were compared to isotropic surfaces obtained by polishing and anisotropic surfaces obtained by machine-tooling. The hOB response on electro-eroded (EE) Ti6A14V surfaces or pure titanium (Ti) surfaces was largely increased when compared to polished or machine-tooled surfaces after 21 days of culture. Moreover, the polygonal morphology of hOB on these EE surfaces was very close to the aspects of hOB in vivo on human bone trabeculae. By a complete description of the surface topography of EE surfaces, we concluded that when the topography was considered below the cell scale, hOB appreciated their isotropic smooth aspect, although when the topography was considered above the cell scale they appreciated their rough isotropic 'landscape' formed by many 'bowl-like nests' favouring cell adhesion and growth. Electro-erosion is a promising method for preparation of bone implant surfaces, as it could easily be applied to preparation of most biomaterials with complex geometries.

The relative influence of the topography and chemistry of TiAl6V4 surfaces on osteoblastic cell behaviour.

Proliferation and adhesion of mouse (MC3T3-E1) osteoblastic cells and primary human osteoblastic cells were carried out on Ti6Al4V titanium alloy samples with varied surface roughnesses. Mechanically or manually polished surfaces were prepared to produce respectively non-oriented or oriented residual polishing grooves. Sand-blasted surfaces were prepared using 500 microm or 3 mm alumina particles. Surface roughness parameters showed a negative correlation in comparison to proliferation and adhesion parameters. X-ray microprobe chemical surface microanalysis showed complete disturbance of the surface element composition of the Ti6Al4V alloy following sand-blasting treatment. An AlOx-enriched layer was observed on sample surfaces. This may lead to the suspicion that the concomittant effect of surface roughness amplitude and AlOx surface concentration has an effect on osteoblastic cell proliferation and adhesion. These findings show the significance of chemical surface analysis after any surface treatment of titanium-based implants before any biological use.

An unscaled parameter to measure the order of surfaces: a new surface elaboration to increase cells adhesion.

We present a new parameter to quantify the order of a surface. This parameter is scale-independent and can be used to compare the organization of a surface at different scales of range and amplitude. To test the accuracy of this roughness parameter versus a hundred existing ones, we created an original statistical bootstrap method. In order to assess the physical relevance of this new parameter, we elaborated a great number of surfaces with various roughness amplitudes on titanium and titanium-based alloys using different physical processes. Then we studied the influence of the roughness amplitude on in vitro adhesion and proliferation of human osteoblasts. It was then shown that our new parameter best discriminates among the cell adhesion phenomena than others' parameters (Average roughness (Ra em leader )): cells adhere better on isotropic surfaces with a low order, provided this order is quantified on a scale that is more important than that of the cells. Additionally, on these low ordered metallic surfaces, the shape of the cells presents the same morphological aspect as that we can see on the human bone trabeculae. The method used to prepare these isotropic surfaces (electroerosion) could be undoubtedly and easily applied to prepare most biomaterials with complex geometries and to improve bone implant integration. Moreover, the new order parameter we developed may be particularly useful for the fundamental understanding of the mechanism of bone cell installation on a relief and of the formation of bone cell-material interface.