Response of Osteoblasts on Amine-Based Nanocoatings Correlates with the Amino Group Density.

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C).

A Biophysical Model for Curvature-Guided Cell Migration.

The latest experiments have shown that adherent cells can migrate according to cell-scale curvature variations via a process called curvotaxis. Despite identification of key cellular factors, a clear understanding of the mechanism is lacking. We employ a mechanical model featuring a detailed description of the cytoskeleton filament networks, the viscous cytosol, the cell adhesion dynamics, and the nucleus. We simulate cell adhesion and migration on sinusoidal substrates. We show that cell adhesion on three-dimensional curvatures induces a gradient of pressure inside the cell that triggers the internal motion of the nucleus. We propose that the resulting out-of-equilibrium position of the nucleus alters cell migration directionality, leading to cell motility toward concave regions of the substrate, resulting in lower potential energy states. Altogether, we propose a simple mechanism explaining how intracellular mechanics enable the cells to react to substratum curvature, induce a deterministic cell polarization, and break down cells basic persistent random walk, which correlates with latest experimental evidences.

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.

Nucleus deformation of SaOs-2 cells on rhombic µ-pillars.

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.

In silico analysis shows that dynamic changes in curvature guide cell migration over long distances.

In vitro experiments have shown that cell scale curvatures influence cell migration; cells avoid convex hills and settle in concave valleys. However, it is not known whether dynamic changes in curvature can guide cell migration. This study extends a previous in-silico model to explore the effects over time of changing the substrate curvature on cell migration guidance. By simulating a dynamic surface curvature using traveling wave patterns, we investigate the influence of wave height and speed, and find that long-distance cell migration guidance can be achieved on specific wave patterns. We propose a mechanistic explanation of what we call dynamic curvotaxis and highlight those cellular features that may be involved. Our results open a new area of study for understanding cell mobility in dynamic environments, from single-cell in vitro experiments to multi-cellular in vivo mechanisms.

Fabrication and characterization of thin self-rolling film for anti-inflammatory drug delivery.

Thin films have been identified as an alternative approach for targeting sensitive site as drug delivery tool. In this work, the preparation of self-rolling thin films to form tubes for wound healing and easy placement (e.g. in the colon via colonoscopy) have been studied. We explored the use of thin films as a protective dressing combined to local release of an anti-inflammatory in order to improve drug efficacy and limit the side effects of the oral route. Non-cytotoxic poly(ethylene) glycol and poly(lactic acid) photo-crosslinkable star copolymers were used for rapid UV crosslinking of bilayered films loaded with prednisolone. The films, crosslinked under UV lamp without the need of photoinitiator, are optimized and compared in terms of water uptake, swelling ratio, final tube diameter and morphology, anti-inflammatory drug loading and release. Our studies showed the spontaneous rolling of bilayer constructs directly after immersion in water. Tubular geometry allows application of the patch through minimally invasive procedures such as colonoscopy. Moreover, the rolled-up bilayers highlighted efficient release of encapsulated drug following Fickian diffusion mechanism. We also confirmed the anti-inflammatory activity of the released anti-inflammatory drug that inhibits the pro-inflammatory cytokine IL-1ß in RAW 264.7 macrophages stimulated by Escherichia coli (E. coli).

Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales.

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.

Bioresorbable Bilayered Elastomer/Hydrogel Constructs with Gradual Interfaces for the Fast Actuation of Self-Rolling Tubes.

In the biomedical field, self-rolling materials provide interesting opportunities to develop medical devices suitable for drug or cell encapsulation. However, to date, a major limitation for medical applications is the use of non-biodegradable and non-biocompatible polymers that are often reported for such applications or the slow actuation witnessed with degradable systems. In this work, biodegradable self-rolling tubes that exhibit a spontaneous and rapid actuation when immersed in water are designed. Photo-crosslinkable hydrophilic and hydrophobic poly(ethylene glycol)-poly(lactide) (PEG-PLA) star-shaped copolymers are prepared and used to prepare bilayered constructs. Thanks to the discrete mechanical and swelling properties of each layer and the cohesive/gradual nature of the interface, the resulting bilayered films are able to self-roll in water in less than 30 s depending on the nature of the hydrophilic layer and on the shape of the sample. The cytocompatibility and degradability of the materials are demonstrated and confirm the potential of such self-rolling resorbable biomaterials in the field of temporary medical devices.

In Vitro and In Vivo Cell-Interactions with Electrospun Poly (Lactic-Co-Glycolic Acid) (PLGA): Morphological and Immune Response Analysis.

Random electrospun three-dimensional fiber membranes mimic the extracellular matrix and the interfibrillar spaces promotes the flow of nutrients for cells. Electrospun PLGA membranes were analyzed in vitro and in vivo after being sterilized with gamma radiation and bioactivated with fibronectin or collagen. Madin-Darby Canine Kidney (MDCK) epithelial cells and primary fibroblast-like cells from hamster's cheek paunch proliferated over time on these membranes, evidencing their good biocompatibility. Cell-free irradiated PLGA membranes implanted on the back of hamsters resulted in a chronic granulomatous inflammatory response, observed after 7, 15, 30 and 90 days. Morphological analysis of implanted PLGA using light microscopy revealed epithelioid cells, Langhans type of multinucleate giant cells (LCs) and multinucleated giant cells (MNGCs) with internalized biomaterial. Lymphocytes increased along time due to undegraded polymer fragments, inducing the accumulation of cells of the phagocytic lineage, and decreased after 90 days post implantation. Myeloperoxidase+ cells increased after 15 days and decreased after 90 days. LCs, MNGCs and capillaries decreased after 90 days. Analysis of implanted PLGA after 7, 15, 30 and 90 days using transmission electron microscope (TEM) showed cells exhibiting internalized PLGA fragments and filopodia surrounding PLGA fragments. Over time, TEM analysis showed less PLGA fragments surrounded by cells without fibrous tissue formation. Accordingly, MNGC constituted a granulomatous reaction around the polymer, which resolves with time, probably preventing a fibrous capsule formation. Finally, this study confirms the biocompatibility of electrospun PLGA membranes and their potential to accelerate the healing process of oral ulcerations in hamsters' model in association with autologous cells.

Tunable antibacterial coatings that support mammalian cell growth.

Bacterial infections present an enormous problem causing human suffering and cost burdens to healthcare systems worldwide. Here we present novel tunable antibacterial coatings which completely inhibit bacterial colonization by Staphylococcus epidermidis but allow normal adhesion and spreading of osteoblastic cells. The coatings are based on amine plasma polymer films loaded with silver nanoparticles. The method of preparation allows flexible control over the amount of loaded silver nanoparticles and the rate of release of silver ions.

Size-Dependent Internalization Efficiency of Macrophages from Adsorbed Nanoparticle-Based Monolayers.

Functional coatings based on the assembly of submicrometric or nanoparticles are found in many applications in the biomedical field. However, these nanoparticle-based coatings are particularly fragile since they could be exposed to cells that are able to internalize nanoparticles. Here, we studied the efficiency of RAW 264.7 murine macrophages to internalize physisorbed silica nanoparticles as a function of time and particle size. This cell internalization efficiency was evaluated from the damages induced by the cells in the nanoparticle-based monolayer on the basis of scanning electron microscopy and confocal laser scanning microscopy observations. The internalization efficiency in terms of the percentage of nanoparticles cleared from the substrate is characterized by two size-dependent regimes. Additionally, we highlighted that a delay before internalization occurs, which increases with decreasing adsorbed nanoparticle size. This internalization is characterized by a minimal threshold that corresponds to 35 nm nanoparticles that are not internalized during the 12-h incubation considered in this work.

New Phenotype and Mineralization of Biogenic Iron Oxide in Magnetotactic Bacteria.

Many magnetotactic bacteria (MTB) biomineralize magnetite crystals that nucleate and grow inside intracellular membranous vesicles originating from invaginations of the cytoplasmic membrane. The crystals together with their surrounding membranes are referred to as magnetosomes. Magnetosome magnetite crystals nucleate and grow using iron transported inside the vesicle by specific proteins. Here, we tackle the question of the organization of magnetosomes, which are always described as constituted by linear chains of nanocrystals. In addition, it is commonly accepted that the iron oxide nanocrystals are in the magnetite-based phase. We show, in the case of a wild species of coccus-type bacterium, that there is a double organization of the magnetosomes, relatively perpendicular to each other, and that the nanocrystals are in fact maghemite. These findings were obtained, respectively, by using electron tomography of whole mounts of cells directly from the environment and high-resolution transmission electron microscopy and diffraction. Structure simulations were performed with the MacTempas software. This study opens new perspectives on the diversity of phenotypes within MTBs and allows to envisage other mechanisms of nucleation and formation of biogenic iron oxide crystals.

Topographically induced self-deformation of the nuclei of cells: dependence on cell type and proposed mechanisms.

Osteosarcoma-derived cell lines (SaOs-2, MG63) have recently been shown to deform their nucleus considerably in response to surface topography. Such a deformation had not been described previously. Here we present results on additional cell lines, including cancerous (OHS4, U2OS), immortalized (F/STRO-1(+)A and FHSO6) and healthy cells (HOP). The cancerous cells were found to deform extensively, the immortalized cells showed small deformations, whereas the healthy cells showed deformation only at short incubation times. These results suggest a strong link between the malignant transformation of cells and the state of the cytoskeletal network. We propose mechanisms to explain the deformation in which the cytoskeleton either pushes down on the nucleus during spreading or pulls it down upon adhesion to the pillars.

Topographical curvature is sufficient to control epithelium elongation.

How biophysical cues can control tissue morphogenesis is a central question in biology and for the development of efficient tissue engineering strategies. Recent data suggest that specific topographies such as grooves and ridges can trigger anisotropic tissue growth. However, the specific contribution of biologically relevant topographical features such as cell-scale curvature is still unclear. Here we engineer a series of grooves and ridges model topographies exhibiting specific curvature at the ridge/groove junctions and monitored the growth of epithelial colonies on these surfaces. We observe a striking proportionality between the maximum convex curvature of the ridges and the elongation of the epithelium. This is accompanied by the anisotropic distribution of F-actin and nuclei with partial exclusion of both in convex regions as well as the curvature-dependent reorientation of pluricellular protrusions and mitotic spindles. This demonstrates that curvature itself is sufficient to trigger and modulate the oriented growth of epithelia through the formation of convex "topographical barriers" and establishes curvature as a powerful tuning parameter for tissue engineering and biomimetic biomaterial design.

Surface Texturization of Breast Implants Impacts Extracellular Matrix and Inflammatory Gene Expression in Asymptomatic Capsules.

BACKGROUND: Texturing processes have been designed to improve biocompatibility and mechanical anchoring of breast implants. However, a high degree of texturing has been associated with severe abnormalities. In this study, the authors aimed to determine whether implant surface topography could also affect physiology of asymptomatic capsules. METHODS: The authors collected topographic measurements from 17 different breast implant devices by interferometry and radiographic microtomography. Morphologic structures were analyzed statistically to obtain a robust breast implant surface classification. The authors obtained three topographic categories of textured implants (i.e., "peak and valleys," "open cavities," and "semiopened cavities") based on the cross-sectional aspects. The authors simultaneously collected 31 Baker grade I capsules, sorted them according to the new classification, established their molecular profile, and examined the tissue organization. RESULTS: Each of the categories showed distinct expression patterns of genes associated with the extracellular matrix (Timp and Mmp members) and inflammatory response (Saa1, Tnsf11, and Il8), despite originating from healthy capsules. In addition, slight variations were observed in the organization of capsular tissues at the histologic level. CONCLUSIONS: The authors combined a novel surface implant classification system and gene profiling analysis to show that implant surface topography is a bioactive cue that can trigger gene expression changes in surrounding tissue, even in Baker grade I capsules. The authors' new classification system avoids confusion regarding the word "texture," and could be transposed to implant ranges of every manufacturer. This new classification could prove useful in studies on potential links between specific texturizations and the incidence of certain breast-implant associated complications.

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography.

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, their largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

In Vitro Degradation of Electrospun Poly(Lactic-Co-Glycolic Acid) (PLGA) for Oral Mucosa Regeneration.

Poly(lactic-co-glycolic acid) (PLGA) has been used in the field of tissue engineering as a scaffold due to its good biocompatibility, biodegradability and mechanical strength. With the aim to explore the degradability of PLGA electrospun nonwoven structures for oral mucosa tissue engineering applications, non-irradiated and gamma irradiated nonwovens were immersed in three different solutions, in which simulated body fluid (SBF) and artificial saliva are important for future oral mucosa tissue engineering. The nonwovens were immersed for 7, 15 and 30 days in SBF, culture media (DMEM) and artificial saliva at 37 °C. Before immersion in the solutions, the dosage of 15 kGy was applied for sterilization in one assay and compared with non-irradiated samples at the same timepoints. Samples were characterized using different techniques such as scanning electron microscopy (SEM), differential scanning calorimetric (DSC) and gel permeation chromatography (GPC) to evaluate the nonwoven degradation and Fourier-transform infrared spectroscopy (FTIR) to evaluate the chain scissions. Our results showed that PLGA nonwovens were constituted by semicrystalline fibers with moderate degradation properties up to thirty days. The non-irradiated samples exhibited slower kinetics of degradation than irradiated nonwovens. For immersion times longer than 7 days in the three different solutions, the mean diameter of irradiated fibers stayed in the same range, but significantly different from the control sample. On non-irradiated samples, the degradation kinetics was slower and the plateau in the diameter value was only attained after 30 days of immersion in the fluids. Plasticization (fluid absorption into the fiber structure) occurred in the bulk material, as confirmed by a decrease in Tg observed by DSC analyses of non-irradiated and irradiated nonwovens, in comparison with the respective controls. In addition, artificial saliva showed a higher capacity of influencing PLGA crystallization than SBF and DMEM. FTIR analyses showed typical PLGA chemical functional groups changes. These results will be important for future application of those PLGA electrospun nonwovens for oral mucosa regeneration.

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.

Dynamic adaptation of mesenchymal stem cell physiology upon exposure to surface micropatterns.

Human mesenchymal stem (hMSCs) are defined as multi-potent colony-forming cells expressing a specific subset of plasma membrane markers when grown on flat tissue culture polystyrene. However, as soon as hMSCs are used for transplantation, they are exposed to a 3D environment, which can strongly impact cell physiology and influence proliferation, differentiation and metabolism. Strategies to control in vivo hMSC behavior, for instance in stem cell transplantation or cancer treatment, are skewed by the un-physiological flatness of the standard well plates. Even though it is common knowledge that cells behave differently in vitro compared to in vivo, only little is known about the underlying adaptation processes. Here, we used micrometer-scale defined surface topographies as a model to describe the phenotype of hMSCs during this adaptation to their new environment. We used well established techniques to compare hMSCs cultured on flat and topographically enhanced polystyreneand observed dramatically changed cell morphologies accompanied by shrinkage of cytoplasm and nucleus, a decreased overall cellular metabolism, and slower cell cycle progression resulting in a lower proliferation rate in cells exposed to surface topographies. We hypothesized that this reduction in proliferation rate effects their sensitivity to certain cancer drugs, which was confirmed by higher survival rate of hMSCs cultured on topographies exposed to paclitaxel. Thus, micro-topographies can be used as a model system to mimic the natural cell micro-environment, and be a powerful tool to optimize cell treatment in vitro.

Role of the Nucleus as a Sensor of Cell Environment Topography.

The proper integration of biophysical cues from the cell vicinity is crucial for cells to maintain homeostasis, cooperate with other cells within the tissues, and properly fulfill their biological function. It is therefore crucial to fully understand how cells integrate these extracellular signals for tissue engineering and regenerative medicine. Topography has emerged as a prominent component of the cellular microenvironment that has pleiotropic effects on cell behavior. This progress report focuses on the recent advances in the understanding of the topography sensing mechanism with a special emphasis on the role of the nucleus. Here, recent techniques developed for monitoring the nuclear mechanics are reviewed and the impact of various topographies and their consequences on nuclear organization, gene regulation, and stem cell fate is summarized. The role of the cell nucleus as a sensor of cell-scale topography is further discussed.