Hassan Atmani

Osteogenetic Properties of Electrospun Nanofibrous PCL Scaffolds Equipped With Chitosan-Based Nanoreservoirs of Growth Factors

Bioactive implants intended for rapid, robust, and durable bone tissue regeneration are presented. The implants are based on nanofibrous 3D-scaffolds of bioresorbable poly-ϵ-caprolactone mimicking the fibrillar architecture of bone matrix. Layer-by-layer nanoimmobilization of the growth factor BMP-2 in association with chitosan (CHI) or poly-L-lysine over the nanofibers is described. The osteogenetic potential of the scaffolds coated with layers of CHI and BMP-2 is demonstrated in vitro, and in vivo in mouse calvaria, through enhanced osteopontin gene expression and calcium phosphate biomineralization. The therapeutic strategy described here contributes to the field of regenerative medicine, as it proposes a route toward efficient repair of bone defects at reduced risk and cost level.

Nanostructured polyelectrolyte multilayer drug delivery systems for bone metastasis prevention.

Polyelectrolyte multilayers (PEM) are well established nanoarchitectures with numerous potential applications, in particular as biomaterial coatings. They may exhibit specific biological properties in terms of controlled cell activation or local drug delivery. Here, in a new approach for bone metastasis prevention, we employed poly-l-lysine covalently grafted with beta-cyclodextrin as a polycationic vector (PLL-CD) for the antitumor bisphosphonate drug risedronate (RIS). Molar ratio for maximum loading of the PLL-CD vector with RIS was determined by Raman microspectroscopy. The efficacy of RIS at inhibiting cancer cell invasion in vitro was strongly enhanced upon complexation, whatever PLL-CD:RIS complexes were in solution or embedded into PEM nanoarchitectures. Complexes in solution also clearly prevented cancer-induced bone metastasis in animals. Incorporation of the complexes into PEM nanoarchitectures covering bone implants appears of interest for in situ prevention of bone metastasis after ablation.

Evidence of different growth regimes coexisting within biomimetic Layer-by-Layer films

Layer-by-Layer (LbL) films are extensively studied as promising (bio)functional coatings. Although many of their structural features are known, the self-assembly mechanism governing their buildup is still under debate. In this work, we describe the initial buildup and structure of biomimetic LbL films comprised of chondroitin sulfate A (ChS) and poly(L-lysine) (PLL), by means of a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Optical Waveguide Lightmode Spectroscopy (OWLS), Fourier Transform Infrared Spectroscopy (ATR-FTIR) and Atomic Force Microscopy (AFM). While the QCM-D and ATR-FTIR reveal a supralinear, so-called “exponential” growth regime, OWLS surprisingly indicates a linear growth regime. Based on a thorough discussion of the experimental data, we conclude from this apparent contradiction that, shortly after the beginning of the buildup, an inner “dense” zone and an outer “diffuse” zone governed by different growth regimes coexist within the film. This observation constitutes an experimental validation of the structural model assuming a dense, gradually restructuring internal zone, proposed in the literature to explain the exponential-to-linear regime transition of exponentially growing LbL films.

Tunable Protein-Resistance of Polycation-Terminated Polyelectrolyte Multilayers

The prevention of nonspecific protein adsorption is a crucial prerequisite for many biomedical and biotechnological applications. Therefore, the design of robust and versatile methods conferring optimal protein-resistance properties to surfaces has become a challenging issue. Here we report the unexpected case of polycation-ending polyelectrolyte multilayers (PEM) that efficiently prevented the adsorption of a negatively charged model protein, glucose oxidase (GOX). PEM films were based on two typical weak poyelectrolytes: poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). No chemical modification of the polyelectrolytes was required and tunable GOX adsorption was possible by simply changing the buildup pH conditions. Protein-resistance properties are attributed to high film hydration becoming the predominant factor over electrostatic interactions. We explain this effect by oscillations of the internal PAA ionization state throughout the buildup, which results in an excess of carboxylic acid groups within the film. This excess acts as a reservoir of potential carboxylate groups compensating the outer PAH positive charges. Partial results indicated that the system was also resistant to the adsorption of a positively charged protein, lysozyme. Control of the internal ionization of weak polyelectrolyte multilayers might open a route toward simple tuning of protein adsorption. These results should help to rationalize the design of biomaterials, biosensors, or protein separation devices.

Genipin-Cross-Linked Layer-by-Layer Assemblies: Biocompatible Microenvironments To Direct Bone Cell Fate

The design of biomimetic coatings capable of improving the osseointegration of bone biomaterials is a current challenge in the field of bone repair. Toward this end, layer-by-layer (LbL) films composed of natural components are suitable candidates. Chondroitin sulfate A (CSA), a natural glycosaminoglycan (GAG), was used as the polyanionic component because it promotes osteoblast maturation in vivo. In their native state, GAG-containing LbL films are generally cytophobic because of their low stiffness. To stiffen our CSA-based LbL films, genipin (GnP) was used as a natural cross-linking agent, which is much less cytotoxic than conventional chemical cross-linkers. GnP-cross-linked films display an original combination of microscale topography and tunable mechanical properties. Structural characterization was partly based on a novel donor/acceptor Förster resonance energy transfer (FRET) couple, namely, FITC/GnP, which is a promising approach for further inspection of any GnP-cross-linked system. GnP-cross-linked films significantly promote adhesion, proliferation, and early and late differentiation of preosteoblasts.

Protein-triggered instant disassembly of biomimetic Layer-by-Layer films.

Layer-by-Layer (LbL) coatings are promising tools for the biofunctionalization of biomaterials, as they allow stress-free immobilization of proteins. Here, we explore the possibility to immobilize phosvitin, a highly phosphorylated protein viewed as a model of bone phosphoproteins and, as such, a potential promotive agent of surface-directed biomineralization, into biomimetic LbL architectures. Two immobilization protocols are attempted, first, using phosvitin as the polyanionic component of phosvitin/poly-(L-lysine) films and, second, adsorbing it onto preformed chondroitin sulfate/poly-(L-lysine) films. Surprisingly, it is neither possible to embed phosvitin as the constitutive polyanion of the LbL architectures nor to adsorb it atop preformed films. Instead, phosvitin triggers instant massive film disassembly. This unexpected, incidentally detected behavior constitutes the first example of destructive interactions between LbL films and a third polyelectrolyte, a fortiori a protein, which might open a route toward new stimuli-responsive films for biosensing or drug delivery applications. Interestingly, additional preliminary results still indicate a promotive effect of phosvitin-containing remnant films on calcium phosphate deposition.

Mechano-chemical control of cell behaviour by elastomer templates coated with biomimetic Layer-by-Layer nanofilms

Within the field of biomaterials, the control of cell–surface interactions is generally addressed through surface chemistry modification. In this respect, Layer-by-Layer (LbL) coatings constitute promising versatile tools. Most cells are mechanosensitive, therefore the stiffness of their microenvironment should also be considered as a crucial parameter when designing polymer-based biomaterials. Here, we report on the combination of mechanics and surface chemistry of LbL-treated polydimethylsiloxane substrates of tunable stiffness to control cell behaviour. Biomimetic LbL films consist of poly-L-lysine (PLL) and chondroitin-4-sulfate (CSA). Stiffness, surface chemistry, wettability and topography of bare and LbL-treated polydimethylsiloxane were analysed. We demonstrated that cells adhered and grew up on all the substrates with significant promotive effects of PLL-terminated films and stiffer substrates. Pre-osteoblasts differentiation was enhanced onto PLL-terminated films irrespective of stiffness and onto CSA-terminated films in a mechanical dependent manner. These results open outlooks within the field of bone tissue engineering, as they demonstrate that osteoconductive surfaces can be obtained by combining uncrosslinked biomimetic LbL films and the intrinsic mechanical properties of the substrates, that is, without any commonly used, potentially cytotoxic chemical crosslinking.

Anomalous permeation of the [P5W30O110]15− polyoxoanion in polyelectrolyte multilayer films

The integration of polyoxoanions into functional thin films is of major interest to increase their use as catalysts or to provide new properties to the films. The incorporation of polyoxoanions in polyelectrolyte multilayer (PEM) films has been widely used for such aims. In most investigations, the polyoxoanions were used as a component of the layering process and there is only limited work investigating the interactions of polyoxoanions with preformed PEM films. Herein, we investigated the incorporation of the 30-tungsto-5-phosphate [P5W30O110]15− polyoxoanion into PEM films made from hyaluronic acid (HA) and poly(allylamine hydrochloride) (PAH) as a function of the polyoxoanions concentration. Surprisingly, we found an increased amount of incorporated [P5W30O110]15− upon a decrease in its bulk concentration. Confocal Raman microscopy allowed us to investigate the concentration profile of the polyoxoanion across films of about 3 μm in thickness. Homogeneous distribution was only found for films fed with polyoxoanions at the lowest bulk concentration, namely 1.2 × 10−6 M, whereas the films fed with solutions at higher concentrations (1.2 × 10−4 M) showed local enrichment in POM at the film–solution interface. We explain the lower amount of incorporated polyoxoanion at high bulk concentration by the formation of a polyoxoanion-rich barrier reducing further diffusion of the anions in the deeper part of the films.

Concomitant dehydration mechanisms in single crystals of α,α-trehalose

The dehydration behaviour of alpha,alpha-trehalose (alpha-D-glucopyranosyl alpha-D-glucopyranoside) dihydrate single crystals is investigated by thermomicroscopy, Raman microscopy, and differential scanning calorimetry. The results show at a given stage the simultaneous presence of two polymorphic forms, amorphous material, and movement of a fluid phase. The study also underlines that the characterization of the average phase by conventional XRPD and DSC techniques is not sufficient to describe the dehydration mechanisms of alpha,alpha-trehalose particles. Moreover, it confirms that the dehydration behaviour is mainly driven by heterogeneities and the rate of water loss.

Fibronectin-based multilayer thin films

Thin films mimicking the structure and composition of the extra-cellular matrix (ECM) are potentially attractive as biomaterials for cell contacting applications. Layer-by-layer (LbL) assembly of a biological polycation, poly(l-lysine) (PLL), and a common ECM protein, fibronectin (Fn), was employed here to construct nanoscale, ECM mimicking films. Incremental film thickness and interfacial charge magnitude are observed to diminish with layer number, resulting in sub-linear film growth scaling and saturation after about 10 layers. Infrared spectroscopy and electron microscopy together reveal the formation of Fn containing aggregates, whose presence correlates with diminished charge reversal and suppressed LbL assembly. PLL-Fn films induce a significantly greater murine MC3T3-E1 pre-osteoblastic cell proliferation, while maintaining a much higher proportion of Fn in the molecular (as opposed to fibrillar) state, compared to a Fn monolayer, suggesting the enhanced Fn content of these ECM-mimicking films to significantly, and positively, affect cell behavior.