Reverse Mechanotransduction: Driving Chromatin Compaction to Decompaction Increases Cell Adhesion Strength and Contractility.

Mechanical extracellular signals elicit chromatin remodeling via the mechanotransduction pathway, thus determining cellular function. However, the reverse pathway is an open question: does chromatin remodeling shape cells, regulating their adhesion strength? With fluidic force microscopy, we can directly measure the adhesion strength of epithelial cells by driving chromatin compaction to decompaction with chromatin remodelers. We observe that chromatin compaction, induced by performing histone acetyltransferase inhibition or ATP depletion, leads to a reduction in nuclear volume, disrupting actin cytoskeleton and focal adhesion assembly, and ultimately decreases in cell adhesion strength and traction force. Conversely, when chromatin decompaction is drived by removing the remodelers, cells recover their original shape, adhesion strength, and traction force. During chromatin decompaction, cells use depolymerized proteins to restore focal adhesion assemblies rather than neo-synthesized cytoskeletal proteins. We conclude that chromatin remodeling shapes cells, regulating adhesion strength through a reverse mechanotransduction pathway from the nucleus to the cell surface involving RhoA activation.

Escherichia coli Biofilm Formation, Motion and Protein Patterns on Hyaluronic Acid and Polydimethylsiloxane Depend on Surface Stiffness.

The surface stiffness of the microenvironment is a mechanical signal regulating biofilm growth without the risks associated with the use of bioactive agents. However, the mechanisms determining the expansion or prevention of biofilm growth on soft and stiff substrates are largely unknown. To answer this question, we used PDMS (polydimethylsiloxane, 9-574 kPa) and HA (hyaluronic acid gels, 44 Pa-2 kPa) differing in their hydration. We showed that the softest HA inhibited Escherichia coli biofilm growth, while the stiffest PDMS activated it. The bacterial mechanical environment significantly regulated the MscS mechanosensitive channel in higher abundance on the least colonized HA-44Pa, while Type-1 pili (FimA) showed regulation in higher abundance on the most colonized PDMS-9kPa. Type-1 pili regulated the free motion (the capacity of bacteria to move far from their initial position) necessary for biofilm growth independent of the substrate surface stiffness. In contrast, the total length travelled by the bacteria (diffusion coefficient) varied positively with the surface stiffness but not with the biofilm growth. The softest, hydrated HA, the least colonized surface, revealed the least diffusive and the least free-moving bacteria. Finally, this shows that customizing the surface elasticity and hydration, together, is an efficient means of affecting the bacteria's mobility and attachment to the surface and thus designing biomedical surfaces to prevent biofilm growth.

Insensitivity of dental pulp stem cells migration to substrate stiffness.

Dental pulp stem cells (DPSCs) are a promising cell source for regeneration of dental pulp. Migration is a key event but influence of the microenvironment rigidity (5 kPa at the center of dental pulp to 20 GPa for the dentin) is largely unknown. Mechanical signals are transmitted from the extracellular matrix to the cytoskeleton, to the nuclei, and to the chromatin, potentially regulating gene expression. To identify the microenvironmental influence on migration, we analyzed motility on PDMS substrates with stiffness increasing from 1.5 kPa up to 2.5 MPa. We found that migration speed slightly increases as substrate stiffness decreases in correlation with decreasing focal adhesion size. Motility is relatively insensitive to substrate stiffness, even on a bi-rigidity PDMS substrate where DPSCs migrate without preferential direction. Migration is independent of both myosin II activity and YAP translocation after myosin II inhibition. Additionally, inhibition of Arp2/3 complex leads to significant speed decrease for all rigidities, suggesting contribution of the lamellipodia in the migration. Interestingly, the chromatin architecture remains stable after a 7-days exposure on the PDMS substrates for all rigidity. To design scaffold mimicking dental pulp environment, similar DPSCs migration for all rigidity, leaves field open to choose this mechanical parameter.

Enzyme assisted peptide self-assemblies trigger cell adhesion in high density oxime based host gels.

Peptide supramolecular self-assemblies are recognized as important components in responsive hydrogel based materials with applications in tissue engineering and regenerative medicine. Studying the influence of hydrogel matrices on the self-assembly behavior of peptides and interaction with cells is essential to guide the future development of engineered biomaterials. In this contribution, we present a PEG based host hydrogel material generated by oxime click chemistry that shows cellular adhesion behavior in response to enzyme assisted peptide self-assembly (EASA) within the host gel. This hydrogel prepared from poly(dimethylacrylamide-co-diacetoneacrylamide), poly(DMA-DAAM) with high molar fractions (49%) of DAAM and dialkoxyamine PEG cross-linker, was studied in the presence of embedded enzyme alkaline phosphatase (AP) and a non-adhesive cell behavior towards NIH 3T3 fibroblasts was observed. When brought into contact with a Fmoc-FFpY peptide solution (pY: phosphorylated tyrosine), the gel forms intercalated Fmoc-FFY peptide self-assemblies upon diffusion of Fmoc-FFpY into the cross-linked hydrogel network as was confirmed by circular dichroism, fluorescence emission spectroscopy and confocal microscopy. Nevertheless, the mechanical properties do not change significantly after the peptide self-assembly in the host gel. This enzyme assisted peptide self-assembly promotes fibroblast cell adhesion that can be enhanced if Fmoc-F-RGD peptides are added to the pre-gelator Fmoc-FFpY peptide solution. Cell adhesion results mainly from interactions of cells with the non-covalent peptide self-assemblies present in the gel despite the fact that the mechanical properties are very close to those of the native host gel. This result is in contrast to numerous studies which showed that the mechanical properties of a substrate are key parameters of cell adhesion. It opens up the possibility to develop a diverse set of hybrid materials to control cell fate in culture due to tailored self-assemblies of peptides responding to the environment provided by the host guest gel.

Chromatin de-condensation by switching substrate elasticity.

Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.

The tumor suppressor CDX2 opposes pro-metastatic biomechanical modifications of colon cancer cells through organization of the actin cytoskeleton.

The vast majority of cancer deaths are caused by the formation of metastases rather than the primary tumor itself. Despite this clinical importance, the molecular and cellular events that support the dissemination of cancer cells are not yet fully unraveled. We have previously shown that CDX2, a homeotic transcription factor essential for gut development, acts as a colon-specific tumor suppressor and opposes metastasis. Here, using a combination of biochemical, biophysical, and immunofluorescence techniques, we further investigated the mechanisms promoted by CDX2 that might antagonize tumor cell dissemination. We found that CDX2 expression regulates the transcription of RHO GEFs, thereby activating RHO signaling cascades that lead to reorganization of the actin cytoskeleton and enhanced adherent junctions. Accordingly, we observed by atomic force microscopy (AFM) that colon cancer cells expressing CDX2 are less deformable, a feature that has been shown to correlate with poor metastatic potential. Thus, this study illustrates how the loss of expression of a transcription factor during colon cancer progression modifies the biomechanical characteristics of tumor cells and hence facilitates invasion and metastasis.

Cell guidance into quiescent state through chromatin remodeling induced by elastic modulus of substrate.

Substrate stiffness is known to strongly influence the fate of adhering cells. Yet, little is known about the influence of the substrate stiffness on chromatin. Chromatin integrates a multitude of biochemical signals interpreted by activation or gene silencing. Here we investigate for the first time the organization of chromatin of epithelial cells on substrate with various mechanical properties. On stiff substrates (100-200 kPa), where cells preferentially adhere, chromatin is mainly found in its euchromatin form. Decreasing the Young modulus to 50 kPa is correlated with a partial shift from euchromatin to heterochromatin. On very soft substrates (≪10 kPa) this is accompanied by cell lysis. On these very soft substrates, histone deacetylase inhibition by adding a drug preserves acetylated histone and thus maintains the euchromatin form, thereby keeping intact the nuclear envelope as well as a residual intermediate filament network around the nucleus. This allows cells to survive in a non-adherent state without undergoing proliferation. When transfer on a stiff substrate these cells retain their capacity to adhere, to spread and to enter a novel mitotic cycle. A similar effect is observed on soft substrates (50 kPa) without need of histone deacetylase inhibition. These new results suggest that on soft substrates cells might enter in a quiescence state. Cell quiescence may thus be triggered by the Young modulus of a substrate, a major result for strategies focusing on the design of scaffold in tissue engineering.

Titanium microbead-based porous implants: bead size controls cell response and host integration.

Openly porous structures in implants are desirable for better integration with the host tissue. Sintered microbead-based titanium implants for oto-rhinolaryngology applications, which create an environment where the cells can migrate in the areas between the microbeads, are developed. This structure promotes fibrovascular tissue formation within the implant in vivo. In this study, it is determine to what extent these events can be controlled by changing the physical environment of the implants both in vitro and in vivo. By cell tracking, it is observed that the size of the beads and the distance between the neighboring beads significantly affect the ability of cells to develop cell-to-cell contacts and to bridge the pores. Live cell staining shows that as the bead size gets smaller, the probability to observe cells that fill the porous areas is higher. This also affects the initial attachment and distribution of the cells and collagen secretion by fibroblasts. Obtaining a fast coverage of the system also enables co-culture systems where, the number and the distribution of the second cell type are boosted by the presence of the first. This concept is utilized to increase the attachment of vascular endothelial cells by an initial layer of fibroblasts. By decreasing the bead diameter, the overall colonization of the implant can be significantly increased in vivo. The effect of bead size has a similar pattern both in rats and rabbits, with faster colonization of smaller bead-based structures. Using smaller beads would improve clinical outcomes as faster integration facilitates the attainment of functionality by the implant.

Contribution of soft substrates to malignancy and tumor suppression during colon cancer cell division.

In colon cancer, a highly aggressive disease, progression through the malignant sequence is accompanied by increasingly numerous chromosomal rearrangements. To colonize target organs, invasive cells cross several tissues of various elastic moduli. Whether soft tissue increases malignancy or in contrast limits invasive colon cell spreading remains an open question. Using polyelectrolyte multilayer films mimicking microenvironments of various elastic moduli, we revealed that human SW480 colon cancer cells displayed increasing frequency in chromosomal segregation abnormalities when cultured on substrates with decreasing stiffness. Our results show that, although decreasing stiffness correlates with increased cell lethality, a significant proportion of SW480 cancer cells did escape from the very soft substrates, even when bearing abnormal chromosome segregation, achieve mitosis and undergo a new cycle of replication in contrast to human colonic HCoEpiC cells which died on soft substrates. This observation opens the possibility that the ability of cancer cells to overcome defects in chromosome segregation on very soft substrates could contribute to increasing chromosomal rearrangements and tumor cell aggressiveness.

Multi-scale modification of metallic implants with pore gradients, polyelectrolytes and their indirect monitoring in vivo.

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Modification of macroporous titanium tracheal implants with biodegradable structures: tracking in vivo integration for determination of optimal in situ epithelialization conditions.

Previously, we showed that macroporous titanium implants, colonized in vivo together with an epithelial graft, are viable options for tracheal replacement in sheep. To decrease the number of operating steps, biomaterial-based replacements for epithelial graft and intramuscular implantation were developed in the present study. Hybrid microporous PLLA/titanium tracheal implants were designed to decrease initial stenosis and provide a surface for epithelialization. They have been implanted in New Zealand white rabbits as tracheal substitutes and compared to intramuscular implantation samples. Moreover, a basement membrane like coating of the implant surface was also designed by Layer-by-Layer (LbL) method with collagen and alginate. The results showed that the commencement of stenosis can be prevented by the microporous PLLA. For determination of the optimum time point of epithelialization after implantation, HPLC analysis of blood samples, C-reactive protein (CRP), and Chromogranin A (CGA) analyses and histology were carried out. Following 3 weeks the implant would be ready for epithelialization with respect to the amount of tissue integration. Calcein-AM labeled epithelial cell seeding showed that after 3 weeks implant surfaces were suitable for their attachment. CRP readings were steady after an initial rise in the first week. Cross-linked collagen/alginate structures show nanofibrillarity and they form uniform films over the implant surfaces without damaging the microporosity of the PLLA body. Human respiratory epithelial cells proliferated and migrated on these surfaces which provided a better alternative to PLLA film surface. In conclusion, collagen/alginate LbL coated hybrid PLLA/titanium implants are viable options for tracheal replacement, together with in situ epithelialization.

The control of chromosome segregation during mitosis in epithelial cells by substrate elasticity.

Materials of defined elasticity, including synthetic material scaffolds and tissue-derived matrices, can regulate biological responses of cells and in particular adhesion, migration, growth and differentiation which are essential parameters for tissue integration. These responses have been extensively investigated in interphase cells, but little is known whether and how material elasticity affects mitotic cells. We used polyelectrolyte multilayer films as model substrates with elastic modulus ranging from Eap = 0 up to Eap = 500 kPa and mitotic PtK2 epithelial cells to address these important questions. Soft substrates (Eap < 50 kPa) led to abnormal morphology in chromosome segregation, materialized by chromatin bridges and chromosome lagging. Frequency of these damages increased with decreasing substrate stiffness and was correlated with a pro-apoptotic phenotype. Mitotic spindle was not observed on soft substrates where formation of chromatin damages is due to low ß1-integrin engagement and decrease of Rac1 activities. This work constitutes the first evidence that soft substrates hinder epithelial cell division. In perspective, our findings emphasize the prime incidence of the material elasticity on the fate of the phenotype, especially of stem cells in the mitotic phase.

Development of surgical protocol for implantation of tracheal prostheses in sheep.

This article documents experiments performed in ewes to design an artificial larynx. The artificial larynx is composed of a hollow, porous tube that elongates the trachea and is capped with a valve that acts as a laryngeal sphincter. Through an industrial collaboration, our team developed a porous biomaterial that can be colonized by cervical tissues. This biomaterial has been used in animals to replace part of the trachea, but it is meant to eventually substitute for laryngeal cartilage. The tracheal prosthesis is a hollow cylindrical tube composed of titanium microbeads. We performed a study in large animals to establish an optimal surgical protocol for tracheal replacement in humans. The study included 11 sheep (n = 11) and compared 5 methods of implantation. We successfully established an optimal three-step surgical protocol to make the porous-titanium tracheal prosthesis functional: (1) large lumen endoprosthetics, (2) colonization by the peripheral tissues, and (3) endoprosthetic epithelialization. This study is the first step in developing an artificial larynx because it successfully identifies a biomaterial capable of extending the trachea to allow it to open at the junction of the upper aerodigestive tracts.

Hybrid titanium/biodegradable polymer implants with an hierarchical pore structure as a means to control selective cell movement.

UNLABELLED: In order to improve implant success rate, it is important to enhance their responsiveness to the prevailing conditions following implantation. Uncontrolled movement of inflammatory cells and fibroblasts is one of these in vivo problems and the porosity properties of the implant have a strong effect on these. Here, we describe a hybrid system composed of a macroporous titanium structure filled with a microporous biodegradable polymer. This polymer matrix has a distinct porosity gradient to accommodate different cell types (fibroblasts and epithelial cells). The main clinical application of this system will be the prevention of restenosis due to excessive fibroblast migration and proliferation in the case of tracheal implants. METHODOLOGY/PRINCIPAL FINDINGS: A microbead-based titanium template was filled with a porous Poly (L-lactic acid) (PLLA) body by freeze-extraction method. A distinct porosity difference was obtained between the inner and outer surfaces of the implant as characterized by image analysis and Mercury porosimetry (9.8±2.2 µm vs. 36.7±11.4 µm, p≤0.05). On top, a thin PLLA film was added to optimize the growth of epithelial cells, which was confirmed by using human respiratory epithelial cells. To check the control of fibroblast movement, PKH26 labeled fibroblasts were seeded onto Titanium and Titanium/PLLA implants. The cell movement was quantified by confocal microscopy: in one week cells moved deeper in Ti samples compared to Ti/PLLA. CONCLUSIONS: In vitro experiments showed that this new implant is effective for guiding different kind of cells it will contact upon implantation. Overall, this system would enable spatial and temporal control over cell migration by a gradient ranging from macroporosity to nanoporosity within a tracheal implant. Moreover, mechanical properties will be dependent mainly on the titanium frame. This will make it possible to create a polymeric environment which is suitable for cells without the need to meet mechanical requirements with the polymeric structure.

Dynamic aspects of films prepared by a sequential deposition of species: perspectives for smart and responsive materials.

The deposition of surface coatings using a step-by-step approach from mutually interacting species allows the fabrication of so called "multilayered films". These coatings are very versatile and easy to produce in environmentally friendly conditions, mostly from aqueous solution. They find more and more applications in many hot topic areas, such as in biomaterials and nanoelectronics but also in stimuli-responsive films. We aim to review the most recent developments in such stimuli-responsive coatings based on layer-by-layer (LBL) depositions in relationship to the properties of these coatings. The most investigated stimuli are based on changes in ionic strength, temperature, exposure to light, and mechanical forces. The possibility to induce a transition from linear to exponential growth in thickness and to change the charge compensation from "intrinsic" to "extrinsic" by controlling parameters such as temperature, pH, and ionic strength are the ways to confer their responsiveness to the films. Chemical post-modifications also allow to significantly modify the film properties.

[Research solutions to find the conception of artificial larynx]. / Les voies de recherche pour la conception d'une prothàse laryngée.

OBJECTIVE: The aim of this work is to present requirements and actual solutions to produce a laryngeal prosthesis. METHOD: Data were collected after a literature review and in the light of our work in that field. RESULTS: The conception of an artificial larynx requires biocompatibility studies to find the ideal biomaterial capable of being integrated in cervical environment and upper airway tracts. A hollow tube extending the trachea, surrounded by cervical tissue, and covered with epithelium on the endoluminal part would allow connecting the trachea to the tongue base. The biomaterial should be rigid, inert, non cytotoxic, and smooth to fulfill the physical necessities of the implant and to optimize its tolerance and colonization. Among all biomaterials tested, porous titanium seems to be the most interesting. To prevent aspiration, the creation of a laryngeal sphincter located over the superior part of the hollow tube is required. An opening and closing system, coordinated to respiration and swallowing, could be considered with valves. In proportion to the unavoidable wear and tear of the mechanism, the conception of a removable sphincter seems necessary. Moreover, this system could harbour a phonatory valve to generate the fundamental laryngeal tone. CONCLUSION: Although any laryngeal sphincter is currently available, the biocompatibility studies already performed allow disposing of biomaterial to create a laryngeal prosthesis.

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells.

Actin cytoskeleton forms a physical connection between the extracellular matrix, adhesion complexes and nuclear architecture. Because tissue stiffness plays key roles in adhesion and cytoskeletal organization, an important open question concerns the influence of substrate elasticity on replication and transcription. To answer this major question, polyelectrolyte multilayer films were used as substrate models with apparent elastic moduli ranging from 0 to 500 kPa. The sequential relationship between Rac1, vinculin adhesion assembly, and replication becomes efficient at above 200 kPa because activation of Rac1 leads to vinculin assembly, actin fiber formation and, subsequently, to initiation of replication. An optimal window of elasticity (200 kPa) is required for activation of focal adhesion kinase through auto-phosphorylation of tyrosine 397. Transcription, including nuclear recruitment of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), occurred above 50 kPa. Actin fiber and focal adhesion signaling are not required for transcription. Above 50 kPa, transcription was correlated with alphav-integrin engagement together with histone H3 hyperacetylation and chromatin decondensation, allowing little cell spreading. By contrast, soft substrate (below 50 kPa) promoted morphological changes characteristic of apoptosis, including cell rounding, nucleus condensation, loss of focal adhesions and exposure of phosphatidylserine at the outer cell surface. On the basis of our data, we propose a selective and uncoupled contribution from the substrate elasticity to the regulation of replication and transcription activities for an epithelial cell model.

The effect of microstructured surfaces and laminin-derived peptide coatings on soft tissue interactions with titanium dental implants.

In the present study, we investigated the dental implant protection from peri-implant inflammation by improving the soft tissue adhesion on the titanium surface. Porous titanium was used to create, at the level of the transmucosal part of the implants (the "neck"), a microstructured 3-dimensional surface that would tightly seal the interface between the implant and soft tissue. Cell-specific adhesion properties were induced via an adhesion peptide derived from laminin-5 coupled to native or cross-linked PLL/PGA multilayered polyelectrolyte films (MPFs), which are used for biomedical device coatings. Porous titanium exhibited good cell-adhesion properties, but the colonisation of the material was further improved by a coating with laminin-5 functionalised MPFs and especially with (PLL/PGA)(6,5)-PGA-peptide film. Focal contact formation was observed on cross-linked architectures, reflecting cell anchorage on these surfaces. In contrast, when seeded on laminin-5-functionalised native films, epithelial cells formed only very diffuse focal contacts, but adhered via hemidesmosome formation. In vivo experiments confirmed that the porous titanium was colonised by cells of soft tissue. Altogether, the results indicate that the microstructure of the implant neck combined with a specific bioactive coating could constitute efficient routes to improve the integration of soft tissue on titanium dental implants, which could significantly protect implants from peri-implant inflammation and enhance long-term implant stabilisation.

Characterization of polyelectrolyte multilayer films on polyethylene terephtalate vascular prostheses under mechanical stretching.

Layer-by-layer (LBL) polyelectrolyte films offer extensive potentials to enhance surface properties of vascular biomaterials. From the time of implantation, PET prostheses are continuously subjected to multiple mechanical stresses such as important distorsions and blood pressure. In this study, three LBL films, namely (1) poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride), (2) poly(L-lysine)/hyaluronan, and (3) poly(L-lysine)/poly(L-glutamic acid) were built on to isolated PET filaments, thread, and vascular prostheses. The three LBL films uniformly covered the surface of the PET samples with rough, totally smooth, and "wrinkled" appearances respectively for (PAH/PSS)(24), (PLL/HA)(24), and (PLL/PGA)(24) systems. We then assessed the behavior of these LBL films, in an aqueous environment [by environmental scanning electronic microscopy (ESEM)], when subjected to unidirectional longitudinal stretches. We found that stretching induces ruptures in the multilayer films on isolated filaments for longitudinal stretches of 14% for (PSS/PAH)(24), 13% for (PLL/PGA)(24), and 30% for (PLL/HA)(24) films. On threads, the rupture limit is enhanced to be respectively 26, 20, and 28%. Most interestingly, we found that on vascular prosthesis no rupture is visible in any of the three multilayers types, even for elongations of 200% (200% undergone by the PET prostheses is representative of those encountered during graft deployment) which by far exceeds elongations observed under physiological conditions (10-20%, blood pressure). In term of mechanical behaviors, these preliminary data constitute a first step toward the possible use of LBL film to coat and functionalize vascular prosthesis.

Immunogold detection of types I and II chondrocyte collagen fibrils: an in situ atomic force microscopic investigation.

Chondrocyte tissue engineering is a major challenge in the field of cartilage repair. The phenotype of chondrocytes consists of cartilage specific proteoglycan and type II collagen. During serial passages, chondrocytes dedifferentiate into cells, presenting a fibroblast-like phenotype consisting predominately of type I collagen synthesis. Observation of native collagen fibers could be visualized by atomic force microscope. Here, we developed an original and useful atomic force microscopy-based immunogold technique allowing biochemical distinction between types I and II collagen fibers. Imaging of 40-nm gold particles staining collagen fibers was performed in tapping mode. Rat 1 fibroblasts and human chondrosarcoma cells were used as positive models for types I and II collagen, respectively. As demonstrated by our data, primary rat chondrocytes adhering for 48 h on a glass substrate synthesize type II collagen native fibers. This technique allows analyses of local areas of the extracellular matrix of fixed cells, providing complementary data about cartilage phenotype. This simple approach could be of major interest for the biologist community in routine laboratory investigations, to localize in situ, macromolecules of the extracellular matrix.