Conferences

A.L. Tohmé, N.A. Saffioti, D.A. Pallarola. Development of microfluidic biosensors for studying epithelial cells processes by impedance spectroscopy.

Poster Presentation: Ibersensor 2024: 13er Congreso Iberoamericano de Sensores, Buenos Aires (Argentina), 21-24th October, 2024

ABSTRACT ➞

Epithelial cells are crucial for the function of tissues and organs, defending against pathogens, enabling nutrient absorption, and maintaining homeostasis. The complex interactions between epithelial cells and the extracellular matrix (ECM) underpin epithelial physiology, while disruptions in these interactions contribute to numerous diseases, including bacterial infections and autoimmune disorders. Traditional in vitro methods to study these barriers fail to replicate the full complexity of the in vivo environment, limiting our understanding of cellular processes and our ability to dynamically monitor epithelial function. To address these limitations, we have aimed to develop innovative biosensors that permit simultaneous Electrochemical Impedance Spectroscopy (ECIS) and microscopic observation of epithelial cell interactions with their environment under biomimetic conditions.

In this work, we fabricated Indium Tin Oxide (ITO) microelectrodes with excellent transparency and characterized them through ECIS and cyclic voltammetry, demonstrating high sensitivity. We also developed an aligner to precisely integrate the microelectrodes with polydimethylsiloxane (PDMS) microfluidic devices, creating biomimetic conditions for epithelial culture. We optimized channel functionalization with ECM proteins and assessed the adhesion of Madin-Darby Canine Kidney (MDCK) cells, a model cell line for epithelium studies. Our ongoing work focuses on optimizing experimental conditions for direct monitoring of cell-to-ECM and cell-to-cell interactions using ECIS. This research presents a novel approach for studying the molecular basis of epithelial pathologies by enabling multiparametric monitoring of epithelial cell interactions.

B. García, B. Su, D. Pallarola. Enhancing bacterial detection with bactericidal nanostructured titania electrodes.

Poster Presentation: Ibersensor 2024: 13er Congreso Iberoamericano de Sensores, Buenos Aires (Argentina), 21-24th October, 2024

ABSTRACT ➞

In the coming years, an increase in joint replacement surgeries is anticipated, with a 10% failure rate prompting surface modifications for bactericidal action and improved implant integration. Despite significant advancements in implant technology, individuals with compromised immune systems remain susceptible to pathogens, increasing the risk of biofilm formation and potential revision surgeries. To address these challenges, current research efforts are focused on nanostructured surfaces, particularly those utilizing titanium. However, electrochemical bacterial biosensing faces limitations due to titanium oxide's high capacitance. This study proposes the fabrication of bactericidal titania electrodes to enhance the sensitivity of microbial detection. By incorporating antimicrobial properties into the electrode design, this approach aims to improve bacterial detection efficiency, contributing to the advancement of implant bioengineering.

P. López-Gómez, N. Mehwish, M.P. Ginebra, C. Mas-Moruno. Self-assembled peptide-based hydrogels with antimicrobial and cell adhesive properties for tissue engineering applications.

Oral Presentation: 8th China-Europe Symposium on Biomaterials in Regenerative Medicine CESB 2024, Nuremberg (Germany), 15-18th September, 2024

ABSTRACT ➞

Peptide-derived hydrogels have attracted significant interest in the field of tissue engineering owing to their distinctive characteristics and broad range of applications. They have the ability to closely replicate the porosity and nanostructure of the native extracellular matrix, facilitating the integration of signaling epitopes for cellular interaction or enabling preparation against bacterial infections more effectively. [1]. In fact, bacterial colonization of biomaterials is a problematic concern in the clinics. Moreover, bacterial infections and the possibility of biofilm formation are one of the major causes of implant failure [2]. In this regard, peptide-based hydrogels can be readily modified with diverse bioconjugation techniques to display a wide variety of biologically relevant signals for enhanced therapeutic function, including enhancing cell adhesion or exhibiting antibacterial properties. Thus, in this work we have designed peptide sequences containing a self-assembling unit, capable of forming a hydrogel in aqueous buffers [3], and diverse bioactivities, including the RGD cell adhesive sequence [4] and an antibacterial peptide derived from the 1-11 region of lactoferrin (LF1-11) [5]. To aid in the gelation process, hyaluronic acid was also used to crosslink the peptidic chains efficiently. The self-assembled peptide-based hydrogels were extensively characterized presenting an optimal three- dimensional fiber-like structure, with interconnected porosity, and adequate physicochemical properties for tissue engineering applications. Moreover, the biological characterization demonstrated the ability of RGD-based hydrogels to drive osteoblast-like (Saos-2) behavior; while LF1-11-derived hydrogels significantly decreased the bacterial colonization and viability in both Gram-positive and Gram-negative models. Overall, our research underscores the potential of self-assembling peptide-based hydrogels as versatile and promising candidates for tissue engineering strategies, offering a three-dimensional environment mimicking the extracellular matrix, promoting cellular adhesion and exhibiting antibacterial properties.

M.K. Akma Bin Darwis, V. Levario-Diaz, E. Ada Cavalcanti-Adam, P. Lavalle, N.E. Vrana, N.B. Sabani, S.H. Binti Ngalim. Development Of Pegda-Based Micropattern Substrate To Study The Interplay Between Surface Topography And Cellular Response For Tissue Engineering Applications

Oral Presentation: 8th AMDI-UNAIR International Postgraduate Research and Innovation Colloquium (AUPC2024), Penang (Malaysia), 11th - 12th September, 2024.

ABSTRACT ➞

The main highlight in the field of tissue engineering is the orchestration of strategies in line with the development of biofunctionalized scaffolds to recapitulate the generation of fully functional neotissues. Poly(ethylene glycol) diacrylate (PEGDA) hydrogel is known as a biocompatible substrate and offers an excellent mechanical tunability which is suitable to be applied in biomedical applications. Due to its high resistance to protein adsorption, PEGDA is cell non-adhesive, therefore it is suitable to be used as a blank slate in micropatterns. Here, PEGDA-based hydrogel was developed by adopting a micropatterning approach to fabricate substrate with topographic cues to investigate its integration for bone regeneration. Ultraviolet/ozone and polydimethylsiloxane (PDMS) approaches were adapted to develop PEGDA hydrogel with microtopography and biofunctionalized them with fibronectin using stamping and depositing methods. It was found that the combination of PDMS and fibronectin deposition is easier and more efficient in developing well-functionalized patterned hydrogel based on cell alignment and adhesion test. The C2C12 cells were found to behave distinctly when encountered different micropatterns motifs that proved geometrical cues significantly affecting cellular behaviour. Future-wise, this hydrogel will be tested for its feasibility to be integrated for bone implant application through C2C12 differentiation and immune cells integration.

S. Alfonsi, R. Spuri, P. Milon, D. Petrelli. Plastics as vehicles for antibiotic resistance genes in a river of Central Italy.

Poster Presentation: ASM Microbe, Atlanta (USA), June 13-17, 2024.

ABSTRACT ➞

Plastics originating from industrial processes and plastic debris’ fragmentation, represents an emerging health threat in aquatic environments. By providing a suitable surface for bacterial adhesion, plastic bits promote biofilm formation by bacteria from aquatic communities, and can act, even over a long distance, as means of transport of pathogens and their repertoire of biosynthetic genes. Spreading of antibiotic resistance genes (ARGs) may be mediated by Class 1 Integrons, which are mobile genetic elements involved in the transfer and exchange of resistance genes. In this study, we have analysed artificial plastic substrates placed in an Italian river to investigate the composition of associated biofilm and their role as carrier of antibiotic resistance genes. (...) The off-site analysis of plastic fragments included sonication for cells detachment; cultivation of post-sonicated solution in selective agar medium supplemented with Cephotaxime (1mg/L), isolation and identification of Gram- negative resistant strains using biochemical methods (API® 20E/NE, Biomérieux), antimicrobial susceptibility test of isolates according to EUCAST guidelines, and genomic DNA extraction and amplification to detect Class 1 Integron Gene Cassettes (GCs). (...) On polyethylene fragments, six strains of Aeromonas hydrophyla were isolated, showing resistances to ceftazidime (100%), cefepime (90%), levofloxacin (66,6%), and trimethoprim-sulfamethoxazole (50%). Class 1 integron GCs revealed resistances linked to aminoglycosides, chloramphenicol, trimethoprim-sulfamethoxazole and metallo-beta-lactamases. These results provide a detailed characterization of multidrug resistant Gram-negative bacteria, able to produce biofilms on polypropylene and polyethylene fragments floating on river water and identify genetic elements potentially involved in the ARGs acquisition.

N.E. Vrana. The Routes of Incorporation of Immunomodulatory Properties for Multifunction Supramolecular Biomaterial-based Systems

Invited Talk: 12th World Biomaterials Congress (WBC 2024), Daegu (Korea), May 26-31, 2024..

PRESENTATION ➞

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L. Oliver-Cervelló, H. Martin-Gómez, C. Gonzalez-Garcia, E. A. Cavalcanti-Adam, H.-W. Kim, M. Salmeron-Sanchez, M. P. Ginebra, C. Mas-Moruno. Biomimetic multifunctional peptides promote synergistic integrin and growth factor signaling on biomaterials

Poster Presentation: 12th World Biomaterials Congress (WBC 2024), Daegu (Korea), May 26-31, 2024..

ABSTRACT ➞

Cell–material interactions are regulated by mimicking the extracellular matrix (ECM) on the surface of biomaterials. In this regard, reproducing the cellular microenvironment that promotes integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. However, the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entails clinical risks. Thus, the aim of this study was to combine integrin- binding ligands together with osteogenic peptides derived from BMP-2 to recapitulate the healing microenvironment of bone and foster its regeneration.

The wrist epitope of BMP-2 was screened to identify peptides with osteogenic potential. The DWIVA sequence was selected and combined with the cell adhesive peptide RGD using a tailor-made synthetic platform which provides chemical and spatial control over the single binding of integrins and BMP receptors. The biomimetic interfaces were able to engage in integrin-GF signaling and activated the BMP-dependent signaling via p38. Further, analogues of such multifunctional platform were designed and showed potential to synergistically promote human mesenchymal stem cells (MSCs) adhesion and osteogenic differentiation, as well as formation of new bone in vivo on titanium implants. Subsequently, polyethylene glycol (PEG)-based hydrogels were functionalized with the multifunctional peptides and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and enhanced MSCs spreading and osteogenic differentiation, thus expanding the scope of these peptides to dynamic 3D systems. These results prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics. This biomimetic strategy holds promise in bone tissue engineering, without the need for exogenous GFs.

Y. Li, C. Guilbaud-Chéreau, C. Calligaro, N.E. Vrana, P. Lavalle. Antimicrobial, antiviral, and anti-inflammatory coatings: implementation of innovative production tools

Poster Presentation: 12th World Biomaterials Congress (WBC 2024), Daegu (Korea), May 26-31, 2024..

ABSTRACT ➞

Root canal treatment is a procedure that consists of removing an infection from the dental pulp. During the root canal treatment, a small opening is made in the treated tooth to gain access to the canal. Then, the pulp is removed and the canal is cleaned by irrigation with sodium hypochlorite. A temporary filling is used to seal the opening hole in the tooth. After the root canal heals, your dentist replaces the temporary filling with a permanent one but infectious agents may still be present. In this context we tested the addition of antimicrobial coatings onto actual temporary filling using manual dipping process with hyaluronic acid and polycations and performed antimicrobial measurement against M. luteus, S. aureus and E. faecalis. The cytotoxic properties of the coated samples against selected cell lines were performed to confirm their potential use in clinic. In parallel, highly viscous gels made of hyaluronic acid and polycation were tested as innovative disinfectant filler. Rheological study was performed on the solutions to determine their viscosity an shear stress properties. We observed that manual dipping of temporary filling in hyaluronic acid followed by poly(L-arginine) or poly-epsilon(L-lysine) at low concentration of 0.5 mg/mL have high antimicrobial activity. Gel coating with the same composition are also strongly effective. The biocompatibility of the coated samples can be improved by modulating the coating time and concentration. As an alternative to hypochlorite sodium cleaning, hyaluronic acid viscous gels containing polycation are efficient against S. aureus, have pseudoplastic fluid properties and their viscosity can be modulated by changing the hyaluronic acid concentration or chain size. In conclusion we improved the use of temporary fillings by adding an active antimicrobial agent and we developed an alternative to hypochlorite sodium with the use of poly-arginine/hyaluronic acid viscous hydrogel as a more biocompatible disinfectant.

B. García, B. Su, D. Pallarola. Fabrication of titanium oxide nanopatterns with potential bactericidal activity

Poster Presentation: V Jornadas de Jóvenes Bionanocientíficxs (JOBION). 3rd November, 2023. Buenos Aires (Argentina).

POSTER (PART) ➞

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N. Garcia-de-Albeniz, A. Ahmed, D.W. Müller, F. Mücklich, E. Jimenez-Piqué, C. Mas-Moruno. Micro and nano-scale topographies fine-tune cell and bacterial response on dental zirconia.

Oral Presentation: SIBB2023 - XLV Congreso de la Sociedad Ibérica de Biomecánica y Biomateriales. November 2-3rd, 2023, Barcelona (Spain).

ABSTRACT ➞

Introduction: Zirconia-based materials have become increasingly popular in the development of metal-free tooth-coloured dental implants due to their superior aesthetic appearance, high biocompatibility and outstanding mechanical properties. However, implant failure due to insufficient osseointegration or peri-implantitis still represents a great concern among the dental community. To overcome those biological complications, surface modification of zirconia has been explored in order to improve the implant-tissue interaction.

(...)

Conclusions: The combination of laser micropatterning and etching represents a powerful strategy to fine tune the biological response at the surface of 3Y-TZP for biomedical applications.

J. Werner, C. Mas-Moruno, D. Pallarola. Functionalization of ITO electrodes with cell adhesive and osteoimmunomodulatory peptides: towards studying in real time osteogenesis in the context of immune-mediated inflammation.

Oral Presentation: SIBB2023 - XLV Congreso de la Sociedad Ibérica de Biomecánica y Biomateriales. November 2-3rd, 2023, Barcelona (Spain).

ABSTRACT ➞

Introduction: The immune system plays a key role in bone regeneration. Excessive production of pro-inflammatory cytokines by immune cells induces bone resorption and affects osteogenesis, leading to implant failure. Therefore, developing coatings that modulate the inflammatory response to create a beneficial bone immune microenvironment that promotes osteogenesis is crucial. Furthermore, studying in real-time and non-invasively the interaction of cells with such coatings is essential to gain a detailed understanding of their effect on cellular behavior. In this regard, electrochemical impedance spectroscopy (EIS) stands out for its unique sensitivity in studying multiple cellular functions. Accordingly, with the final goal of studying osteogenesis on real time by EIS, we focused on developing novel multifunctional coatings combining the adhesive sequence RGD and the osteoimmunomodulatory peptide OGP10-14, their immobilization on indium tin oxide (ITO) surfaces, and a preliminary study of mesenchymal stem cells (MSCs) response on the functionalized surfaces.

(...)

Conclusions: Preliminary results indicate that it is possible to create a coating based on the RGD and OGP10-14 peptides capable of improving the interaction of MSCs with ITO surfaces. Our next studies will involve investigating the osteoimmunomodulatory and osteogenic potential of the coated electrodes by EIS. In this regard, studies focusing on the effect of the coatings on the response of macrophages are currently in progress.

③ PRIZE WINNER 

B. García, B. Su, D. Pallarola. Fabrication of titania nanopatterns with potential bactericidal activity. 

Poster Presentation: LatinXChem2023 Twitter Poster Conference.  October, 16-17th, 2023. Virtual Event.

TWEET ➞

Bacterial infections are a global health concern, particularly for implanted patients. This study propose the use of fingerprint-type titania nanostructures on the surface of titanium implants. These nanostructures were created by applying a thin film of PS-b-P2VP block copolymer, followed by solvent vapor annealing, anodization, and oxygen plasma treatment. A detailed topographical characterization by AFM and preliminary antimicrobial effectiveness testing is presented.

B. García, B. Su, D. Pallarola. Fabricación de nanopatrones de óxido de titanio con potencial actividad bactericida

Poster Presentation: XXII Encuentro de Superficies y Materiales Nanoestructurados (NANO 2023). September 6-8th , 2023. Bahía Blanca, Bs As (Argentina).

ABSTRACT ➞

Bacterial infections continue to be a problem for the world population today. For To minimize them, it is necessary to take precautions and care, especially if these are patients who require implants. For years we have worked on different alternatives that allow us to optimize the designs and materials used, in order to improve the integration of the implant. In this context, in this work proposes the manufacture of nanostructures with bactericidal or bacteriostatic activity to apply them in titanium implant parts[1] . These nanostructures form a pattern similar to that of a fingerprint. Its manufacturing consists of depositing on titanium a thin film of the block copolymer polystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP) [2] and subsequent “annealing” with acetone vapor in order to rearrange the blocks to the desired structure. The nanostructured polymer film then acts as a guide during an anodizing process giving lead to the growth of titanium oxide that copies the fingerprint pattern. Finally, a treatment with Oxygen plasma removes organic material. The dimensions of the nanopatterns could be determined using atomic force microscopy (AFM). For the PS block, a width of (87±11) nm was obtained, while for the P2VP block a width of (92±12) nm. After the anodizing process, it was determined that the TiO2 structures have a width of (30±4) nm and a height of (42±8) nm. Preliminary trials are currently underway using S. aureus to check bactericidal activity or adhesion inhibition capacity bacteria on these nanostructured surfaces.

Bo Su. Bio-inspired nanostructured
antimicrobial surfaces for 3D printed titanium implants.

Highlight Lecture:
FEMS EUROMAT 2023 Congress.September 3-7th, 2023. Hybrid Conference. Frankfurt am Mein (Germany) & Online. 

PROGRAMME ➞

N. Garcia-de-Albeniz, A. Ahmed, D.W. Müller, F. Mücklich, E. Jimenez-Piqué, C. Mas-Moruno. Enhancement of the biological properties of 3Y-TZP by topographical modification approaches.

Oral Presentation:
FEMS EUROMAT 2023 Congress.September 3-7th, 2023. Hybrid Conference. Frankfurt am Mein (Germany) & Online. 

ABSTRACT ➞

Zirconia-based materials have become increasingly popular in the development of metal-free tooth- coloured dental implants due to their superior aesthetic appearance, high biocompatibility and outstanding mechanical properties. However, implant failure due to insufficient osseointegration or peri-implantitis still represent a great concern among the dental community. To overcome those biological complications, surface modification of zirconia has been explored in order to improve the implant-tissue interaction. In this light, the presented work aims to investigate the effect of topographical modification strategies on the biological properties of 3Y-TZP, in terms of cell and bacterial response. Concretely, the surface of 3Y-TZP was modified by two different approaches: ultrashort pulsed direct laser interference patterning (USP-DLIP) and chemical etching treatment. On the one side, USP-DLIP utilizing a femtosecond pulsed laser source was used to introduce microscale line-like periodic surface patterns on the surface of 3Y-TZP. On the other side, hydrofluoric acid etching was performed to produce homogeneous sub microscale roughness. The resulting topography and surface damage were assessed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The microstructure and wettability changes were characterized by X-Ray diffraction (XRD) and contact angle technique, respectively. Furthermore, human mesenchymal stem cell (hMSC) response was evaluated in terms of cell adhesion, proliferation and differentiation. Finally, bacterial adhesion to the modified surfaces was investigated using Staphylococcus aureus and Pseudomonas aeruginosa strains. The topographical features obtained after the two surface modifications performed in this work significantly influenced the biological response. The periodic microgrooves generated by the laser treatment allowed hMSCs attachment and favoured cell alignment and migration, and induced bacteria confinement inside of the grooves. In the case of the chemical etching, cell adhesion, proliferation and differentiation were increased, while antibacterial properties are displayed. Thus, the combination of laser micropatterning and etching represents a powerful strategy to fine tune the biological response at the surface of 3Y-TZP for biomedical applications.

P. López-Gómez, L. Oliver-Cervelló, H. Martin-Gómez, M.P. Ginebra, C. Mas-Moruno. Alginate hydrogels with antimicrobial and cell adhesive properties for bone
tissue engineering.

Oral Presentation:
FEMS EUROMAT 2023 Congress.September 3-7th, 2023. Hybrid Conference. Frankfurt am Mein (Germany) & Online.

ABSTRACT ➞

Hydrogels with cell adhesive properties are a promising approach to promote bone tissue regeneration. Among the different polymers available to design hydrogels, alginate has been widely used, due to its high versatility and availability, biocompatibility, application to different fabrication processes and the ease in tuning its intrinsic properties. However, alginate is a bioinert material and thus, it does not naturally possess adhesive cues to promote cell attachment. In addition, bacterial colonization of biomaterials is also a problematic concern in the clinics. Indeed, bacterial infections and the possibility of biofilm formation are one of the major causes of implant failure, representing about 20% of failures. Thus, addressing bioactivity of hydrogels as well as inhibiting bacterial colonization are crucial aspects to ensure the success of an implant. To manage both issues, an alginate hydrogel modified with a multifunctional peptide containing the well-known RGD cell adhesive motif in combination with an antibacterial peptide derived from the 1-11 region of lactoferrin (LF) was developed. The RGD-LF branched peptide was successfully linked to the alginate by carbodiimide chemistry, as demonstrated by 1 H NMR and fluorescence experiments. The functionalized alginate hydrogel presented good physicochemical properties (porosity, swelling and rheological behavior). Moreover, biological characterization demonstrated the ability of the modified hydrogels to promote ALP activity of human bone marrow derived mesenchymal stem cells (hBMMSCs) as well as to decrease bacterial viability and the possible early colonization of the hydrogel in both Gram- positive and Gram-negative models. In summary, the functionalized hydrogels presented in this work, with the dual capacity to promote cell adhesion while preventing bacterial contamination, would have potential application in bone tissue engineering applications.

Bo Su. Nanostructured antimicrobial surfaces for medical implants.

Invited Talk: 31st International Materials Research Congress (IMRC2023). August 13-18, 2023. Cancún (Mexico).

ABSTRACT ➞

Cell-instructive surfaces that deter bacterial biofilm formation while encouraging host tissue integration are highly desirable for applications such as medical implants where there is often a competition between tissue cell growth and bacterial colonisation at their surfaces. It has been known that nanotopography is a potent modulator to both eukaryotic and prokaryotic cells under complex physiological conditions. Inspired by cicada and dragonfly wings whose surfaces have shown to exhibit bactericidal nanotopographies, we have produced biomimetic protruding nanostructures on titanium and polymer substrates using a range of facile fabrication techniques. In this talk, an overview of our recent development in the design of the nanostructured antimicrobial surfaces and the understanding of the mechano-bactericidal mechanisms will be provided. Advantages of nanotopography-induced bactericidal surfaces in overcoming the issue of growing antimicrobial resistance will be highlighted. The translation of the nanostructured surfaces to 3D printed titanium scaffolds/implants will be discussed regarding the improved mechanical match (to minimise the stress shielding) and enhanced cell infiltration (to facilitate the tissue integration). Finally, the effect of enzyme functionalised nanostructures on antibiofilm performance and stem cells responses of 3D printed titanium implants will be presented to demonstrate the potential of such cell-instructive surfaces for implant application.

M.P. Ginebra. Bioinspired calcium phosphates as osteoinductive biomaterials.

Highlight Lecture:
Materials Science and Engineering Congress (MSE). September 27-29th, 2022. Hybrid conference.  Darmstadt (Germany) & Online.

ABSTRACT ➞

Bioinspiration is a powerful approach when designing functional biomaterials. Synthetic bone substitutes have been used for more than 40 years in the clinics. However, after decades of research, they have not been able to outperform autografts, which are still the gold standard. Recently, novel biomimetic routes have opened unprecedented possibilities in terms of tuning the nanostructure and composition of hydroxyapatite, imitating more closely the mineral phase of bone. Moreover, tuning the size, shape and composition of hydroxyapatite nanocrystals has proven to be an efficient way to promote the osteoinductive and osteogenic properties of the material.

V. Jayawarna, M. Dalby, M. Salmeron-Sanchez. Acrylate plasma coated 3D printed polycaprolactone scaffolds for bone regeneration

Oral Presentation:
Materials Science and Engineering Congress (MSE). September 27-29th, 2022. Hybrid conference.  Darmstadt (Germany) & Online.

ABSTRACT ➞

Three dimensional (3D) porous scaffolds with favourable osteogenic ability and mechanical properties are promising candidates for bone repair and regeneration. Scaffolds geometry, porosity and topography in combination with chemical composition and surface coating are known to influence cell attachment, proliferation, differentiation and subsequently osteointegration of biomaterials. Taken this evidence base into account, the aim of our study is to evaluate the bioactivity of a novel 3D printed polycaprolactone (PCL) scaffold with nanometer thick polymer coating designed for use as a bone implants to heal the mind blast injury. Here we used an inductively coupled plasma system to modify 3D scaffold with thin coatings of poly (ethyl acrylate) (PEA). Our lab has previously shown the outstanding functional properties of PEA, that induce a fibrillar conformation of fibronectin (FN) adsorbed on its surface, biomimetically exposing its integrin and growth factor-binding domains, and in turn allowing an efficient and synergistic presentation of growth factors in vitro and in vivo (1, 2). The efficiency of plasma polymerisation as well as FN adsorption and interaction with bone morphogenetic protein-2 (BMP-2) on PEA coated PCL scaffolds were evaluated. We have then studied the in vitro ability of PEA coated PCL scaffolds to facilitate cell proliferation and osteoblast differentiation when the scaffolds were subjected to varying scaffolds pore geometry accomplished by manipulating the advancing angle between printed layers. Alkaline phosphatase assays, PCR analysis, immunofluorescence and calcium mineralisation studies revealed that the PCL scaffolds coated with PEA, FN and BMP-2 enhanced the osteogenic differentiation of hMSCs. Thereafter, the chorioallantoic membrane (CAM) assay confirmed biocompatibility of the PCL material, PEA coating and growth factor coating, and the ability of the coatings to support blood vessel formation in a dynamic biological environment. In addition, the coated scaffolds were subcutaneously implanted into rats to assess early-stage osteogenesis and vascularization. We propose that our functionalised 3D scaffolds exhibit potential technique for bone regenerative therapies.

Ll. Oliver-Cervelló, H. Martin-Gómez, N. Mandakhbayar, Y.W. Jo, E.A. Cavalcanti-Adam, H.W. Kim, M.P. Ginebra, J.H. Lee, C. Mas-Moruno. Recreating bone extracellular matrix on titanium substrates by multifunctional biomimetic peptide coatings.

Oral Presentation:
Materials Science and Engineering Congress (MSE). September 27-29th, 2022. Hybrid conference.  Darmstadt (Germany) & Online.

ABSTRACT ➞

The non-bioactivity of titanium can be improved by installing biomolecules on titanium surface. In this regard, mimicking bone extracellular matrix (EMC) can be possible by reproducing integrin and growth factor (GF) signaling. In this work, multifunctional biomimetic peptides containing the well-known RGD motif (or its cyclic analog, cRGD) and a BMP-2-derived peptide (DWIVA or cDWIVA) in a chemically and spatially defined manner were engineered. Such peptides were further used to coat titanium substrates. The successful functionalization of the substrates was assessed by different physicochemical methods. Then, the biological performance of the multifunctional-modified titanium was evaluated. In this regard, the biomimetic peptides were able to enhance MSCs adhesion and osteogenic differentiation in comparison to controls. Moreover, such biomimetic approach was validated in vivo in a model of rat calvarial defect, promoting an improvement of new bone growth and reduced formation of fibrous tissue on functionalized titanium implants.

C. Mas-Moruno, Ll. Oliver-Cervelló, H. Martín-Gómez. Chemical tools to functionalize biomaterials with multifunctional bioactive peptides

Poster Presentation:
36th European Peptide Symposium & 12th International Peptide Symposium. August 28th - September 2, 2022. Sitges (Spain).

ABSTRACT ➞

Recreating the healing microenvironment is a major goal in tissue engineering. To this end, the functionalization of biomaterials with cell instructive molecules from the extracellular matrix stands out as a powerful strategy to regulate the growth and regeneration of tissue.

In this poster, the design and synthesis of a biomimetic multifunctional peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif, derived from bone morphogenetic protein (BMP)-2, is discussed. This approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors, and has been shown on model substrates to improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. In subsequent studies, conformationally constrained analogues have been designed to better mimic the structural requirements needed for achieving optimal integrin-growth factor signaling.

To functionalize clinically relevant materials with this type of molecules, the development of a novel click chemistry-based approach is also discussed. In detail, this methodology is based on the solid-phase conjugation of specific anchoring units onto resin-bound azido-functionalized peptides by using click chemistry. Such a chemical ‘toolkit’ has been used to coat biomaterials of different nature with the RGD/DWIVA peptide, showcasing the efficiency and versatility of this new approach.

A.E. Sanchez, V. Barrenechea, K. Peñaranda, J.A. Nakamoto, M. Vargas-Reyes, L. Amado , A. Giuliodori, A. Fabbretti, P. Milon. Complementary mechanisms of ribosome inhibition by known antibiotics.

Oral Presentation:
Ribosome Structure & Function (Ribosomes 22). 10-14th July. 2022. Bourdeaux (France).

ABSTRACT ➞

Many commercial and clinically relevant antibiotics target the bacterial ribosome, and a subset of these exert their inhibitory function by binding and functionally compromising the small 30S ribosomal subunit. Albeit a plethora of biochemical, structural, and genetic data is available, complementary mechanisms of translation inhibition appear to have transited unnoticed. This work uses FRET-based rapid kinetics coupled with computational modelling to study how antibiotics that bind the 30S subunit perturb the binding and conformational landscapes of bacterial translation initiation complexes (IC) and their intermediates. Our data shows that Tetracyclines promote compaction of IF3 and result in slower transitions to translation. Particularly, Tigecycline appears to hold IF1 in the resulting 70S IC by direct interactions with the factor. Whether the increased IF1 occupation in the A-site contributes to the known mechanisms of translation elongation inhibition in the cell remains to be studied. On the contrary, Kanamycin and Streptomycin promote an extended IF3 configuration, resulting in higher dissociation rates of the factor and 70S formation for cognate and non-cognate initiation codons. Remarkably, Streptomycin drastically reduces the affinity of IF1 for early 30S initiation complex; however, late 30S complexes remain capable of retaining the factor. Albeit IF1 and Streptomycin occupy the A-site, structural modelling of the 30S IC shows that they do not share interaction points with the ribosome, suggesting that the antibiotic enhances 70S formation by long-range conformational changes on the 30S, likely perturbing the IF1/IF3-dependent molecular network. In the presented work, the Milon Lab will discuss the findings mentioned above and more recent observations for other 30S antibiotics, altogether suggesting complementary inhibitory mechanisms that could be exploited to develop and/or screen new antibiotics. Among these, the development of aptamers against the 30S IC will be shown and discussed.

Alicia Arica, Roberto Alcántara, Pohl Milón, Miguel Quiliano. Identification of Ribosome Inhibitors using Massive Pharmacophore-Based Screening and Cloud Computing.

Poster Presentation:
Ribosome Structure & Function (Ribosomes 22). 10-14th July. 2022. Bourdeaux (France).

ABSTRACT ➞

Antibiotic-resistant bacteria are a global public health concern. Hence, there is a significant necessity to identify new antibiotics. Iboxamycin is a broad-spectrum antimicrobial compound that is orally bioavailable, safe, and effective in treating both Gram-positive and Gram-negative bacterial infections. The antibiotic targets the 50S subunit showing a mechanism of action similar to Lincomycin and Clindamycin. In the present study, we set up a cloud-based high-throughput screening platform that uses a structure-based pharmacophore model based on Iboxamycin scaffold. Our platform can screen the ChEMBL28 library of 2,240,223 molecules in less than six hours. The screening led to 827 candidates with the potential to bind at the Iboxamycin binding site. Deeper analysis of the matching features reduced this number to 49 candidates, which were further subjected to molecular docking analysis. The calculated binding affinities and their structural features are being analyzed considering: i) Hydrogen bond interactions with A2058, G2505, m2A2503, A2059, G2061; and ii) surface complementarity. The final candidates will be validated using fully reconstituted in vitro translation, rapid kinetics, and in vivo experiments. Altogether, the combination of pharmacophore-based screening and cloud computing provides an efficient and scalable platform for identifying new compounds targeting the bacterial ribosome.

Katherin Peñaranda, Ana Sanchez-Castro, Nicolle Pereira, Lesia Tello, Wilfredo Evangelista, Rebecca Corrigan, Pohl Milon. Aptamer-mediated blocking of translational factors.

Poster Presentation:
Ribosome Structure & Function (Ribosomes 22). 10-14th July. 2022. Bourdeaux (France).

ABSTRACT ➞

Translation of the mRNA requires the coordinated action of several protein factors at different ribosomal sites and times. During ribosome biogenesis and translation initiation, Ribosome Associated-GTPases (RA-GTPases) and translation initiation factors (IFs) ensure both the correct ribosome assembly and decoding of the initiation codon, respectively. The complexity of the system is further increased since many translational factors are composed of multiple domains. Here, we develop aptamers for RA-GTPases of S. aureus and for initiation factors IF2 and IF3 of E. coti, aiming for factor or domain-specific blocking of their functions. Next-generation sequencing coupled with molecular modelling allowed us to identify over 25 aptamer candidates. Aptamer-target complexes were built using molecular docking and examined using molecular dynamics (MD) simulatms. MD trajectories allowed us to identify the most stable complexes. Based on available 3D ribosomal structures, aptamer-mediated inhibition was postulated and further studied by FRET coupled with pre-steady state kinetic and biochemical assays. Aptamers against the N terminal domain of IF3 showed over 100-fold reduction in the binding kinetics to the 30S subunit, delayed 30S 1C formation, and did not affect 70S 1C formation. On the Other hand, an aptamer localized between the G3 and CI domains of IF2 resulted in poorly 70S 1C formation. Aptamers against RA-GTPases are currently being biochernically tested. Altogether. the presented results support the use of aptamers to study factor- or domain-specific functions in nulti-ligand and complex systems as the translational apparatus.

E. Lebaudy, P. Lavalle.  Polyethylene glycol diacry-late/poly epsilon L-lysine hydrogels for wound healing applications.

Oral Presentation:
NANOinBIO 2022 Conference. 30th May - 5th June, Le Gosier, Guadeloupe (French Caribbean).

ABSTRACT ➞

Health-care associated infections (HAIs) are big issues in medical science. They represent nowadays 6% of hospital patients in Europe. One of the major causes of those diseases are the use of medical devices (MDs)1. Moreover, the increase of diabetic ulcers shows the importance of HAIs issues. The administration of antibiotics is a conventional method to fight against bacteria but this solution is no more viable in long run because of the emergence of bacteria resistance to antibiotics. Thus, new solutions need to be found in biomaterials science to decrease the infection risks. Read more (page 45).

X. Liu. Bacterial surface appendages modulate physical killing induced by nanoflake-coated titanium.

Poster Presentation:
8th Thesinge Biofilm Conference 2022, May 23-24th, 2022, Groningen (The Netherlands).

PROGRAMME ➞

Ph. Lavalle. Homopolypeptides as building blocks for supramolecular coatings and hydrogels with antimicrobial properties.

Workshop:
Pre-clinical development of antimicrobial peptides International Workshop and Symposium. 13th May 2022. Strasbourg (France).

BANNER ➞

E. Ada Cavalcanti-Adam. Local control of receptor-mediated adhesion for the regulation of cellular and molecular forces. 

Force Talk:
King's College London. 4th May 2022. Online Conference. 

ABSTRACT ➞

Mechanical and chemical properties of the extracellular environment regulate cell adhesion and migration. Designing materials and tools which allow the local control of cell-matrix and cell-cell adhesion is of great interest to address how specific receptor-ligand interactions affect cell mechanotransduction. I will present the development of surface functionalisation strategies to control integrin clustering for the generation of molecular and cellular forces during adhesion. Such surface functionalisation strategies applied to hydrogels also allow to elucidate the crosstalk between integrins and growth factors in the regulation of cell adhesion and mechanotransduction signaling. In the last part of my presentation, I will introduce the application of opto-chemical and DNA-based tools to control the assembly and disassembly of E-cadherin-mediated cell-cell junctions and the impact on collective cell adhesion dynamics and forces.

S. Hawa Ngalim, R. Nath, E. Ada Cavalcanti-Adam. Understanding C2C12 cells' mad signaling upon presentation of BMP 2/7 chimera or individual BMPs in a temporal or spacial controlled system. 

Oral Presentation:
6th world congress of the Tissue Engineering & Regenerative Medicine Internat. Society (TERMIS 2021). Online Conference.

ABSTRACT ➞

C2C12 cell line (mouse myoblast cell line) that differentiates into myotubes (muscle cell) upon confluency or transdifferentiates into osteoblasts (bone cells) upon Bone Morphogenetic (BMP) 2 induction. Such characteristic has made C2C12 a stem cell-like cell line that is ideal for myotubes/osteoblast research model. Current literature shows BMP2 and BMP7 heterodimers are present and important in the early mouse embryo and Xenopus embryo development (1-2). It is not yet clear how spatially controlled BMP2/7 chimera presentation, in soluble and immobilised forms, influence C2C12 cells morphology and cell signalling any differently. As a start, we compare Smad 1/5/9 signalling activity when C2C12 cells were presented to a soluble form of the BMP: sBMP2/7 chimera, sBMP2 or sBMP7 alone. Only 7.5nM BMP2 and BMP2/7 chimera while 22.5nM BMP7 were needed to exert significant differences (**P < 0.005) in Smad 1/5/9 activities with their respective negative control within 45 mins. Next, diblockcopolymer micelle nanolithography (BCMN) technique was used to generate spatially controlled of approximately 8 nm gold nanoparticles and they were biofunctionalised after. Two different types of spacings were generated: 49.00 +/- 1.95 nm with an order of 0.5, and 95.82 +/- 0.92 nm with an order of 0.70. Quantitation of BMP immobilisation via mu N-hydroxysuccinamide reaction was done on gold monolayer of quartz crystal microbalance – dissipation (QCM-D). Next, C2C12 cells’ morphology on the immobilised BMPs, Smad 1/5/9 expressions and involvement of certain integrin subunits will be tested.

Ll. Oliver-Cervelló, H. Martin-Gomez, M. P. Gine-bra, C. Mas-Moruno. The power of peptides to mimic bone extracellular matrix: development of a multifunctional engineered scaffold. 

Oral Presentation:
World Biomaterials Congress (WBC 2020). Dec 11th–15th, 2020. Online Conference.

ABSTRACT ➞

Mimicking bone extracellular matrix (ECM) on biomaterials is essential to regulate cell-material interactions. This may be achieved reproducing integrin and growth factor (GF) signaling. To avoid the clinical risks associated with the use of bone morphogenetic protein 2 (BMP-2), synthetic osteogenic domains may be an alternative way to promote integrin-GF signaling. Herein, we have thus identified sequences derived from the wrist and knuckle epitopes of BMP-2 and evaluated their osteogenic potential. Subsequently, the most active sequences have been combined with integrin-binding RGD peptides within an engineered biomimetic multifunctional scaffold, aiming at exploiting integrin and GF signaling. Such scaffold has been further used to functionalize titanium surfaces to recreate bone ECM. The feasibility of this strategy has been corroborated by means of cell adhesion and osteodifferentiation assays with mesenchymal stem cells (MSCs).

H. Martin-Gomez, I. Sánchez, Ll. Oliver-Cervelló, V. Marchán, C. Mas-Moruno. A click-based chemical toolkit to functionalize biomaterials. 

Oral Presentation:
World Biomaterials Congress (WBC 2020). Dec 11th–15th, 2020. Online Conference.

ABSTRACT ➞

In biomaterials science, peptides and peptidomimetics are widely used to functionalize material surfaces in order to confer bioactivity to otherwise inert substrates. The attachment of biomolecules to biomaterials is commonly achieved using specific anchors with chemical affinity for the substrates. Well-known examples include the use of thiols to bind gold substrates or catechol groups to bind titanium and other metallic oxides. However, this implies that each synthesized peptide can be used only for a narrow range of materials. Thus, in the majority of cases, changing the material of study requires synthesizing the same peptide with a distinct anchor, resulting in time-consuming and repetitive procedures. To solve this, here we report on the development of a novel and versatile click-based solid-phase synthetic strategy to prepare peptidic coatings suitable for functionalizing a variety of biomaterials (a chemical toolkit). In detail, we focused on the synthesis of a model peptide and the optimization of the copper-catalysed azide-alkyne cycloaddition reaction to introduce to the peptidic backbone distinct anchoring groups in an efficient and modular fashion. Subsequently, model substrates of different nature commonly used as biomaterials were functionalized with the peptidic toolkit by simply selecting the appropriate anchor. The feasibility of this method was validated by physicochemical surface characterization and cellular assays.

E. Ada Cavalcanti-Adam. Surface control of adhesion and growth factor mecha-nical and chemical cross-talk.

Keynote Lecture:
13th IBEC Symposium. Online conference: 28th October, 2020

ABSTRACT ➞

Mechanical and chemical cues present in the extracellular environment regulate cell adhesion- mediated responses, such as migration, proliferation and differentiation. To address how local changes in the extracellular environment regulate cell responses through specific receptor-ligand interactions material surfaces can be designed to combine adhesive ligands and growth factors at the nanoscale. I will present surface functionalization strategies to control integrin clustering and cellular adhesion forces. The nanoscale presentation of integrin ligands is also combined with growth factors, namely bone morphogenetic proteins (BMPs), to modulate cell osteodifferentiation. In this talk, I will also discuss the synergistic effect of BMPs and mechanical cues on osteogenic signaling and mechanotransduction, elucidating the interdependency of Smad 1/5 and YAP/TAZ signaling.

Lluis Oliver-Cervelló, H. Martin-Gómez, F. Nouredi-nne, E.A. Cavalcanti-Adam, M.P. Ginebra, C. Mas-Moruno. . The Capacity of RGD and DWIVA Peptidic Biointerface to Transdiffe-renciate C2C12 Myoblasts into Osteogenic Lineage.

Flash Presentation:
13th IBEC Symposium. Online conference: 28th October, 2020

ABSTRACT ➞

Recreating the healing microenvironment of bone tissue is particularly important to ensure the success of biomaterials. In this regard, surface biofunctionalization with integrin-binding molecules combined with growth factor derived sequences is an interesting approach. Herein, a multifunctional peptidic biointerface containing the RGD cell adhesive motif together with the DWIVA peptide (derived from the wrist epitope of BMP-2), was used to study the synergistic promotion of integrin/GF signaling on C2C12 myoblasts. Biological characterization with C2C12 myoblasts showed the potential of this biointerface to promote cell adhesion (increasing the number of attached cells and their projected area), transdifferentiation to osteogenic lineage (by inhibiting myotube formation) and BMP-2 dependent signaling through Smad independent pathway (as shown by the high expression of p38).

Nihal Engin Vrana. Personalised, multifunctio-nal coatings for medical devices against infections and adverse immune reactions.

Keynote Lecture:
Materials Science and Engineering Conference. 22-25th Sept. 2020. Originally in Darmstadt (Germany), held virtually.

ABSTRACT ➞

The personalized medicine approach has already paid dividends in patient-specific chemotherapy and other pharmaceutical applications. In the case of medical devices, the personalization is mainly based on the anatomical fit of the implants. However, the personalization of the implant interface to control better the host response to the implanted materials and also to diminish the risks such as infection is only recently come to be considered. Another important aspect is the ability to resolve several problems using only one coating. In this context, we have been developing multi-component coatings based on supramolecular assemblies where each component brings in an inherent biological activity while contributing to the physicochemical properties of the coating. As such assemblies can be produced by iterative processes, the coatings can be modified with respect to the patients' immunoprofiles. In this talk, a personalized coating development workflow including immunoprofiling and coating optimization using polypeptides and polysaccharides will be presented.

Judit Buxadera-Palomero, H. Martin-Gómez, Ll. Oliver-Cervelló, M.P. Ginebra, C. Mas-Moruno. Design of multifunctional peptide plat-forms with antimicrobial and cell adhesive properties.

Oral Presentation:
Materials Science and Engineering Conference. 22-25th Sept. 2020. Originally in Darmstadt (Germany), held virtually.

ABSTRACT ➞

Implant failure due to bacterial infection and incomplete integration represents a major issue in the fields of orthopedics and dentistry. To overcome this problem, in this study we present the design and development of peptidic platforms that simultaneously reduce bacterial adhesion while enhancing eukaryotic cell adhesion. Such dual activity is achieved by combining the human lactoferrin 1-11 peptide (Lf1-11) with the cell binding motif RGD within a branched molecular scaffold. Furthermore, the peptides contain a catechol group as anchoring unit and poly(ethylene glycol) (PEG) spacers. Different spacer lengths were studied, in order to assess the correlation between the conformation and accessibility of the peptides and the biological response. While the anchoring motif provides a single step immobilization on the titanium surface, the versatility of the synthetic routes allowed for the study of different molecular configurations. The peptidic platforms significantly improved the adhesion of human mesenchymal stem cells (MSCs) compared to control titanium, but similar values of cell adhesion were observed among all the studied peptides. Interestingly, cell spreading was higher on the surfaces coated with the peptides with a shorter spacer, indicating that above a certain level of length and flexibility of the spacer, the availability of the peptide for cell receptors is compromised. Read more...