Harnessing Wharton's jelly stem cell differentiation into bone-like nodule on calcium phosphate substrate without osteoinductive factors.

An important aim of bone regenerative medicine is to design biomaterials with controlled chemical and topographical features to guide stem cell fate towards osteoblasts without addition of specific osteogenic factors. Herein, we find that sprayed bioactive and biocompatible calcium phosphate substrates (CaP) with controlled topography induce, in a well-orchestrated manner, Wharton's jelly stem cells (WJ-SCs) differentiation into osteoblastic lineage without any osteogenic supplements. The resulting WJ-SCs commitment exhibits features of native bone, through the formation of three-dimensional bone-like nodule with osteocyte-like cells embedded into a mineralized type I collagen. To our knowledge, these results present the first observation of a whole differentiation process from stem cell to osteocytes-like on a synthetic material. This suggests a great potential of sprayed CaP and WJ-SCs in bone tissue engineering. These unique features may facilitate the transition from bench to bedside and the development of successful engineered bone. STATEMENT OF SIGNIFICANCE: Designing materials to direct stem cell fate has a relevant impact on stem cell biology and provides insights facilitating their clinical application in regenerative medicine. Inspired by natural bone compositions, a friendly automated spray-assisted system was used to build calcium phosphate substrate (CaP). Sprayed biomimetic solutions using mild conditions led to the formation of CaP with controlled physical properties, good bioactivity and biocompatibility. Herein, we show that via optimization of physical properties, CaP substrate induce osteogenic differentiation of Wharton's jelly stem cells (WJ-SCs) without adding osteogenic supplement factors. These results suggest a great potential of sprayed CaP and WJ-SCs in bone tissue engineering and may facilitate the transition from bench to beside and the development of clinically successful engineered bone.

Polysaccharide films built by simultaneous or alternate spray: a rapid way to engineer biomaterial surfaces.

We investigated polysaccharide films obtained by simultaneous and alternate spraying of a chitosan (CHI) solution as polycation and hyaluronic acid (HA), alginate (ALG), and chondroitin sulfate (CS) solutions as polyanions. For simultaneous spraying, the film thickness increases linearly with the cumulative spraying time and passes through a maximum for polyanion/CHI molar charge ratios lying between 0.6 and 1.2. The size of polyanion/CHI complexes formed in solution was compared with the simultaneously sprayed film growth rate as a function of the polyanion/CHI molar charge ratio. A good correlation was found. This suggests the importance of polyanion/polycation complexation in the simultaneous spraying process. Depending on the system, the film topography is either liquid-like or granular. Film biocompatibility was evaluated using human gingival fibroblasts. A small or no difference is observed in cell viability and adhesion between the two deposition processes. The CHI/HA system appears to be the best for cell adhesion inducing the clustering of CD44, a cell surface HA receptor, at the membrane of cells. Simultaneous or alternate spraying of CHI/HA appears thus to be a convenient and fast procedure for biomaterial surface modifications.

Simultaneous spray coating of interacting species: general rules governing the poly(styrene sulfonate)/poly(allylamine) system.

Simultaneous spraying of two solutions of interacting species onto a substrate held vertically leads to the formation of nanometer-sized coatings. Here we investigate the simultaneous spraying of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) solutions leading to the formation of a film composed of PSS/PAH complexes. The thickness of this film increases linearly with the cumulative spraying time. For a given spraying rate of PAH (respectively PSS), the growth rate of the film depends strongly upon the PSS/PAH ratio and passes through a maximum for a PSS/PAH ratio lying between 0.55 and 0.8. For a PSS/PAH ratio that is maintained constant, the growth speed of the film increases linearly with the spraying rate of polyelectrolyte of both solutions. Using X-ray photoelectron spectroscopy, we find that the film composition is almost independent of the PSS/PAH (spayed) ratio, with composition very close to 1:1 in PSS:PAH film. The 1:1 PSS:PAH composition is explained by the fact that the simultaneous spraying experiments are carried out with salt-free solutions; thus, electroneutrality in the film requires exact matching of the charges carried by the polyanions and the polycations. Zeta potential measurements reveal that, depending on whether the PSS/PAH spraying rate ratio lies below or above the optimal spraying rate ratio, the film acquires a positive or a negative excess charge. We also find that the overall film morphology, investigated by AFM, is independent of the spraying rate ratio and appears to be composed of nanometer-sized grains which are typically in the 100 nm range.

Active multilayered capsules for in vivo bone formation.

Interest in the development of new sources of transplantable materials for the treatment of injury or disease has led to the convergence of tissue engineering with stem cell technology. Bone and joint disorders are expected to benefit from this new technology because of the low self-regenerating capacity of bone matrix secreting cells. Herein, the differentiation of stem cells to bone cells using active multilayered capsules is presented. The capsules are composed of poly-L-glutamic acid and poly-L-lysine with active growth factors embedded into the multilayered film. The bone induction from these active capsules incubated with embryonic stem cells was demonstrated in vitro. Herein, we report the unique demonstration of a multilayered capsule-based delivery system for inducing bone formation in vivo. This strategy is an alternative approach for in vivo bone formation. Strategies using simple chemistry to control complex biological processes would be particularly powerful, as they make production of therapeutic materials simpler and more easily controlled.

Three-dimensional sprayed active biological gels and cells for tissue engineering application.

Complex three-dimensional structures can "a priori" be built layer-by-layer with a large number of different components, including various cell types, polyelectrolytes, drugs, proteins, peptides or DNA. Our approach is based on the spraying of such elements in order to form a highly functionalized and structured biomaterial. The proposed route will allow the control at the surface and in depth the distribution of the different included elements (matrix and cells).The main objective of this work concerns the buildup of biomaterials aimed to reconstruct biological tissue. The proposed ways are highly innovative and consist in a simple and progressive spraying of all the elements constituting finally the biomaterial.We report here that it is possible (i) to build an alginate gel by alternate spraying of alginate and Ca(2+); (ii) to spray active alginate gel and cells; (iii) to build layer-by-layer an active reservoir under and on the top of this sprayed gel and cells; (iv) to follow the activity of these sprayed cells with time; (v) to propose a three-dimensional sprayed structure for tissue engineering application.

Changes in silicon elastomeric surface properties under stretching induced by three surface treatments.

Poly(dimethylsiloxane) (PDMS) substrates are used in many applications where the substrates need to be elongated and various treatments are used to regulate their surface properties. In this article, we compare the effect of three of such treatments, namely, UV irradiation, water plasma, and plasma polymerization, both from a molecular and from a macroscopic point of view. We focus our attention in particular on the behavior of the treated surfaces under mechanical stretching. UV irradiation induces the substitution of methyl groups by hydroxyl and acid groups, water plasma leads to a silicate-like layer, and plasma polymerization causes the formation of an organic thin film with a major content of anhydride and acid groups. Stretching induces cracks on the surface both for silicate-like layers and for plasma polymer thin coatings. This is not the case for the UV irradiated PDMS substrates. We then analyzed the chemical composition of these cracks. In the case of water plasma, the cracks reveal native PDMS. In the case of plasma polymerization, the cracks reveal modified PDMS. The contact angles of plasma polymer and UV treated surfaces vary only very slightly under stretching, whereas large variations are observed for water plasma treatments. The small variation in the contact angle values observed on the plasma polymer thin film under stretching even when cracks appear on the surface are explained by the specific chemistry of the PDMS in the cracks. We find that it is very different from native PDMS and that its structure is somewhere between Si(O2) and Si(O3). This is, to our knowledge, the first study where different surface treatments of PDMS are compared for films under stretching.

Influence of the polyelectrolyte molecular weight on exponentially growing multilayer films in the linear regime.

Alternated deposition of polyanions and polycations on a charged solid substrate leads to the buildup of polyelectrolyte multilayer (PEM) films. Two types of PEM films were reported in the literature: films whose thickness increases linearly and films whose thickness increases exponentially with the number of deposition steps. However, it was recently found that, for exponentially growing films, the exponential increase of the film thickness takes place only during the initially deposited pairs of layers and is then followed by a linear increase. In this study, we investigate the growth process of hyaluronic acid/poly(L-lysine) (HA/PLL) and poly(L-glutamic acid)/poly(allylamine) (PGA/PAH) films, two films whose growth is initially exponential, when the growth process enters the linear regime. We focus, in particular, on the influence of the molecular weight (Mw) of the polyelectrolytes. For both systems, we find that the film thickness increment per polyanion/polycation deposition step in the linear growth regime is fairly independent of the molecular weights of the polyelectrolytes. We also find that when the (HA/PLL)n films are constructed with low molecular weight PLL, these chains can diffuse into the entire film during each buildup cycle, even for very thick films, whereas the PLL diffusion of high molecular weight chains is restricted to the upper part of the film. Our results lead to refinement of the buildup mechanism model, introduced previously for the exponentially growing films, which is based on the existence of three zones over the entire film thickness. The mechanism no longer needs all the "in" and "out" diffusing polyanions or polycations to be involved in the buildup process to explain the linear growth regime but merely relies on the interaction between the polyelectrolytes with an upper zone of the film. This zone is constituted of polyanion/polycation complexes which are "loosely bound" and rich in the polyelectrolyte deposited during the former deposition step.

Initial adhesion of endothelial cells on polyelectrolyte multilayer films.

Polyelectrolyte multilayer films were recently investigated to favour attachment of Human Vein Umbilical Endothelial Cells (HUVECs) on non-adhesive surfaces. In this study, we evaluated the initial adhesion of HUVECs after 3 h of seeding on two polyelectrolyte multilayer films ending by poly(D-lysine) (PDL) or poly(allylamine hydrochloride) (PAH). In order to obtain information about initial adhesion of HUVECs, cell morphology as well as the expression of beta1 integrins, specific receptors of adhesion, were evaluated after 3 h of seeding on polyelectrolyte multilayer films. The data were also compared to PDL or PAH monolayers (polyelectrolytes terminating the multilayer architecture). The expression of beta1 integrins was not different, whatever are the studied surfaces. However, HUVECs spreading on polyelectrolyte multilayer films, in particular on PAH ending film, was more important as compared to polyelectrolyte monolayers or glass. In conclusion, the best initial adhesion conditions of HUVECs on polyelectrolyte films could not be elucidated, moreover the results suggested also that beta1 integrins could only play a limited role.

Decellularized umbilical artery treated with thin polyelectrolyte multilayer films: potential use in vascular engineering.

Decellularized allograft tissues have been identified as a potential extracellular matrix scaffold for tissue-engineered vascular substitutes. In order to improve the thromboresistance, it is necessary to pre-coat the intra-luminal vessel surface. Recently a new surface modification technique appeared, based on the alternate adsorption of positive and negative charged polyelectrolytes. Our objective was to develop an alternative vascular scaffold made of decellularized human umbilical arteries treated with a PAH/PSS polyelectrolyte multilayered film. The vessels luminal surfaces covered with the multilayer film were observed by electronic scanning microscopy. Our observations showed that the luminal surface is completely devoid of ECs following treatment with trypsin. A top view of the coated artery indicated that the multilayer uniformly covered internal surface of the vessels. The successful of the multilayer correct deposition and retention on the arterial wall were controlled by confocal microscopy using a fluorescent polyelectrolyte (rhodamine-PAH). The data suggest that decellularized cryopreserved arteries represent a potential scaffold for further vascular tissue engineering efforts. Moreover, the multilayer films can be used to coat biological surfaces and following the terminated layer (PAH or PSS), favour the cell adhesion or cell resistance.

Multiple and time-scheduled in situ DNA delivery mediated by beta-cyclodextrin embedded in a polyelectrolyte multilayer.

The basic premise of gene therapy is that genes can be used to produce in situ therapeutic proteins. The controlled delivery of DNA complexes from biomaterials offers the potential to enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment. Immobilization of the DNA to the substrate to which cells adhere maintains the DNA in the cell microenvironment for subsequent cellular internalization. Here, layer-by-layer (LBL) films made from poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) containing DNA were built in the presence of charged cyclodextrins. The biological activities of these polyelectrolyte films were tested by means of induced production of a specific protein in the nucleus or in the cytoplasm by cells in contact with the films. This type of coating offers the possibility for either simultaneous or sequential interfacial delivery of different DNA molecules aimed at cell transfection. These results open the route to numerous potential applications in patch vaccination, for example.

Polyelectrolyte multilayer film coating and stability at the surfaces of oral prosthesis base polymers: an in vitro and in vivo study.

A new type of coating involving a layer-by-layer technique has been recently reported. This coating is composed of a polyelectrolyte multilayer film that confers specific properties on surfaces to which it is applied. Here, we studied the applicability of such a technique to the coating of oral prostheses, by first testing the construction of polyelectrolyte multilayer films on several polymers used in oral prosthesis bases, and, subsequently, by studying the stability of these coatings in vitro, in human saliva, and in vivo in a rat model. We demonstrated that the multilayered films are able to coat the surfaces of all tested polymers completely, thus increasing their wettability. We also showed that saliva does not degrade the film after 7 days in vitro and after 4 days in vivo. Taken together, our results establish that the layer-by-layer technique is suitable for the coating of oral devices.

Mechanically responsive films of variable hydrophobicity made of polyelectrolyte multilayers.

Mechanically responsive surfaces that allow to switch reversibly from a hydrophobic to a hydrophilic substrate are reported. The surfaces are constituted of polyelectrolyte multilayers deposited on modified charged silicone sheets. n bilayers of poly(allylamine)-Nafion (PAH-Naf) and m bilayers of poly(allylamine)-poly(acrylic acid) (PAH-PAA) composed the multilayers. A (PAH-Naf)(n) film possesses a water contact angle of around 105 degrees, whereas the contact angle of a (PAH-Naf)(4)-(PAH-PAA)(m) multilayer is around 50 degrees. When such a film with m < 5 and terminated by PAA is stretched out, its water contact angle increases up to around 100 degrees. Successive elongation/retraction cycles allow the water contact angle to alternate reversibly between 100 and 57 degrees indicating the reversible mechanical responsive nature of the film.

Behaviour of endothelial cells seeded on thin polyelectrolyte multilayered films: a new biological scaffold.

The surface modification using thin polyelectrolyte multilayered films was proposed as a new scaffold material for different cell lines. In this study, we evaluated the possible use of polyelectrolyte multilayers as surface modification for the development of endothelial cells. In order to control the behaviour of endothelial cells, cell viability by MTT assay was studied. Moreover, the endothelial cell phenotype was checked and the expression of a leukocyte adhesion molecule (ICAM-1) was quantified. The behaviour of the cells on two polyelectrolyte multilayers was compared to cells on polystyrene, and two polyelectrolyte monolayers (terminating the multilayer architectures). The results have shown a better cell viability on the polyelectrolyte multilayers, inducing a higher cell number compared to polyelectrolyte monolayers after 1 and 3 days of culture. Moreover, the cells showed a normal morphology of cytoskeleton. The phenotype of the endothelial cells was kept and a low level of leukocyte adhesion molecules was observed. In conclusion, the polyelectrolyte multilayers can be considered as a potential surface modification procedure to enhance the development of endothelial cells on hydrophobic substrate and which can be applied to vascular tissue engineering.

Layer by layer self-assembled polyelectrolyte multilayers with embedded phospholipid vesicles obtained by spraying: integrity of the vesicles.

In a previous paper (Michel, M.; Vautier, D.; Voegel, J.-C.; Schaaf, P.; Ball, V. Langmuir 2004, 20, 4835), we showed that phospholipid vesicles can be incorporated into poly(glutamic-acid)/poly(allylamine) (PGA/PAH) multilayered polyelectrolyte films built by the alternated dipping of a surface in polyanion and polycation solutions. AFM imaging, quartz crystal microbalance, and ellipsometry suggested that the vesicles remain intact when adhering on the surface. In the present paper, we show that such films can also be realized by spraying both the polyelectrolyte solutions and the vesicles onto the surface. Using such vesicles filled with ferrocyanide ions, we prove by cyclic voltammetry that the sprayed vesicles remain intact when embedded in the multilayers. We show that multilayers containing two distinct layers of intact vesicles separated by several polyanion/polycation bilayers can also be constructed. Polyelectrolyte multilayers containing layers of phospholipid vesicles could act as reservoirs for drug or other biologically active molecules in controlled release bioactive coatings.

Dipping versus spraying: exploring the deposition conditions for speeding up layer-by-layer assembly.

Polyelectrolyte film fabrication by successive spraying of polycation and polyanion solutions is described and compared to classic dipping. The poly(styrenesulfonate)/poly(allylamine) system is examined in detail. The influence of various parameters such as spraying time, polyelectrolyte concentration, and effect of film drying during multilayer construction is investigated. It is found that film deposition by spraying is easily controlled and very reliable. The thickness of the multilayers grows linearly with the number of deposition cycles similarly to what is observed when dipping substrates or when polyelectrolyte solutions flow over a surface. The assembly of films is very fast and leads to films with small surface roughness as estimated by atomic force microscopy and X-ray reflectometry. Spray deposition allows achieving regular multilayer growth even under conditions for which dipping fails to produce homogeneous films (e.g., extremely short contact times). Moreover, because drainage constantly removes a certain quantity of the excess material arriving at the surface, one can even skip the rinsing step and, thus, speed up even further the whole buildup process.

Multivalent ion/polyelectrolyte exchange processes in exponentially growing multilayers.

We show, in this paper that multivalent ferrocyanide anions can penetrate into exponentially growing (PGA/PAH)n multilayer films whatever the nature of the last deposited layer. These ions are not able to diffuse out of the film when it is brought in contact with a pure buffer solution. However, the contact of this film with a poly(allylamine) (PAH) or a poly(L-glutamic acid) (PGA) solution leads to the release of ferrocyanide ions from the multilayer. It is shown that the release of ferrocyanide anions, when the film is in contact with a PGA solution, is due to the diffusion of the PGA chains into the film so that an exchange between ferrocyanide ions and PGA chains takes place inside the film. On the other hand, PAH chains do not diffuse into PGA/PAH multilayers. When the film is then brought in contact with a PAH solution, the PAH chains from the solution are expected to strongly interact with the ferrocyanide ions and thus induce a diffusion mechanism of the multivalent anions out of the film, the film/solution interface playing the role of a sink for these ions. This work thus shows that interactions between multivalent ions and exponentially growing films are much more complex than expected at first sight and that polyelectrolyte multilayers must be seen as dynamic entities in which diffusion and exchange processes can take place.

Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization.

Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic surgery. In the present study, we developed a new strategy based on the insertion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into polyelectrolyte multilayer films built by the alternate deposition of polyanions and polycations. Quartz crystal microbalance and streaming potential measurements were used to follow step by step the construction of the multilayer films and embedding of the defensin within the films. Antimicrobial assays were performed with two strains: Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium). The inhibition of E. coli D22 growth at the surface of defensin-functionalized films was found to be 98% when 10 antimicrobial peptide layers were inserted in the film architecture. Noticeably, the biofunctionalization could be achieved only when positively charged poly(l-lysine) was the outermost layer of the film. On the basis of the results of bacterial adhesion experiments observed by confocal or electron microscopy, these observations could result from the close interaction of the bacteria with the positively charged ends of the films, which allows defensin to interact with the bacterial membrane structure. These results open new possibilities for the use of such easily built and functionalized architectures onto any type of implantable biomaterial. The modified surfaces are active against microbial infection and represent a novel means of local host protection.

Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials.

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.