An Elastin-derived composite matrix for enhanced vascularized and innervated bone tissue reconstruction: from material development to preclinical evaluation.

Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, we developed a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin-like polypeptides (ELPs) grafted with laminin-derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation led to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, we performed longitudinal micro-CT analysis, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP-based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP-based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration. This article is protected by copyright. All rights reserved.

Solution behavior and encapsulation properties of fatty acid-elastin-like polypeptide conjugates.

Developing new biomaterials is an active research area owing to their applications in regenerative medicine, tissue engineering and drug delivery. Elastin-like polypeptides (ELPs) are good candidates for these applications because they are biosourced, biocompatible and biodegradable. With the aim of developing ELP-based micelles for drug delivery applications we have synthesized 15 acyl-ELP compounds by conjugating myristic, palmitic, stearic, oleic or linoleic acid to the N-terminus of three ELPs differing in molar mass. The ELP-fatty acid conjugates have interesting solution behavior. They form micelles at low temperatures and aggregate above the cloud point temperature (Tcp). The critical micelle concentration depends on the fatty acid nature while the micelle size is mainly determined by the ELP block length. We were able to show that ELPs were better hydrated in the micelles than in their individual state in solution. The micelles are stable in phosphate-buffered saline at temperatures below the Tcp, which can vary between 20 °C and 38 °C depending on the length or hydrophilicity of the ELP. Acyl-ELP micelles were loaded with the small hydrophobic molecule Nile red. The encapsulation efficiency and release kinetics showed that the best loading conditions were achieved with the largest ELP conjugated to stearic acid.

Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Development of a cell-free and growth factor-free hydrogel capable of inducing angiogenesis and innervation after subcutaneous implantation.

Despite significant progress in the field of biomaterials for bone repair, the lack of attention to the vascular and nervous networks within bone implants could be one of the main reasons for the delayed or impaired recovery of bone defects. The design of innovative biomaterials should improve the host capacity of healing to restore a functional tissue, taking into account that the nerve systems closely interact with blood vessels in the bone tissue. The aim of this work is to develop a cell-free and growth factor-free hydrogel capable to promote angiogenesis and innervation. To this end, we have used elastin-like polypeptides (ELPs), poly(ethylene glycol) (PEG) and increasing concentrations of the adhesion peptide IKVAV (25% (w/w) representing 1.7 mM and 50% (w/w) representing 4.1 mM) to formulate and produce hydrogels. When characterized in vitro, hydrogels have fine-tunable rheological properties, microporous structure and are biocompatible. At the biological level, 50% IKVAV composition up-regulated Runx2, Osx, Spp1, Vegfa and Bmp2 in mesenchymal stromal cells and Tek in endothelial cells, and sustained the formation of long neurites in sensory neurons. When implanted subcutaneously in mice, hydrogels induced no signals of major inflammation and the 50% IKVAV composition induced higher vessel density and formation of nervous terminations in the peripheral tissue. This novel composite has important features for tissue engineering, showing higher osteogenic, angiogenic and innervation potential in vitro, being not inflammatory in vivo, and inducing angiogenesis and innervation subcutaneously. STATEMENT OF SIGNIFICANCE: One of the main limitations in the field of tissue engineering remains the sufficient vascularization and innervation during tissue repair. In this scope, the development of advanced biomaterials that can support these processes is of crucial importance. Here, we formulated different compositions of Elastin-like polypeptide-based hydrogels bearing the IKVAV adhesion sequence. These compositions showed controlled mechanical properties, and were degradable in vitro. Additionally, we could identify in vitro a composition capable to promote neurite formation and to modulate endothelial and mesenchymal stromal cells gene expression, in view of angiogenesis and osteogenesis, respectively. When tested in vivo, it showed no signs of major inflammation and induced the formation of a highly vascularized and innervated neotissue. In this sense, our approach represents a potential advance in the development of new strategies to promote tissue regeneration, taking into account both angiogenesis and innervation.

Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications.

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.

Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains.

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.

Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation.

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media.

Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals.

Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/ß-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration.

Use of the human hepcidin gene to build a positive-selection vector for periplasmic expression in Escherichia coli.

Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process. The oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hormone hepcidin 25 (Hep25) fused to the maltose-binding protein (MBP) resulted in cell death. This toxicity was not observed when MBP-Hep25 accumulated in the bacterial cytoplasm, or when Hep25 was addressed to the periplasm without the MBP tag. We then modified the periplasmic expression vector pMALp2E to create pMALp2EH, a positive-selection vector with Hep25 as counterselection gene.

Recombinant production and purification of short hydrophobic Elastin-like polypeptides with low transition temperatures.

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. We report herein the recombinant expression of three hydrophobic ELPs (VPGIG)n with variable lengths (n = 20, 40, 60) and sub-ambient transition temperatures. These ELPs were purified from the cytoplasmic soluble fraction of Escherichia coli by inverse transition cycling, and their exact molecular weight was confirmed by various mass spectrometry techniques. Transition temperatures of ELP20, ELP40, and ELP60 were measured at 18.6 °C, 12.4 °C and 11.7 °C, respectively.

Purification process of recombinant monoclonal antibodies with mixed mode chromatography.

An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass.

Production and purification of recombinant human hepcidin-25 with authentic N and C-termini.

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZαA vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale.

Quantitative Side-Chain Modifications of Methionine-Containing Elastin-Like Polypeptides as a Versatile Tool to Tune Their Properties.

Tuning the lower critical solution temperature (LCST) of temperature-responsive recombinant elastin-like polypeptides has usually been achieved by designing different protein sequences, in terms of amino acid composition and length, implying tedious molecular cloning steps. In the present work, we have explored the chemoselective alkylation of methionine as an easy means to modify elastin repeat side chains and easily modulate the LCST of the polypeptides. Such a versatile synthetic method shall practically be exploited to modulate any properties of recombinant polymers.

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies.

BACKGROUND: Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth. RESULTS: Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated. CONCLUSIONS: Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.

Selection of Pseudomonas aeruginosa reference genes for RT-qPCR analysis from sputum of cystic fibrosis patients.

The prerequisite to monitor gene expression is the selection of reference genes for normalization of RT-qPCR results. Using 13 sputum samples collected from 9 CF patients, we demonstrated that PA2875 and PA3340 are better reference genes than the previously used clpX and oprL genes.

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation.

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

Importance of the disulfide bridges in the antibacterial activity of human hepcidin.

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a ß-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.