[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The goal of this work was to study the thermal stabilization of calcium apatites in which the Ca2+ ions were substituted for Sr2+ in increasing concentrations via ionic co-substitutions. Two distinct standard syntheses were proposed for comparative purposes: one using counter-ions that were not easily incorporated into the apatite structure (NH4+/NO3−) and one using counter-ions that can be easily incorporated into the structure (Na+/Cl−). After calcination, only the apatites synthesized in the presence of NH4+/NO3− presented phase transformation. In contrast, the apatites synthesized in the presence of Na+/Cl− formed a solid solution after calcination, with Na+, Ca2+, Sr2+ and Cl− sharing the same apatite lattice. Wavelength dispersive X-ray fluorescence spectroscopy (WDXRF), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and temperature-programmed desorption (TPD) techniques showed that the counter-ions present during the syntheses that are associated with CO32− play an important role in the thermal stabilization of the apatites.
Materials Science and Engineering B 05/2015; 199. DOI:10.1016/j.mseb.2015.05.003 · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To discriminate the most important physicochemical parameters for bone reconstruction, the inflammatory potential of seven nanoporous hydroxyapatite powders synthesized by hard or soft templating was evaluated both in vitro and in vivo.
After physical and chemical characterization of the powders, we studied the production of inflammatory mediators by human primary monocytes after 4 and 24 h in contact with powders, and the host response after 2 weeks implantation in a mouse critical size defect model.
In vitro results highlighted increases in the secretion of TNF-α, IL-1, -8, -10 and proMMP-2 and -9 and decreases in the secretion of IL-6 only for powders prepared by hard templating. In vivo observations confirmed an extensive inflammatory tissue reaction and a strong resorption for the most inflammatory powder in vitro.
These findings highlight that the most critical physicochemical parameters for these nanoporous hydroxyapatite are, the crystallinity that controls dissolution potential, the specific surface area and the size and shape of crystallites.
[Show abstract][Hide abstract] ABSTRACT: It has been previously shown that osteosarcoma (SaOs-2) cells respond to micropillared surfaces consisting of poly-l-lactic acid with strong deformation of the cell body and nucleus. Until now, cell nucleus deformation of SaOs-2 cells was only studied by exposing them to square shaped micropillars in an isotropic pattern. Here we report on experiments of the cell nucleus response of such cells to rhombic structures of different topographies generated from a rubbery polymer, namely poly(n-butyacrylate). It is observed that cells orientate themselves perpendicular to the long axis of the rhombi. While their spreading on the surface is not influenced by the opening angle of the structures, rhombic structures with sharper angles induce stronger deformation of the cells and accordingly more elongated nuclei.
Journal of Materials Science Materials in Medicine 02/2015; 26(2):5427. DOI:10.1007/s10856-015-5427-1 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lifetime of bone implants inside the human body is directly related to their osseointegration. Ideally, future materials should be inspired by human tissues and provide the material structure-function relationship from which synthetic advanced biomimetic materials capable of replacing, repairing or regenerating human tissues can be produced. This work describes the development of biomimetic thin coatings on titanium implants to improve implant osseointegration. The assembly of an inorganic-organic biomimetic structure by UV laser pulses is reported. The structure consists of a nanostructured hydroxyapatite (HA) film grown onto a titanium substrate by pulsed laser deposition (PLD) and activated by a top fibronectin (FN) coating deposited by matrix assisted pulsed laser evaporation (MAPLE). A pulsed KrF* laser source (λ=248 nm, τ=25 ns) was employed at fluences of 7 and 0.7J/cm2 for HA and FN transfer, respectively. Films approximately 1500 and 450 nm thick were obtained for HA and FN, respectively. A new cryogenic temperature-programmed desorption mass spectrometry analysis method was employed to accurately measure the quantity of immobilized protein. We determined that less than 7µg FN per cm2 HA surface is adequate to improve adhesion, spreading, and differentiation of osteoprogenitor cells. We believe that the proposed fabrication method opens the door to combining and immobilizing two or more inorganic and organic materials on a solid substrate in a well-defined manner. The flexibility of this method enables the synthesis of new hybrid materials by simply tailoring the irradiation conditions according to the thermo-physical properties of the starting materials.
[Show abstract][Hide abstract] ABSTRACT: Directional wettability, i.e. the variation of wetting properties, depending on the surface orientation, can be achieved by anisotropic surface texturing. A new high-precision process can produce homogeneous sinusoidal surfaces (in particular, parallel grooves) at the microscale, with a nanoscale residual roughness five orders of magnitude smaller than the texture features. Static wetting experiments have shown that this pattern, even with a very small aspect ratio, can induce a strong variation of the contact angle, depending on the direction of the observation. A comparison with numerical simulations (using Surface Evolver software) shows good agreement and could be used to predict fluid–solid interaction and droplet behaviour on textured surfaces. Two primary mechanisms of directional spreading of water droplets on textured stainless steel surface have been identified. The first one is the mechanical barrier created by the textured surface peaks; this limits spreading in a perpendicular direction to the surface anisotropy. The second one is the capillary action inside of the sinusoidal grooves, which accelerates spreading along the grooves. Spreading has been shown to depend strongly on the history of wetting and internal drop dynamics.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to synthesize and characterize novel nano-silver poly(acrylic acid) (AgNP-PAA) via photoreduction technique, and evaluate the biocompatibility and mechanical strength of the formed glass–ionomer cements (GIC). The polymer in the cement was composed of a long-chain poly(acrylic acid) (PAA) solution and AgNO3 (0, 0.05, 0.10 and 0.50 % wt/wt), and tartaric acid (TA) were added to formulate different final concentrations of silver nanoparticles polyacids. The formulations were UV irradiated and UV-Vis spectroscopy monitored the formation of AgNP. Human gingival fibroblasts HGF-1 (ATCC® CRL-2014™) were cultivated using the 24 h DMEM extract of the GIC. The cements were evaluated by compressive strength (CS) and in vitro indirect cytotoxicity test using an MTT assay. In the groups containing high AgNP levels, CS increased by 32 % compared to the control GIC group. Moreover, the synthesis of AgNP in the polyacids of the GIC increased the biocompatibility of the cements by 52 % compared to the control group. The PAA used in this work are not normally used to pharmaceutical needs. Therefore, we hypothesized that the process of photoredution used to synthesize AgNP in the polyacids helped to increase the biocompatibility of this PAA.
8th Latin American Congress of Artificial Organs, Biomaterials and Tissue Engineering (8th COLAOB), Rossario, Argentina; 08/2014
[Show abstract][Hide abstract] ABSTRACT: Defined protein quantities were embedded in situ in a biodegradable polymer coating during simultaneous laser vaporization of two targets. Fibronectin (FN) and poly-DL-lactide (PDLLA) were transferred and immobilized concomitantly by Combinatorial Matrix Assisted Pulsed Laser Evaporation onto solid substrates. The film surface with gradient of composition was characterized by optical, scanning electron microscopy and profilometry. Micrometric FN packages were visualized in the polymeric matrix by confocal microscopy. The composition of FN was investigated by FUR and tiFTIR analyses in a polymeric matrix with different thickness.
[Show abstract][Hide abstract] ABSTRACT: Highly controlled mixed molecular layers are crucial to study the role of material surface chemistry in biointerfaces, such as bacteria and subsequent biofilms interacting with biomaterials. Silanes with non-nucleophilic functional groups are promising to form self-assembled monolayers (SAMs) due to their low sensitivity to side-reactions. Nevertheless, the real control of surface chemistry, layer structure and organization has not been determined. Here, we report a comprehensive synthesis and analysis of undecyltrichlorosilane- and 11-bromoundecyltrichlorosilane-based mixed SAMs on silicon substrates. The impact of the experimental conditions on the control of surface chemistry, layer structure and organization was investigated by combining survey and high-resolution X-ray photoelectron spectroscopy analysis, wettability measurements and ellipsometry. The most appropriate conditions were first determined for elaborating highly reproducible, but easily made, pure 11-bromoundecyltrichlorosilane SAMs. We have demonstrated that the control is maintained on more complex surfaces i.e., surfaces revealing various chemical densities, which were obtained with different ratios of undecyltrichlorosilane and 11-bromoundecyltrichlorosilane. The control is also maintained after bromine to amine group conversion via SN2 bromine-to-azide reactions. The appropriateness of such highly controlled amino- and methyl-group revealing platforms (NH2-X%/CH3) for biointerface studies was shown by the higher reproducibility of bacterial adhesion on NH2-100%/CH3 SAMs compared to bacterial adhesion on molecular layers of overall similar surface chemistry, but less control at the molecular scale.
[Show abstract][Hide abstract] ABSTRACT: Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allows for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which induces both increased cell proliferation and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements.
[Show abstract][Hide abstract] ABSTRACT: Objective: The aim of this study was to use a novel synthesized silver nanoparticle (NAg)-containing polyacid to formulate conventional glass ionomer (GI) and evaluate the effect of NAg on the compressive strength and contact inhibition of Streptococcus mutans to the surface of the formed GI.
Method: Poly(acrylic acid) (PAA) solutions containing or not NAg synthesized in situ were hand mixed to glass powders of Fuji IXTM (GC coorp.). Compressive strength of groups (n=8) were tested according to ISO 9917:2003. Contact inhibition of S. mutans (CIP 103220) incubated in BHI for 20h at 37oC were compared on GI surfaces using a confocal laser scanning microscopy.
Result: The presence of 0.07% NAg in the glass ionomer matrix increased values of compressive strength compared to control (no NAg) (P<0.01). Fluorescent phase-contrast images suggest that NAg are inhibiting S. mutans adhesion due to the reduced amount of bacteria adhered to the surface of NAg groups, and also because of the tendency of agglomeration and changes in cell morphology.
Conclusion: This study developed a novel antibacterial conventional glass-ionomer. Within the limitations of this study, it appears that the experimental cement is a clinically attractive dental restorative due to its high mechanical strength and antibacterial function.
Annual Meeting of the IADR Continental European Division 2013; 09/2013
[Show abstract][Hide abstract] ABSTRACT: This work describes the preparation and characterization of porous 3D-scaffolds based on chitosan (CHI), chitosan/silk fibroin (CHI/SF) and chitosan/silk fibroin/hydroxyapatite (CHI/SF/HA) by freeze drying. The biomaterials were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, studies of porosity, pore size, contact angle and biological response of SaOs-2osteoblastic cells were performed. The CHI scaffolds have a porosity of 94.2±0.9%, which is statistically higher than the one presented by CHI/SF/HA scaffolds, 89.7±2.6%. Although all scaffolds were able to promote adhesion, growth and maintenance of osteogenic differentiation of SaOs-2 cells, the new 3D-scaffold based on CHI/SF/HA showed a significantly higher cell growth at 7days and 21days and the level of alkaline phosphatase at 14 and 21days was statistically superior compared to other tested materials.
[Show abstract][Hide abstract] ABSTRACT: Introduction Experimental Methods The Interaction of Bone Cells with Micropillars The Deformation of Skin Cells as a Function of Their Malignancy The Deformation of Fibroblasts of Different Cellular Ages Discussion Conclusions Acknowledgments References
[Show abstract][Hide abstract] ABSTRACT: Calcium phosphates, particularly hydroxyapatite
Ca10(PO4)6(OH)2 (HA), arewidely used for bone regeneration
due to their biocompatibility and good resorption properties.
However, their performance upon implantation is improved
when they are associated with bioactive molecules such as
growth factors. Using mesoporous HA leads to improved
protein adsorption and release kinetics because the diameter
of the mesopores (2–50 nm) is in the same range as their size.
We prepared this type of material by the nanocasting method
using three different templates: a silica foam and two carbon
templates derived fromit using propylene or sucrose as carbon
source. We investigated the influence of the template, the
calcination temperature and of the conditions during template
removal. We obtained HA materials with a surface area of up
to 90 m2 g-1 and with an intergranular mesopore volume of
up to 0.4 cm3 g-1. In this paper,we showfor the first time that
the synthesis of mesoporous HA from a mesoporous silica
foam template allows eliminating the template at lower temperatures
(in an alkaline medium), thus preventing the sintering
of the HA. These materials have interesting properties
for drug delivery applications. The protein adsorption and
release capacities of these HAs were tested with two model
proteins, bovine serum albumin (BSA), and Cytochrome C.
These materials are an important milestone for future bone
regeneration systems based on HA associated with human
growth factor proteins.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Materials Science Materials in Medicine 04/2013; 24(7). DOI:10.1007/s10856-013-4927-0 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have recently developed new 3D hydroxyapatite/collagen (50/50 wt%) scaffolds using a biomimetic synthesis approach. The first in vitro tests performed in static culture showed a limited cell colonization and survival inside the scaffolds. The current study evaluated in dynamic culture the scaffold changes and colonization by human immortalized osteoprogenitor STRO-1A cells. The stability of our scaffolds in the different culture conditions (static, low flow, high flow) was validated by the maintenance of the pore diameter and interconnectivity over 21 d. The colonization and the viability of STRO-1A cells inside the scaffolds were further evaluated on histological sections. It was demonstrated that only the high flow-rate allowed cell survival after 7 d and a complete scaffold colonization. Moreover, the colonization and viability was different in function of the scaffold position inside the perfusion container. The differentiation markers (alkaline phosphatase activity, type I procollagen and osteocalcin synthesis) of STRO-1A cells were analyzed in the culture medium after 7, 14 and 21 d. The low flow-rate increased significantly the three markers compared with static conditions. In contrast, markers were reduced in high flow-rate compared with low flow-rate. To explain this surprising result, we hypothesized that the different molecules were actually adsorbed on the scaffold because of the closed circuit used in the high flow-rate conditions. In summary, this study provides original results on the influence of flow rate but mostly of the circuit used (open/closed) on the structural modifications and cell colonization of collagen-HA scaffolds.