Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

Some Plant Defense Stimulators can induce IL-1ß production in human immune cells in vitro.

Among Plant Protection Products (PPP), a new emerging category of pesticides act by stimulating plant defense in order to improve plant resistance against microbial pathogens. Given that these compounds, the so-called Plant Defense Stimulators (PDS) act on innate immunity, we tested, using an in vitro approach on human mononuclear leucocytes (PBMC), the potential toxicity (XTT assay) and inflammatory effects (production of IL-1ß) of 4 PPP belonging to different chemical families. We found that two products (LBG-01F34® and Regalis®) did not induce any cytotoxicity or IL-1 ß production. The product BION-50 WG®, that contains Acibenzolar-S-methyl (ASM) and silica particles did not present any cytotoxicity but induced a significant increase in the production of the inflammatory cytokine IL-1 ß. Finally, Vacciplant® that contains laminarin, was highly cytotoxic and pro-inflammatory. It induced a strong production of IL-1 ß when used at a concentration in the culture medium, as low as 0.02 mg/mL. We also tested the potential toxic effect of these 4 PPP on 4 days old zebra fish larvae. After 24 h of exposure, our results indicate that Vacciplant® induced zebra fish larvae mortality at concentration of 20 µg/mL. LBG did not induced significant mortality at concentrations up to 1 mg/mL whereas Regalis was lethal for 0,3 mg/mL concentrations and BION-50 WG began to induce mortality at 2,5 mg/mL. Our results indicate possible effects of PDS on IL-1ß production in human cells and fish survival, calling for more studies on the potential noxious side effects of these compounds.

zHSF1 modulates zper2 expression in zebrafish embryos.

HSF1 is a transcription factor that plays a key role in circadian resetting by temperature. We have used zebrafish embryos to decipher the roles of zHsf1, heat and light on zper2 transcription in vivo. Our results show that heat shock (HS) stimulated zper2 expression in the dark but has no cumulative effect combined with light. After light exposition, zper2 expression was 2.7 fold increased threefold in the hsf1-morphants in comparison to control embryos. Our results show that zHsf1 plays a positive role in HS-driven expression of zper2 in the dark but seems to act as an attenuator in the presence light.

Resveratrol-Induced Xenophagy Promotes Intracellular Bacteria Clearance in Intestinal Epithelial Cells and Macrophages.

Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and the modulating inflammatory response. Several infectious and immune disorders are associated with autophagy defects, suggesting that stimulation of autophagy in these diseases should be beneficial. Here, we show that resveratrol is able to boost xenophagy, a selective form of autophagy that target invasive bacteria. We demonstrated that resveratrol promotes in vitro autophagy-dependent clearance of intracellular bacteria in intestinal epithelial cells and macrophages. These results were validated in vivo using infection in a transgenic GFP-LC3 zebrafish model. We also compared the ability of resveratrol derivatives, designed to improve the bioavailability of the parent molecule, to stimulate autophagy and to induce intracellular bacteria clearance. Together, our data demonstrate the ability of resveratrol to stimulate xenophagy, and thereby enhance the clearance of two invasive bacteria involved life-threatening diseases, Salmonella Typhimurium and Crohn's disease-associated Adherent-Invasive Escherichia coli. These findings encourage the further development of pro-autophagic nutrients to strengthen intestinal homeostasis in basal and infectious states.

Inflammatory Effects of the Plant Protection Product Stifenia (FEN560) on Vertebrates.

Plant defense stimulators (PDSs) rely on the activation of plant innate immunity in order to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03-1 mg mL-1) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1ß inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1ß. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1ß and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection.

Biofilms of Lactobacillus plantarum and Lactobacillus fermentum: Effect on stress responses, antagonistic effects on pathogen growth and immunomodulatory properties.

Few studies have extensively investigated probiotic functions associated with biofilms. Here, we show that strains of Lactobacillus plantarum and Lactobacillus fermentum are able to grow as biofilm on abiotic surfaces, but the biomass density differs between strains. We performed microtiter plate biofilm assays under growth conditions mimicking to the gastrointestinal environment. Osmolarity and low concentrations of bile significantly enhanced Lactobacillus spatial organization. Two L. plantarum strains were able to form biofilms under high concentrations of bile and mucus. We used the agar well-diffusion method to show that supernatants from all Lactobacillus except the NA4 isolate produced food pathogen inhibitory molecules in biofilm. Moreover, TNF-α production by LPS-activated human monocytoid cells was suppressed by supernatants from Lactobacillus cultivated as biofilms but not by planktonic culture supernatants. However, only L. fermentum NA4 showed anti-inflammatory effects in zebrafish embryos fed with probiotic bacteria, as assessed by cytokine transcript level (TNF-α, IL-1ß and IL-10). We conclude that the biofilm mode of life is associated with beneficial probiotic properties of lactobacilli, in a strain dependent manner. Those results suggest that characterization of isolate phenotype in the biofilm state could be additional valuable information for the selection of probiotic strains.

The impact of tumor nitric oxide production on VEGFA expression and tumor growth in a zebrafish rat glioma xenograft model.

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

SMYD3 promotes cancer invasion by epigenetic upregulation of the metalloproteinase MMP-9.

Upregulation of the matrix metalloproteinase (MMP)-9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference-mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression.

Leukemic cell xenograft in zebrafish embryo for investigating drug efficacy.

Zebrafish were proposed as an alternative to mammalian models to assess the efficacy and toxicity of antileukemic drugs. Due to the limited number of transgenic zebrafish leukemia models, we explored human leukemic cell xenograft in zebrafish embryos. Human leukemic cell lines and blast cells sorted from patients with acute myelogenous leukemia were injected 48 hours post-fertilization and remained in the circulation of zebrafish embryos for several days without affecting their development. Imatinib and oxaphorines did not demonstrate any toxicity on normal zebrafish embryos and decreased the leukemic burden in animals xenografted with sensitive leukemic cell lines. Two other molecules, all-trans retinoic acid and the translation inhibitor 4EGI-1, demonstrated teratogenic effects at concentrations shown to be efficient in vitro, which precluded investigation of their antileukemic activity in such models. Altogether, xenografted leukemic cells in zebrafish embryos are a pharmacologically relevant model for screening non-teratogenic drugs.

Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish.

Nonaspanins constitute a family of proteins, also called TM9SF, characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. This family is highly conserved through evolution and comprises three members in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1-TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, furthermore, human TM9SF1 plays a role in the regulation of autophagy and human TM9SF4 in tumor cannibalism. Here we report that the zebrafish genome encodes five members of this family, TM9SF1-TM9SF5, which show high level of sequence conservation with the previously reported members. Expression analysis in zebrafish showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Gene expression could not be regulated by pathogen-associated molecular patterns such as LPS, CpG, or Poly I:C. By bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants, and animals, we confirmed a very conserved protein structure. An evolutionary conserved immunoreceptor tyrosine-based inhibition motif has been detected in the cytoplasmic domain between transmembrane domain (TM) 7 and TM8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5, and at the extreme C-terminal end of TM9SF4. Finally, a conserved TRAF2 binding domain could also be predicted in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This confirms the hypothesis that TM9SF proteins may play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity.

Comparative analysis of zebrafish nos2a and nos2b genes.

Nitric oxide synthase (NOS) produces nitric oxide (NO) from arginine. Three NOS isoforms have been identified in mammals, namely a neuronal (NOS1), an inducible (NOS2) and an endothelial (NOS3) enzyme. In zebrafish genome, one nos1 gene and two nos2 genes (nos2a and nos2b) were observed. We cloned zebrafish nos2a cDNA and compared nos2a and nos2b sequences, expression and inducibility. When analyzed by reverse transcription-PCR, the expression of nos2a remained very low during initial development, then increased at 96 hpf, while nos2b was expressed from 6 hpf and subsequently remained stable. Expression of nos2a is detected in the head, eye and gut regions by WISH experiments performed at 48, 72 and 96 hpf larvae. In adults, nos2a expression varies from one tissue to another whereas nos2b is expressed in all studied tissues. Both nos2 isoforms can be induced by pro-inflammatory or mechanical stresses (tissue injury). In vitro as in vivo stimulations with Poly I:C and lipopolysaccharides (LPS) enhanced more dramatically nos2a than nos2b expression. After tail transection in 4 dpf larvae a strong increase of nos2a and nos2b expression was evidenced in the regeneration site, skin cells and for the nos2b gene in neuromasts. Phylogenetic and syntenic analyses show that nos2b gene was associated with syntenic genes identified for nos2 genes in vertebrate. This is not the case for the nos2a gene, despite zebrafish nos2a presenting the inducible property of a classical vertebrate nos2 isoform. A myristoylation consensus site was detected at the N-terminal extremity of zebrafish Nos2b, a property shared with mammal NOS3 isoforms. Thus, the evolution of nos2 genes in zebrafish provides a typical example of gene divergence after duplication.

A role for the transcription intermediary factor 2 in zebrafish myelopoiesis.

OBJECTIVE: TIF2 is fused with MOZ in the inv(8)(p11q13) acute myeloid leukemia. TIF2, member of the p160 family, is a histone acetyl transferase (HAT). Deletion of p160 genes were performed in mice. Some observations suggest that p160 family members may perform overlapping functions in mice. Therefore, we decided to choose the zebrafish model to study TIF2. The aim of this study was to characterize the role of this HAT during embryonic development. MATERIAL AND METHODS: We use antisense, morpholino-modified oligomers to transiently knockdown tif2 gene, thus determining whether TIF2 plays a role in zebrafish early development. RESULTS: We show that tif2 is involved in embryogenesis and in primitive hematopoiesis. tif2-knockdown zebrafish embryos are smaller than controls, they demonstrate shorter tails, they display notochord deformation and they exhibit U-shaped tail somites. A synthetic RNA encoding human TIF2 rescues the tif2-knockdown phenotype. Analysis of fli1 expression by whole-mount in situ hybridization indicates normal angioblast specification, but altered localization of intersomitic vessels. The posterior intermediate cell mass, in which a part of primitive hematopoiesis occurs, is altered in tif2 morphants and whole-mount in situ hybridization analyses of l-plastin and mpx expression suggest a specific inhibition of granulocytic and macrophagic differentiation at late stages. CONCLUSION: These data indicate an important role for TIF2 in zebrafish primitive myelopoiesis.

Imaging of nitric oxide in a living vertebrate using a diamino-fluorescein probe.

Numerous approaches have been described to identify nitric oxide (NO), a free radical involved in various physiological and pathophysiological processes. One of these approaches is based on the use of chemical probes whose transformation by NO generates highly fluorescent derivatives, permitting detection of NO down to nanomolar concentrations. Here, we show that the cell-permeant diamino-fluorophore 4-amino-5-methylamino-2'-7'-difluoro-fluorescein diacetate (DAF-FM-DA) can be used to detect NO production sites in a living vertebrate, the zebrafish Danio rerio. The staining pattern obtained in larvae includes the bulbus arteriosus, forming bones, the notochord, and the caudal fin. The specificity of the signal was confirmed by its decrease in animals exposed to a NO scavenger or a NO synthase inhibitor and its increase in the presence of a NO donor. Using this method, NO production was observed to change along development in the notochord and the caudal fin whereas it remained stable in the bulbus arteriosus. Local changes in NO production in response to stressful conditions were also detected by this method. Altogether, labeling with DAF-FM DA is an efficient method to monitor changes in NO production in live zebrafish under physiological as well as pathophysiological conditions, suggesting applications to drug screening and molecular pharmacology.

Toll-like receptor gene family and TIR-domain adapters in Danio rerio.

The toll-like family of receptors (TLR) is an ancient pattern recognition receptor family, conserved from insects to mammals. We have identified in zebrafish (Danio rerio) 19 putative TLR variants, the orthologs of mammalian TLR2-5, 7-9, a fish specific receptor type group and three putative splice variants. One receptor is very close to mammalian TLR1, 6 and 10 and seems to be their common ancestor. However, in contrast to the pufferfish, Fugu rubripes, we found two receptors homologous to TLR4, showing that lack of TLR4 is not general for fish. In addition, we identified two members close to mammalian TLR8 and five members close to FuguTLR21 and goldfish TLR, a TLR group which now has only been found in fish. By RT-PCR we showed that all TLR are widely expressed in adult tissues, but also at different stages of development. All these TLRs contain very conserved toll/interleukin-1 receptor (TIR) domains able to interact with TIR-domain of adapter molecules. We demonstrate here that TIR-domain containing adapters MyD88 and SARM are present in zebrafish, showing that TLR adapter molecules are highly conserved in evolution.

3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells.

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.

Polyelectrolyte multilayer films modulate cytoskeletal organization in chondrosarcoma cells.

The aim of this study was to evaluate polyelectrolyte multilayer films as interfaces for implants. Polyelectrolyte multilayers were built up with different terminating layers by alternate deposition of oppositely charged polyelectrolytes on which chondrosarcoma (HCS-2/8) cells were grown in the presence of serum. Films formed by an increasing number of layers were investigated. The terminating layer was made of one of the following polyelectrolytes: poly-sodium-4-styrenesulfonate (PSS), poly-L-glutamic acid (PGA), poly-allylamine hydrochloride (PAH), or poly(L-lysine) (PLL). Cell viability, inflammatory response, adherence, and cytoskeletal organization were studied. Induction of interleukin-8 (IL-8) secretion was detected on PAH and PLL ending polyelectrolyte films. Early cellular adherence was enhanced with PGA, PAH, PLL, and, to a lower extent, PSS terminating layers. Adherence was independent of the number of layers constituting the films. The presence of actin filaments and vinculin focal adhesion spots was observed on PSS or PAH ending films. They were respectively partially and totally absent on PGA and PLL terminating multilayer architectures. For PLL ending films, vinculin and actin organization was clearly dependent on the number of deposited layers. The results of this study suggest that PSS ending multilayered films constitute a good interfacial micro-environment at the material surface for HCS-2/8 cells.

Survival analysis of rats implanted with porous titanium tracheal prosthesis.

BACKGROUND: Surgical treatment of a malignancy in the trachea may lead to a long resection that has to be reconstructed with an artificial prosthesis. However, most of the available prostheses encounter inflammatory rejection and mechanical constraint problems. To improve tracheal rehabilitation a porous titanium prosthesis was developed. The aim of this study was to test the biocompatibility of this novel material. METHODS: Seventeen rats had a partial tracheal prosthesis made of porous titanium inserted in the cervical trachea. The histologic analysis of the tissue surrounding the prosthesis was performed in 11 surviving animals after a period of 15 to 41 days. RESULTS: Fibroblast colonization of titanium pores and a ciliary cylindrical epithelial layer developed on the endoluminal side of the prosthesis and the inflammatory reaction was minimal. CONCLUSIONS: The results of this short-term study validate, from surgical and histologic standpoints, the usefulness of a porous titanium tracheal prosthesis.

Viability, adhesion, and bone phenotype of osteoblast-like cells on polyelectrolyte multilayer films.

The aim of this study was to develop new biocompatible coatings for bone implants by the alternating deposition of oppositely charged polyelectrolytes. Polyelectrolyte films were built up with different terminating layers on which SaOS-2 osteoblast-like cells and human periodontal ligament (PDL) cells were grown. The terminating layer was made of one of the following polyelectrolytes: poly(ethylene imine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), poly(allylamine hydrochloride) (PAH), poly(L-glutamic acid) (PGA), or poly(L-lysine) (PLL). Cell adherence, viability, stability of osteoblast phenotype, and inflammatory response were studied. Adherence and viability were good on all terminating layers except the PEI-terminating layer, which was cytotoxic. Maintenance of osteoblast phenotype marker expression was observed on PSS- and PGA-terminating films for both cell types, whereas downregulation, associated with the induction of Interleukin-8 (IL-8) secretion, was detected on PEI and PAH for both cell types and on PLL for PDL cells. These results suggested a good biocompatibility of PSS- and PGA-ending films for PDL cells and of PSS-, PGA-, and PLL-terminating films for SaOS-2 cells. As a result, polyelectrolyte multilayer films could emerge as new alternatives for implant coatings.