Fibronectin amyloid-like aggregation alters its extracellular matrix incorporation and promotes a single and sparsed cell migration.

Fibronectin (Fn) is an extracellular matrix (ECM) multifunctional glycoprotein essential for regulating cells behaviors. Within ECM, Fn is found as polymerized fibrils. Apart from fibrils, Fn could also form other kind of supramolecular assemblies such as aggregates. To gain insight into the impact of Fn aggregates on cell behavior, we generated several Fn oligomeric assemblies. These assemblies displayed various amyloid-like properties but were not cytotoxic. In presence of the more amyloid-like structured assemblies of Fn, the cell-ECM networks were altered and the cell shapes shifted toward extended mesenchymal morphologies. Additionnaly, the Fn amyloid-like aggregates promoted a single-cell and sparsed migration of SKOV3 cancer cells, which was associated with a relocalization of αv integrins from plasma membrane to perinuclear vesicles. These data pointed out that the features of supramolecular Fn assemblies could represent a higher level of fine-tuning cell phenotype, and especially migration of cancer cells.

Effects of Fibronectin Coating on Bacterial and Osteoblast Progenitor Cells Adherence in a Co-culture Assay.

Bacterial adherence to the surface of implants functionalized with cell-adhesive biomolecules is a critical first step of infection development. This study was designed to determine how the immobilization of human plasmatic fibronectin (pFN) could impact bacterial and osteoblast cells interaction with the surface during concomitant exposition to the two cell-types. Calibrated suspensions of P. aeruginosa PAOI or S. aureus CIP4.83 bacteria and STRO-1+A osteoblast progenitor cells were mixed, co-seeded on glass coverslips coated or not with pFN and incubated at 37 °C. After 3 h of co-culture, the presence of bacteria did not modify the STRO-1+A cells adherence to glass. pFN coating significantly enhanced STRO-1+A cells, CIP4.83 and PAOI adherence to glass and bacterial interaction with STRO-1+A cells. Confocal laser scanning microscopy observations revealed that cells on the pFN-coated substrate exhibited a greater spreading, better organized network of cytoskeletal filaments, and an increased cellular FN expression than cells on the uncoated substrate. The use of fluorescently labeled pFN showed that adherent STRO-1+A cells were able to remodel and to concentrate coated pFN at the cells surface. Thus, the use of FN coating could increase the risk of bacterial adherence to the material surface, acting either directly onto the coating layer or indirectly on adherent osteoblastic cells. This may increase the infection risk in the presence of bacterial contamination.

New synthesis method of HA/P(D,L)LA composites: study of fibronectin adsorption and their effects in osteoblastic behavior for bone tissue engineering.

A novel synthetic method to synthesize hydroxyapatite/poly (D,L) lactic acid biocomposite is presented in this study by mixing only the precursors hydroxyapatite and (D,L) LA monomer without adding neither solvent nor catalyst. Three compositions were successfully synthesized with the weight ratios of 1/1, 1/3, and 3/5 (hydroxyapatite/(D,L) lactic acid), and the grafting efficiency of poly (D,L) lactic acid on hydroxyapatite surface reaches up to 84 %. Scanning electron microscopy and Fourier transform infrared spectroscopy showed that the hydroxyapatite particles were successfully incorporated into the poly (D,L) lactic acid polymer and X ray diffraction analysis showed that hydroxyapatite preserved its crystallinity after poly (D,L) lactic acid grafting. Differential scanning calorimetry shows that Tg of hydroxyapatite/poly (D,L) lactic acid composite is less than Tg of pure poly (D,L) lactic acid, which facilitates the shaping of the composite obtained. The addition of poly (D,L) lactic acid improves the adsorption properties of hydroxyapatite for fibronectin extracellular matrix protein. Furthermore, the presence of poly (D,L) lactic acid on hydroxyapatite surface coated with fibronectin enhanced pre-osteoblast STRO-1 adhesion and cell spreading. These results show the promising potential of hydroxyapatite/poly (D,L) lactic acid composite as a bone substitute material for orthopedic applications and bone tissue engineering.

Lectins as probes for assessing the accessibility of N-linked glycans in relation to the conformational changes of fibronectin.

Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research.

Detection of invisible biological traces in relation to the physicochemical properties of substrates surfaces in forensic casework.

Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many factors influence touch DNA transfer, including the "destination" substrate's surface. The latter's physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates' physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates' intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.

Targeting cell-derived markers to improve the detection of invisible biological traces for the purpose of genetic-based criminal identification.

At a crime scene, investigators are faced with a multitude of traces. Among them, biological traces are of primary interest for the rapid genetic-based identification of individuals. "Touch DNA" consists of invisible biological traces left by the simple contact of a person's skin with objects. To date, these traces remain undetectable with the current methods available in the field. This study proposes a proof-of-concept for the original detection of touch DNA by targeting cell-derived fragments in addition to DNA. More specifically, adhesive-structure proteins (laminin, keratin) as well as carbohydrate patterns (mannose, galactose) have been detected with keratinocyte cells derived from a skin and fingermark touch-DNA model over two months in outdoor conditions. Better still, this combinatory detection strategy is compatible with DNA profiling. This proof-of-concept work paves the way for the optimization of tools that can detect touch DNA, which remains a real challenge in helping investigators and the delivery of justice.

Fibronectin Conformations after Electrodeposition onto 316L Stainless Steel Substrates Enhanced Early-Stage Osteoblasts' Adhesion but Affected Their Behavior.

The implantation of metallic orthopedic prostheses is increasingly common due to an aging population and accidents. There is a real societal need to implement new metal implants that combine durability, good mechanical properties, excellent biocompatibility, as well as affordable costs. Since the functionalization of low-cost 316L stainless steel substrates through the successive electrodeposition of a polypyrrole film (PPy) and a calcium phosphate deposit doped with silicon was previously carried out by our labs, we have also developed a bio-functional coating by electrodepositing or oxidating of fibronectin (Fn) coating. Fn is an extracellular matrix glycoprotein involved in cell adhesion and differentiation. Impacts of either electrodeposition or oxidation on the structure and functionality of Fn were first studied. Thus, electrodeposition is the technique that permits the highest deposition of fibronectin, compared to adsorption or oxidation. Furthermore, electrodeposition seems to strongly modify Fn conformation by the formation of intermingled long fibers, resulting in changes to the accessibility of the molecular probes tested (antibodies directed against Fn whole molecule and Fn cell-binding domain). Then, the effects of either electrodeposited Fn or oxidized Fn were validated by the resulting pre-osteoblast behavior. Electrodeposition reduced pre-osteoblasts' ability to remodel Fn coating on supports because of a partial modification of Fn conformation, which reduced accessibility to the cell-binding domain. Electrodeposited Fn also diminished α5 integrin secretion and clustering along the plasma membrane. However, the N-terminal extremity of Fn was not modified by electrodeposition as demonstrated by Staphylococcus aureus attachment after 3 h of culture on a specific domain localized in this region. Moreover, the number of pre-osteoblasts remains stable after 3 h culture on either adsorbed, oxidized, or electrodeposited Fn deposits. In contrast, mitochondrial activity and cell proliferation were significantly higher on adsorbed Fn compared with electrodeposited Fn after 48 h culture. Hence, electro-deposited Fn seems more favorable to pre-osteoblast early-stage behavior than during a longer culture of 24 h and 48 h. The electrodeposition of matrix proteins could be improved to maintain their bio-activity and to develop this promising, fast technique to bio-functionalize metallic implants.

A biomimetic model of 3D fluid extracellular macromolecular crowding microenvironment fine-tunes ovarian cancer cells dissemination phenotype.

An early fundamental step in ovarian cancer progression is the dissemination of cancer cells through liquid environments, one of them being cancer ascites accumulated in the peritoneal cavity. These biological fluids are highly crowded with a high total macromolecule concentration. This biophysical property of fluids is widely used in tissue engineering for a few decades now, yet is largely underrated in cancer biomimetic models. To unravel the role of fluids extracellular macromolecular crowding (MMC), we exposed ovarian cancer cells (OCC) to high molecular weight inert polymer solutions. High macromolecular composition of extracellular liquid presented a differential effect: i) it impeded non-adherent OCC aggregation in suspension and, decreased their adhesion; ii) it promoted adherent OCC migration by decreasing extracellular matrix deposition. Besides, there seemed to be a direct link between the extracellular MMC and intracellular processes, especially the actin cytoskeleton organization and the nucleus morphology. In conclusion, extracellular fluid MMC orients OCC dissemination phenotype. Integrating MMC seems crucial to produce more relevant mimetic 3D in vitro fluid models to study ovarian dissemination but also to screen drugs.

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin.

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/alphav integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and alphav integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and alphav integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-alphav integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/alphav integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.

Diffusions of sound frequencies designed to target dehydrins induce hydric stress tolerance in Pisum sativum seedings.

Among plant responses to environmentally induced stress modulating protein expression appears to be a key stage in inducible signaling. Our study was focused on an innovative strategy to stimulate plant stress resistance, namely, the use of targeted sequences of specific sound frequencies. The influence of acoustic stimulation on plant protein synthesis was investigated. In our study green peas, Pisum sativum, were cultured under hydric stress conditions with targeted acoustic stimulation. Acoustic sequences targeting dehydrins (DHN) which accumulate in plants in response to dehydration were studied. We experimented on pea seeding with two different sequences of sounds: the first one corresponded to DHN cognate protein and the second one was aimed at the DHN consensus sequence. Shoot elongation after pea seed germination was estimated by fresh weight gain studied in the presence of various conditions of exposure to both sequences of sounds. DHN expression in peas was quantified via ELISA tests and Western-blotting by using specific antibodies. A significant increase in fresh weight in peas grown under exposure to the DHN cognate sound sequence was observed, whereas the consensus sound sequence had no effect on growth. Moreover, the 37kDa DHN amount was increased in peas treated with the consensus acoustic sequence. These results suggest that the expression of DHN could be specifically modulated by a designed acoustic stimulus.

Real-Time Imaging of Bacteria/Osteoblast Dynamic Coculture on Bone Implant Material in an in Vitro Postoperative Contamination Model.

Biomedical implants are an important part of evolving modern medicine but have a potential drawback in the form of postoperative pathogenic infection. Accordingly, the "race for surface" combat between invasive bacteria and host cells determines the fate of implants. Hence, proper in vitro systems are required to assess effective strategies to avoid infection. In this study, we developed a real time observation model, mimicking postoperative contamination, designed to follow E. coli proliferation on a titanium surface occupied by human osteoblastic progenitor cells (STRO). This model allowed us to monitor E. coli invasion of human cells on titanium surfaces coated and uncoated with fibronectin. We showed that the surface colonization of bacteria is significantly enhanced on fibronectin coated surfaces irrespective of whether areas were uncovered or covered with human cells. We further revealed that bacterial colonization of the titanium surfaces is enhanced in coculture with STRO cells. Finally, this coculture system provides a comprehensive system to describe in vitro and in situ bacterial and human cells and their localization but also to target biological mechanisms involved in adhesion as well as in interactions with surfaces, thanks to fluorescent labeling. This system is thus an efficient method for studies related to the design and function of new biomaterials.

Amyloid-like aggregates formation by blood plasma fibronectin.

Fibronectin (FN) is a multifunctional glycoprotein of the extracellular matrix (ECM) playing critical roles in physiological and pathological cell processes like adhesion, migration, growth, and differentiation. These various functions of FN are modulated by its supramolecular state. Indeed, FN can polymerize into different types of assemblies like fibrils and aggregates. However, the mechanism of polymerization and the effects of such assemblies on cell behaviors still remain to be elucidated. Here we show that upon irreversible thermal denaturation, human blood plasma fibronectin forms high molecular weight aggregates. These compact and globular aggregates show amyloid features: they are stabilized by intermolecular b-sheets, they bind Thioflavin T and they are resistant to reducing and denaturing agents. Their characterization by electrospray ionization charge detection mass spectrometry shows that two populations can be distinguished according to the mass and charge density. Despite their amyloid features and the presence of hydrophobic patches on their surface, these aggregates are not toxic for cells. However, their binding abilities to gelatin and RGD are drastically decreased compare to native FN, suggesting possible effects on ECM-cell interactions.

Synergistic effects of BMP-2, BMP-6 or BMP-7 with human plasma fibronectin onto hydroxyapatite coatings: A comparative study.

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. STATEMENT OF SIGNIFICANCE: The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects.

Electrochemical processing of fibrinogen modified-graphite surfaces: effect on plasmin generation from adsorbed plasminogen.

With the aim to improve the fibrinolytic properties of carbons by different biological and electrochemical treatments, we modified graphite surfaces by fibrinogen adsorption and subsequent application of various constant potentials before submitting them to plasminogen adsorption. First, we verified that plasminogen (purified or present in human plasma) could adsorb onto these modified surfaces and that adsorbed plasminogen could be converted by t-PA (the principal physiological activator of plasminogen) to adsorbed plasmin. The catalytic properties of the generated enzyme were characterized in assay solutions containing t-PA, fibrinogen and the chromogenic substrate S-2403 (pyroGlu-Phe-Lys-p-nitroaniline, HCl). Experiments showed that the application of electrical potentials to the fibrinogen coating could indirectly affect the properties of the material. In the case of anodic potentials, the amidolytic activity of the generated plasmin was significantly enhanced. Especially, this activity was 10 times higher at a particular potential value.

Influence of hydroxyapatite microstructure on human bone cell response.

Microstructure of calcium phosphate ceramics has been shown to influence long-term in vitro cellular events like proliferation and differentiation, and to favor bone integration in vivo. As long-term cellular events are known to be dependent of early cell adhesion events, we decided to study the in vitro influence of the microstructure of a microporous hydroxyapatite (mHA) and a nonmicroporous hydroxyapatite (pHA) ceramic on serum protein adsorption and SaOs-2 human bone cells attachment after 30 min, 1, 4, and 24 h and cell growth after 96 h. Plastic coverslips were used as controls. Hydroxyapatite composition of mHA and pHA was confirmed by X-ray diffraction and Fourier transform infra-red spectroscopy. The surface energies of ceramics were calculated from contact-angle measurements in di-iodomethane, water or complete culture medium. The total surface energy was 44.8 mJ/m(2) for pHA and 48.7 mJ/m(2) for plastic. The contact-angle measurement was impossible on mHA likely because they displayed 12% of open microporosity, pHA ceramic exhibiting only closed pores (2.5%). Moreover, the roughness amplitude was largely higher on mHA (Sa = 4.35 microm) than on pHA (Sa = 0.065 microm) and plastic (Sa = 0.042 microm). Three different techniques were used to evaluate protein adsorption on the ceramics. SDS-PAGE of desorbed proteins demonstrated that more proteins desorbed from mHA (66.02 microg/m(2)) than from pHA (17.2 microg/m(2)) or plastic (0.08 microg/m(2)). A new method was used to evaluate in situ the quantity of adsorbed total proteins: the temperature-programmed desorption (TPD) analysis coupled with mass spectrometry. The TPD analysis confirmed that 10-fold more proteins adsorbed on mHA compared with those on pHA. A direct immunolabeling on ceramics revealed than more fibronectin and serum albumin adsorbed on microporous ceramic than on dense ceramic. The morphology of SaOs-2 cells was the same on all the substrates after 30 min. At later time points, cell morphology on mHA was radically different than on other surfaces, with the particularity of the cytoplasmic edge that appeared undistinguishable from the surface. Only the extremity of the cells and lamellipodia were visible. Cells seemed like "adsorbed" by the mHA surface, whereas on plastic and pHA surfaces the cells displayed classical aspects of polygonal spreading. The cells displayed on mHA the highest initial attachment potential after 30 min, 1, 4, 24 h but the lower proliferation potential after four days. This study confirms that a microporous ceramic surface can modulate the adsorption of proteins and further the adhesion and proliferation of human bone cells.

Removal of surface by-products from sintered hydroxyapatite: effect of a chelation treatment on fibronectin adsorption and cell adhesion.

It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fibronectin's known affinity for calcium and the composition of the ceramic itself suggested that calcium release could be the main cause of this aggregation effect. It was then decided to investigate the effect of a surface chelation treatment on fibronectin adsorption, and MG63 cell adhesion, onto porous ceramics of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), and HA/TCP biphasic material (BCP). Those ceramics were immersed in an EDTA solution and the effect of this treatment on the material composition was assayed. X-ray diffraction data showed the presence of alpha- and beta-TCP phases in HA and BCP materials, which were both completely removed by the chelation treatment in the case of HA. On BCP, alpha-TCP was removed and beta-TCP partially dissolved. The TCP material, which was pure beta-TCP, underwent a mass loss, but no change in composition was observed. Adhesion of MG63 cells was overall higher on the fibronectin-coated EDTA-treated HA material, but was especially enhanced on EDTA-treated HA. Changes in surface morphologies, as compared with the use of scanning electron microscopy, did not seem to be related to the effects observed. The EDTA treatment proved to be a very efficient way of removing by-products of HA sintered materials, and thus enhancing the biocompatibility of the material.

Adsorption-induced fibronectin aggregation and fibrillogenesis.

Fibronectin (Fn), a high molecular weight glycoprotein, is a central element of extracellular matrix architecture that is involved in several fundamental cell processes. In the context of bone biology, little is known about the influence of the mineral surface on fibronectin supramolecular assembly. We investigate fibronectin morphological properties induced by its adsorption onto a model mineral matrix of hydroxyapatite (HA). Fibronectin adsorption onto HA spontaneously induces its aggregation and fibrillation. In some cases, fibronectin fibrils are even found connected into a dense network that is close to the matrix synthesized by cultured cells. Fibronectin adsorption-induced self-assembly is a time-dependant process that is sensitive to bulk concentration. The N-terminal domain of the protein, known to be implicated in its self-association, does not significantly inhibit the protein self-assembly while increasing ionic strength in the bulk alters both aggregation and fibrillation. The addition of a non-ionic surfactant during adsorption tends to promote aggregation with respect to fibrillation. Ultimately, fibronectin fibrils appear to be partially structured like amyloid fibrils as shown by thioflavine T staining. Taken together, our results suggest that there might be more than one single organization route involved in fibronectin self-assembly onto hydroxyapatite. The underlying mechanisms are discussed with respect to Fn conformation, Fn/surface and Fn/Fn interactions, and a model of fibronectin fibrillogenesis onto hydroxyapatite is proposed.

Adsorption-induced conformational changes in protein diffusion-aggregation surface assemblies.

Two-dimensional rigid colloid aggregation models may be applied to protein layers when no large conformational change is involved. Yet, following adsorption, several proteins undergo a conformational transition that may be involved in aggregative structures. Our focus here is how a conformational change might influence surface clustering in a diffusion-aggregation model. We propose a model including diffusion, aggregation, and unfolding of proteins that are randomly adsorbed onto a surface. Our model allows simulating the case where protein-protein interaction favors unfolding and the case where this interaction prevents it. We study the effect of a simple disk-to-rod unidirectional unfolding and investigate the morphology of the resulting clusters in the diffusion- and reaction-limited regimes. A rich variety of structures is produced, with fractal dimension differing from that in universal diffusive aggregation models. Increasing unfolding probability shifts the system from the neighbor-induced to the neighbor-prevented unfolding regime. The intermediate structures that arise from our model could be helpful in understanding the assembly of different observed protein structures.

Inorganic-organic thin implant coatings deposited by lasers.

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 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/cm(2) 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 7 µg FN per cm(2) 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.

Pre-osteoblasts on poly(L-lactic acid) and silicon oxide: Influence of fibronectin and albumin adsorption.

Cell adhesion and subsequent viability are critical initial steps in biomaterial-tissue integration and are strongly dependent on the material properties and the presence of matrix proteins. In the present study MC3T3-E1 osteoblast-like cell behavior on silicon oxide (SO) and poly(L-lactic acid) (PLLA) substrates has been examined, with a focus on the influence of the adhesive protein fibronectin and the non-adhesive protein albumin adsorbed on the substrates. Quartz crystal microgravimetry showed adsorption of fibronectin and albumin to be nearly identical on SO and PLLA. Subsequent exposure a previously adsorbed fibronectin layer to albumin decreased the rigidity of the adsorbed layer without any measurable increase in adsorbed mass. Cell adhesion and spreading were significantly enhanced on both SO and PLLA substrates coated with fibronectin or with fibronectin and albumin, compared with uncoated or albumin-coated substrates. The only statistically significant difference between the two substrates in these assays was increased spreading on PLLA compared with SO in the presence of fibronectin and albumin. Cell proliferation was significantly higher on SO compared with PLLA after 7 days culture, but depended on the presence of fibronectin only in the PLLA system. In contrast, mitochondrial activity was higher on PLLA than on SO, and was enhanced by fibronectin on both substrates. PLLA substrates coated with fibronectin and subsequently exposed to albumin exhibited the highest level of cell differentiation, as assayed via alkaline phosphatase activity. These results demonstrate the importance of adsorbed proteins on osteoblast-like cell-surface interactions.