Teeth are established as targets for vitamin D metabolites but have retained less attention than the well-defined bone cell metabolism. Here we report that VDRgem mice, which bear a point-mutated vitamin D receptor (VDR) unresponsive to the endogenous bioactive vitamin D, are more severely affected and have a greater severity of tooth and alveolar bone defects than VDR-null mice. Transcriptional profiling of postnatal day 5 VDRgem and VDR-null mutant incisors revealed vitamin D ligand signaling targets that promote mineralization, induce alveolar bone growth, and assure dentin integrity. RNA-sequencing analysis of VDRgem mutants uniquely showed defects in developmental targets- notably Hedgehog and Wnt signaling reductions. Micro computed tomography (μCT) and microscopy analysis of adult VDRgem mice unravels severely reduced alveolar bone density, with abnormal root and pulp morphology. VDRgem mutants have severely hypocalcified alveolar bone and dentin- comparatively worse than VDR-null mice- as confirmed by scanning electron microscopy and mineral analysis. This genetically confirmed that vitamin D, acting through its receptor, enables bone and tooth to accumulate enough calcium and phosphorous for structural integrity. In addition, enamel structure is defective in VDRgem mice, displaying hypomineralized areas. Collectively, the severe defects in VDRgem tooth and alveolar bone demonstrate the importance of vitamin D actions at early stages of tooth development.

Cartilage, particularly hyaline cartilage, is essential for structural and functional integrity in otorhinolaryngological region (nose, ears, larynx, and trachea) but exhibits limited regenerative capacity due to its avascular nature. Current clinical strategies, including microfracture, autologous chondrocyte implantation, and cartilage grafting remain limited by poor integration, donor site morbidity, and fibrocartilage formation rather than hyaline cartilage. In parallel, most clinically investigated scaffolds rely on xenogeneic collagen, raising concerns regarding batch-to-batch variability, immunogenicity, regulatory burden, and sourcing. Together, these limitations highlight the need for more clinically translatable autologous, biomimetic, and scalable biomaterials. Here, we report a proof-of-concept albumin-based bilayered scaffold for cartilage tissue engineering, using a salt-assisted compaction process without the use of chemical crosslinkers, based on albumin self-assembly. This scaffold combines a porous layer to support cell infiltration and cartilage-like formation, and a smooth layer to support later the regeneration of cutaneous (auricular) or respiratory (tracheal or nasal) epithelium in situ. In this study, human nasal chondrocytes seeded in the scaffold showed proliferation, maintained viability and were associated with the production of cartilage-like extracellular matrix rich in type II collagen and aggrecan. Following subcutaneous implantation in nude mice, the scaffold showed progressive degradation, tissue integration, and features consistent with hyaline-like cartilage formation. Overall, this work suggests that albumin-based bilayered scaffolds may represent a promising approach for cartilage repair and may be advantageous for applications in nasal, auricular, and craniofacial reconstruction. Further studies in orthotopic models will be required to evaluate their functional performance and clinical relevance.

Current biodegradable materials are facing many challenges when used for the design of implantable devices because of shortcomings such as toxicity of crosslinking agents and degradation derivatives, limited cell adhesion, and limited immunological compatibility. Here, a class of materials built entirely of stable protein is designed using a simple protocol based on salt-assisted compaction of albumin, breaking with current crosslinking strategies. Salt-assisted compaction is based on the assembly of albumin in the presence of high concentrations of specific salts such as sodium bromide. This process leads, surprisingly, to water-insoluble handable materials with high preservation of their native protein structures and Young's modulus close to that of cartilage (0.86 MPa). Furthermore, these materials are non-cytotoxic, non-inflammatory, and in vivo implantations (using models of mice and rabbits) demonstrate a very slow degradation rate of the material with excellent biocompatibility and absence of systemic inflammation and implant failure. Therefore, these materials constitute promising candidates for the design of biodegradable scaffolds and drug delivery systems as an alternative to conventional synthetic degradable polyester materials.

Poly(3,4)ethylene-dioxythiophene (PEDOT), mainly associated with poly(styrenesulfonate) (PSS), is widely used to create thin conductive films or, more recently, conductive 3D porous materials as scaffolds for tissue engineering. Such conductive materials allow the direct delivery of electrical, electrochemical, and electromechanical stimuli to cells. Typically, surfactants are combined with the PEDOT:PSS dispersion to design conductive films by gelation. To the best of our knowledge, no foams have been reported using PEDOT:PSS/surfactant mixtures and a simple drying process. In this article, to develop a cytocompatible and conductive 3D porous material by a simple process, a PEDOT:PSS/cationic surfactant mixture was used to design solid foams by simple foaming followed by a drying step. We exploit the electrostatic interactions between negatively charged commercial PEDOT:PSS and positively charged N-octyltrimethylammonium bromide (C8TAB) surfactant to develop conductive solid foams via the solidification of liquid foams. The stable and homogeneous liquid PEDOT:PSS/C8TAB foams were dried to obtain solid and conductive foams with a high conductivity of 2.05 +/- 0.80 S/cm. The high conductivity of the bulk PEDOT:PSS/C8TAB thin film, at 17 +/- 4 S/cm, confirmed a doping effect of C8TAB. This increased conductivity in the presence of the surfactants is probably due to an increased interpenetration of the PEDOT:PSS particles during film formation. The resulting foams are highly conductive and noncytotoxic, allowing the proliferation of murine fibroblasts. These foams have great potential for biomedical applications.

Postoperative infections are the most common complications faced by surgeons after implant surgery. To address this issue, an emerging and promising approach is to develop antimicrobial coatings using antibiotic substitutes. We investigated the use of polycationic homopolypeptides in a layer-by-layer coating combined with hyaluronic acid (HA) to produce an effective antimicrobial shield. The three peptide-based polycations used to make the coatings, poly(l-arginine) (PAR), poly(l-lysine), and poly(l-ornithine), provided an efficient antibacterial barrier by a contact-killing mechanism against Gram-positive, Gram-negative, and antibiotic-resistant bacteria. Moreover, this activity was higher for homopolypeptides containing 30 amino-acid residues per polycation chain, emphasizing the impact of the polycation chain length and its mobility in the coatings to deploy its contact-killing antimicrobial properties. However, the PAR-containing coating emerged as the best candidate among the three selected polycations, as it promoted cell adhesion and epithelial monolayer formation. It also stimulated nitric oxide production in endothelial cells, thereby facilitating angiogenesis and subsequent tissue regeneration. More interestingly, bacteria did not develop a resistance to PAR and (PAR/HA) also inhibited the proliferation of eukaryotic pathogens, such as yeasts. Furthermore, in vivo investigations on a (PAR/HA)-coated hernia mesh implanted on a rabbit model confirmed that the coating had antibacterial properties without causing chronic inflammation. These impressive synergistic activities highlight the strong potential of PAR/HA coatings as a key tool in combating bacteria, including those resistant to conventional antibiotics and associated to medical devices.

Inspired by the eumelanin aggregates in human skin, polydopamine nanoparticles (PDA NPs) are promising nanovectors for biomedical applications, especially because of their biocompatibility. We synthesized and characterized fluorescent PDA NPs of 10–25 nm diameter based on a protein containing a lysine–glutamate diad (bovine serum albumin, BSA) and determined whether they can penetrate and accumulate in bacterial cells to serve as a marker or drug nanocarrier. Three fluorescent PDA NPs were designed to allow for tracking in three different wavelength ranges by oxidizing BSA/PDA NPs (Ox-BSA/PDA NPs) or labelling with fluorescein 5-isothiocyanate (FITC-BSA/PDA NPs) or rhodamine B isothiocyanate (RhBITC-BSA/PDA NPs). FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs penetrated and accumulated in both cell wall and inner compartments of Escherichia coli (E. coli) cells. The fluorescence signals were diffuse or displayed aggregate-like patterns with both labelled NPs and free dyes. RhBITC-BSA/PDA NPs led to the most intense fluorescence in cells. Penetration and accumulation of NPs was not accompanied by a bactericidal or inhibitory effect of growth as demonstrated with the Gram-negative E. coli species and confirmed with a Gram-positive bacterial species (Staphylococcus aureus). Altogether, these results allow us to envisage the use of labelled BSA/PDA NPs to track bacteria and carry drugs in the core of bacterial cells.

The difficulty involved in the treatment of many tumours due to their recurrence and resistance to chemotherapy is tightly linked to the presence of cancer stem cells (CSCs). This CSC sub-population is distinct from the majority of cancer cells of the tumour bulk. Indeed, CSCs have increased mitochondrial mass that has been linked to increased sensitivity to mitochondrial targeting compounds. Thus, a platinum-based polyethylenimine (PEI) polymer-drug conjugate (PDC) was assessed as a potential anti-CSC therapeutic since it has previously displayed mitochondrial accumulation. Our results show that CSCs have increased specific sensitivity to the PEI carrier and to the PDC. The mechanism of cell death seems to be necrotic in nature, with an absence of apoptotic markers. Cell death is accompanied by the induction of a protective autophagy. The interference in the balance of this pathway, which is highly important for CSCs, may be responsible for a partial reversion of the stem-like phenotype observed with prolonged PEI and PDC treatment. Several markers also indicate the cell death mode to be capable of inducing an anti-cancer immune response. This study thus indicates the potential therapeutic perspectives of polycations against CSCs.

Resistance to antifungal therapy of Candida albicans and non-albicans Candida strains, frequently associated with oral candidosis, is on the rise. In this context, host-defense peptides have emerged as new promising candidates to overcome antifungal resistance. Thus, the aim of this study was to assess the effectiveness against Candida species of different Catestatin-derived peptides, as well as the combined effect with serum albumin. Among Catestatin-derived peptides, the most active against sensitive and resistant strains of C. albicans, C. tropicalis and C. glabrata was the D-isomer of Cateslytin (D-bCtl) whereas the efficiency of the L-isomer (L-bCtl) significantly decreases against C. glabrata strains. Images obtained by transmission electron microscopy clearly demonstrated fungal membrane lysis and the leakage of the intracellular material induced by the L-bCtl and D-bCtl peptides. The possible synergistic effect of albumin on Catestatin-derived peptides activity was investigated too. Our finding showed that bovine serum albumin (BSA) when combined with the L- isomer of Catestatin (L-bCts) had a synergistic effect against Candida albicans especially at low concentrations of BSA; however, no synergistic effect was detected when BSA interacted with L-bCtl, suggesting the importance of the C-terminal end of L-bCts (GPGLQL) for the interaction with BSA. In this context in vitro D-bCtl, as well as the combination of BSA with L-bCts are potential candidates for the development of new antifungal drugs for the treatment of oral candidosis due to Candida and non-Candida albicans, without detrimental side effects.

Cancer stem cells (CSCs) represent a difficult to treat cellular niche within tumours due to their unique characteristics, which give them a high propensity for resistance to classical anti-cancer treatments and the ability to repopulate the tumour mass. An attribute that may be implicated in the high rates of recurrence of certain tumours. However, other characteristics specific to these cells, such as their high dependence on mitochondria, may be exploited for the development of new therapeutic agents that are effective against the niche. As such, a previously described phosphorescent N-heterocyclic carbene iridium(III) compound which showed a high level of cytotoxicity against classical tumour cell lines with mitochondria-specific effects was studied for its potential against CSCs. The results showed a significantly higher level of activity against several CSC lines compared to non-CSCs. Mitochondrial localisation and superoxide production were confirmed. Although the cell death involved caspase activation, their role in cell death was not definitive, with a potential implication of other, non-apoptotic pathways shown. A cytostatic effect of the compound was also displayed at low mortality doses. This study thus provides important insights into the mechanisms and the potential for this class of molecule in the domain of anti-CSC therapeutics.

The synthesis of surgical adhesives is based on the need to design glues that give rise to strong and fast bonds without cytotoxic side effects. A recent trend in surgical adhesives is to use gel-forming polymers modified with catechol groups, which can undergo oxidative crosslinking reactions and are strongly adhesive to all kinds on surfaces in wet conditions. We previously showed that blending gelatin with catechol can yield strong adhesion when the catechol is oxidized by a strong oxidant. Our previous work was limited to the study of the variation in the sodium periodate concentration. In this article, for an in-depth approach to the interactions between the components of the gels, the influence of the gelatin, the sodium periodate and dopamine/(pyro)catechol concentration on the storage (G′) and loss (G″) moduli of the gels, as well as their adhesion on steel, have been studied by shear rheometry. The hydrogels were characterized by infrared and UV-Vis spectroscopy and the size of their pores visualized by digital microscopy and SEM after freeze drying but without further additives. In terms of adhesion between two stainless steel plates, the optimum was obtained for a concentration of 10% w/v in gelatin, 10 mM in sodium periodate, and 20 mM in phenolic compounds. Below these values, it is likely that crosslinking has not been maximized and that the oxidizing environment is weakening the gelatin. Above these values, the loss in adhesiveness may result from the disruption of the alpha helixes due to the large number of phenolic compounds as well as the maintenance of an oxidizing environment. Overall, this investigation shows the possibility to design strongly adhesive hydrogels to metal surfaces by blending gelatin with polyphenols in oxidative conditions.

Extrusion bioprinting is a relevant 3D technology to create biological systems in regenerative medicine, pharmaceutical development and cancer research. Bioink is the necessary component to incorporate the cells that will be printed by extrusion bioprinting. However, bioinks and extrusion printing can generate shear stresses mechanically unfavorable for cell survival. We thus developed a bioink, based on methacrylated collagen and hyaluronic acid, in combination with porous poly(D,L-lactic-co-glycolic acid) solid microscaffolds to protect cells against mechanical stress during extrusion printing. We found that porosities of the microscaffolds allowed human chondosarcoma cells to colonize the structure. Moreover, metabolic activity of these chondrosarcoma cells, fibroblast cells, and dental pulp stem cells (DPSCs) incorporated within bioink (before printing) increased 4-fold in presence of a polylysine- or collagen-coated microscaffolds compared with those cultured without microscaffolds. Their survival increased by 10% either by hand deposition or by bioprinting extrusion (bioprinter BioBot®Basic) compared to cells in bioink without microscaffolds. In addition to the mechanoshield properties provided by microscaffolds, they allow the migration of DPSCs stem cells towards HCS-2/8 cancer cells after 7 days of co-culture in an organoid created by bioprinting extrusion while without microscaffolds the cells aggregated and remained static.

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

In bone tissue engineering, stem cells are known to form inhomogeneous bone-like nodules on a micrometric scale. Herein, micro- and nano-infrared (IR) micro-spectroscopies were used to decipher the chemical composition of the bone-like nodule. Histological and immunohistochemical analyses revealed a cohesive tissue with bone-markers positive cells surrounded by dense mineralized type-I collagen. Micro-IR gathered complementary information indicating a non-mature collagen at the top and periphery and a mature collagen within the nodule. Atomic force microscopy combined to IR (AFM-IR) analyses showed distinct spectra of "cell" and "collagen" rich areas. In contrast to the "cell" area, spectra of "collagen" area revealed the presence of carbohydrate moieties of collagen and/or the presence of glycoproteins. However, it was not possible to determine the collagen maturity, due to strong bands overlapping and/or possible protein orientation effects. Such findings could help developing protocols to allow a reliable characterization of in vitro generated complex bone tissues.

Secreted extracellular matrix components which regulate craniofacial development could be reactivated and play roles in adult wound healing. We report a patient with a loss-of-function of the secreted matricellular protein SMOC2 (SPARC related modular calcium binding 2) presenting severe oligodontia, microdontia, tooth root deficiencies, alveolar bone hypoplasia, and a range of skeletal malformations. Turning to a mouse model, Smoc2-GFP reporter expression indicates SMOC2 dynamically marks a range of dental and bone progenitors. While germline Smoc2 homozygous mutants are viable, tooth number anomalies, reduced tooth size, altered enamel prism patterning, and spontaneous age-induced periodontal bone and root loss are observed in this mouse model. Whole-genome RNA-sequencing analysis of embryonic day (E) 14.5 cap stage molars revealed reductions in early expressed enamel matrix components ( Odontogenic ameloblast-associated protein ) and dentin dysplasia targets ( Dentin matrix acidic phosphoprotein 1 ). We tested if like other matricellular proteins SMOC2 was required for regenerative repair. We found that the Smoc2-GFP reporter was reactivated in adjacent periodontal tissues 4 days after tooth avulsion injury. Following maxillary tooth injury, Smoc2 −/− mutants had increased osteoclast activity and bone resorption surrounding the extracted molar. Interestingly, a 10-day treatment with the cyclooxygenase 2 (COX2) inhibitor ibuprofen (30 mg/kg body weight) blocked tooth injury-induced bone loss in Smoc2 −/− mutants, reducing matrix metalloprotease (Mmp)9 . Collectively, our results indicate that endogenous SMOC2 blocks injury-induced jaw bone osteonecrosis and offsets age-induced periodontal decay.

Diaphragm repair after congenital diaphragmatic hernia is associated with hernia recurrence due to prosthesis failure. Expanded polytetrafluoroethylene (e-PTFE), a synthetic non-degradable biomaterial, is currently used for those diaphragmatic defect repairs. The drawback of e-PTFE is its poor wettability that leads to coating difficulties, bonding that could favor implant integration. However, polydopamine (PDA) can be deposited as well on organic as on inorganic substrates. Therefore, we assessed the biological responses of a clinically used e-PTFE biomaterial treated with PDA in two different manners: one impregnated with PDA and the other coated with a one side PDA film. Mechanical properties of the raw e-PTFE, the PDA soaked biomaterial and the PDA coated surface were characterized by colloidal probe atomic force microscopy. Behaviors of primary human fibroblasts and Wharton’s jelly stem cells were investigated by electron microscopy. Findings reveal that the mechanical properties at the microscopic scale are not modified by the PDA treatments. Cells spread onto both PDA functionalized substrates. In addition, microscopic observations disclose numerous focal cell contacts, evidencing cell attachment, and cytoplasmic projections particularly with the nanoscale PDA coating. Results clearly suggest that PDA in general but above all the PDA coating enhance cellular colonization of the implant material.

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

Latent‐transforming growth factor beta‐binding protein 3 (LTBP‐3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor‐β (TGF‐β). To investigate the role of LTBP‐3 in tooth formation we performed micro‐computed tomography (micro‐CT), histology, and scanning electron microscopy analyses of adult Ltbp3‐/‐ mice. The Ltbp3‐/‐ mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation‐stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous‐like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP‐3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF‐β signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3‐/‐ mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.

Congenital diaphragmatic hernia is a severe disease requiring diaphragm replacement mostly with expanded polytetrafluoroethylene. Unfortunately, the recurrence rate is high due to prosthesis failure with significant morbidity for the child. To provide a better understanding of the integration and possible failure processes of the biomaterial implanted in humans, we conducted electron microscopical and mechanical assessments on a prosthesis explant from a child with congenital diaphragmatic hernia presenting a recurrence. Our findings show a major penetration of connective tissue into the expanded polytetrafluoroethylene on the rough side, whereas the smooth side presents few tissue colonization. This penetration is more important in the central area (area A) with large collagen bundles and layers, in comparison to the peripheral areas without prosthesis failure (area B), where few extracellular matrix is produced. The connective tissue penetrates the prosthesis in depth. In contrast, the peripheral areas with prosthesis failure (area C) show very few cells and extracellular matrix, with an oriented organization in comparison to areas A and B. Obviously, the forces applied on the implanted material modulate the cellular behavior of the newly developed tissues. Atomic force microscopic measurements of the biomaterials' surfaces may explain some cellular behaviors according to areas with or without failure.

Abnormalities of enamel matrix proteins deposition, mineralization, or degradation during tooth development are responsible for a spectrum of either genetic diseases termed Amelogenesis imperfecta or acquired enamel defects. To assess if environmental/nutritional factors can exacerbate enamel defects, we investigated the role of the active form of vitamin A, retinoic acid (RA). Robust expression of RA-degrading enzymes Cyp26b1 and Cyp26c1 in developing murine teeth suggested RA excess would reduce tooth hard tissue mineralization, adversely affecting enamel. We employed a protocol where RA was supplied to pregnant mice as a food supplement, at a concentration estimated to result in moderate elevations in serum RA levels. This supplementation led to severe enamel defects in adult mice born from pregnant dams, with most severe alterations observed for treatments from embryonic day (E)12.5 to E16.5. We identified the enamel matrix proteins enamelin (Enam), ameloblastin (Ambn), and odontogenic ameloblast-associated protein (Odam) as target genes affected by excess RA, exhibiting mRNA reductions of over 20-fold in lower incisors at E16.5. RA treatments also affected bone formation, reducing mineralization. Accordingly, craniofacial ossification was drastically reduced after 2 days of treatment (E14.5). Massive RNA-sequencing (RNA-seq) was performed on E14.5 and E16.5 lower incisors. Reductions in Runx2 (a key transcriptional regulator of bone and enamel differentiation) and its targets were observed at E14.5 in RA-exposed embryos. RNA-seq analysis further indicated that bone growth factors, extracellular matrix, and calcium homeostasis were perturbed. Genes mutated in human AI (ENAM, AMBN, AMELX, AMTN, KLK4) were reduced in expression at E16.5. Our observations support a model in which elevated RA signaling at fetal stages affects dental cell lineages. Thereafter enamel protein production is impaired, leading to permanent enamel alterations.

Metal coordination between polyphenols and metal cations like Fe3+ allows to produce conformal homogeneous and robust coatings on a vast variety of materials. The deposition kinetics and the stability of the obtained films are however only poorly investigated. In the present article it is shown that rough, granular but pinhole free coatings up to 50nm in thickness can be obtained in a one pot manner using pyrocatechol (Pyr)/Fe3+ mixtures at different stoichiometries (with Fe3+/Pyr ratios equal to 0.55 or 1.10) provided the deposition time is extended up to 24h. More importantly, we show that these films are dissolved upon oxidation of Pyr in cyclic voltammetry experiments. When the films deposited during short durations are rinsed with buffer and subsequently re-exposed to Pyr containing solution, they undergo partial dissolution most probably through a ligand exchange process. Such a dissolution process does not occur anymore in the same conditions, when the deposition time is increased above 5h. All Pyr-Fe3+ based films can be stabilized by a post-deposition of a polyelectrolyte multilayer film based on the alternated adsorption of poly(allylamine hydrochloride) and the sodium salt of poly(styrene sulfonate). The deposition of 5 bilayers of these polyelectrolytes allows suppressing the dissolution of Pyr-Fe3+ based films produced for short deposition times.