Resorbable engineered barrier membranes for oral surgery applications.

Population aging, reduced economic capacity, and neglecting the treatments for oral pathologies, are the main causal factors for about 3 billion individuals who are suffering from partial/total edentulism or alveolar bone resorption: thus, the demand for dental implants is increasingly growing. To achieve a good prognosis for implant-supported restorations, adequate peri-implant bone volume is mandatory. The Guided Bone Regeneration (GBR) technique is one of the most applied methods for alveolar bone reconstruction and treatment of peri-implant bone deficiencies. This technique involves the use of different types of membranes in association with some bone substitutes (autologous, homologous, or heterologous). However, time for bone regeneration is often too long and the bone quality is not simply predictable. This study aims at engineering and evaluating the efficacy of modified barrier membranes, enhancing their bioactivity for improved alveolar bone tissue regeneration. We investigated membranes functionalized with chitosan (CS) and chitosan combined with the peptide GBMP1α (CS + GBMP1α), to improve bone growth. OsseoGuard® membranes, derived from bovine Achilles tendon type I collagen crosslinked with formaldehyde, were modified using CS and CS + GBMP1α. The functionalization, carried out with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide and sulfo-N-Hydroxysuccinimide (EDC/sulfo-NHS), was assessed through FT-IR and XPS analyses. Biological assays were performed by directly seeding human osteoblasts onto the materials to assess cell proliferation, mineralization, gene expression of Secreted Phosphoprotein 1 (SPP1) and Runt-Related Transcription Factor 2 (Runx2), and antibacterial properties. Both CS and CS + GBMP1α functionalizations significantly enhanced human osteoblast proliferation, mineralization, gene expression, and antibacterial activity compared to commercial membranes. The CS + GBMP1α functionalization exhibited superior outcomes in all biological assays. Mechanical tests showed no significant alterations of membrane biomechanical properties post-functionalization. The engineered membranes, especially those functionalized with CS + GBMP1α, are suitable for GBR applications thanks to their ability to enhance osteoblast activity and promote bone tissue regeneration. These findings suggest a potential advancement in the treatment of oral cavity problems requiring bone regeneration.

Electroporation enhances cell death in 3D scaffold-based MDA-MB-231 cells treated with metformin.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer lacks estrogen, progesterone, and HER2 receptors and hence, is therapeutically challenging. Towards this, we studied an alternate therapy by repurposing metformin (FDA-approved type-2 diabetic drug with anticancer properties) in a 3D-scaffold culture, with electrical pulses. 3D cell culture was used to simulate the tumor microenvironment more closely and MDA-MB-231, human TNBC cells, treated with both 5 mM metformin (Met) and 8 electrical pulses at 2500 V/cm, 10 µs (EP1) and 800 V/cm, 100 µs (EP2) at 1 Hz were studied in 3D and 2D. They were characterized using cell viability, reactive oxygen species (ROS), glucose uptake, and lactate production assays at 24 h. Cell viability, as low as 20 % was obtained with EP1 + 5 mM Met. They exhibited 1.65-fold lower cell viability than 2D with EP1 + 5 mM Met. ROS levels indicated a 2-fold increase in oxidative stress for EP1 + 5 mM Met, while the glucose uptake was limited to only 9 %. No significant change in the lactate production indicated glycolytic arrest and a non-conducive environment for MDA-MB-231 growth. Our results indicate that 3D cell culture, with a more realistic tumor environment that enhances cell death using metformin and electrical pulses could be a promising approach for TNBC therapeutic intervention studies.

Development of 3D melanoma cultures on a hyaluronic acid-based scaffold with synthetic self-assembling peptides: Electroporation enhancement.

Electrochemotherapy (ECT) with bleomycin is an effective antitumor treatment. Still, researchers are investigating new drugs and electroporation conditions to improve its efficacy. To this aim, in vivo assays are accurate but expensive and ethically questionable. Conversely, in vitro assays, although cheaper and straightforward, do not reflect the architecture of the biological tissue because they lack a tridimensional (3D) structure (as in the case of two-dimensional [2D] in vitro assays) or do not include all the extracellular matrix components (as in the case of 3D in vitro scaffolds). To address this issue, 3D in vitro models have been proposed, including spheroids and hydrogel-based cultures, which require a suitable low-conductive medium to allow cell membrane electroporation. In this study, a synthetic scaffold based on hyaluronic acid (HA) and self-assembling peptides (SAPs; EAbuK), condensed with a Laminin-derived adhesive sequence (IKVAV), is proposed as a reliable alternative. We compare SKMEL28 cells cultured in the HA-EAbuK-IKVAV scaffold to the control (HA only scaffold). Three days after seeding, the culture on the HA-EAbuK-IKVAV scaffold showed collagen production. SKMEL28 cells cultured on the HA-EAbuK-IKVAV scaffold started to be electroporated at 400 V/cm, whereas, at the same electric field intensity, those cultured on HA were not. As a reference, 2D experiments showed that electroporation of SKMEL28 cells starts at 600 V/cm using an electroporation buffer and at 800 V/cm in a culture medium, but with very low efficiency (<50 % of cells electroporated). 3D cultures on HA-EAbuK-IKVAV allowed the simulation of a more reliable microenvironment and may represent a valuable tool for studying electroporation conditions. Using Finite Element Analysis (FEA) to compute the transmembrane potential, we detected the influence of inhomogeneity of the extracellular matrix on electroporation effect. Our 3D cell culture electroporation simulations showed that the transmembrane potential increased when collagen surrounded the cells. Of note, in the collagen-enriched HA-EAbuK-IKVAV scaffold, EP was already improved at lower electric field intensities. This study shows the influence of the extracellular matrix on electric conductivity and electric field distribution on cell membrane electroporation and supports the adoption of more reliable 3D scaffolds in experimental electroporation studies.

Covalently Grafted Peptides to Decellularized Pericardium: Modulation of Surface Density.

The covalent functionalization of synthetic peptides allows the modification of different biomaterials (metallic, polymeric, and ceramic), which are enriched with biologically active sequences to guide cell behavior. Recently, this strategy has also been applied to decellularized biological matrices. In this study, the covalent anchorage of a synthetic peptide (REDV) to a pericardial matrix decellularized via Schiff base is realized starting from concentrated peptide solutions (10-4 M and 10-3 M). The use of a labeled peptide demonstrated that as the concentration of the working solution increased, the surface density of the anchored peptide increased as well. These data are essential to pinpointing the concentration window in which the peptide promotes the desired cellular activity. The matrices were extensively characterized by Water Contact Angle (WCA) analysis, Differential Scanning Calorimetry (DSC) analysis, geometric feature evaluation, biomechanical tests, and preliminary in vitro bioassays.

Bioactive PEEK: Surface Enrichment of Vitronectin-Derived Adhesive Peptides.

Polyetheretherketone (PEEK) is a thermoplastic polymer that has been recently employed for bone tissue engineering as a result of its biocompatibility and mechanical properties being comparable to human bone. PEEK, however, is a bio-inert material and, when implanted, does not interact with the host tissues, resulting in poor integration. In this work, the surfaces of 3D-printed PEEK disks were functionalized with: (i) an adhesive peptide reproducing [351-359] h-Vitronectin sequence (HVP) and (ii) HVP retro-inverted dimer (D2HVP), that combines the bioactivity of the native sequence (HVP) with the stability toward proteolytic degradation. Both sequences were designed to be anchored to the polymer surface through specific covalent bonds via oxime chemistry. All functionalized PEEK samples were characterized by Water Contact Angle (WCA) measurements, Atomic Force Microscopy (AFM), and X-ray Photoelectron Spectroscopy (XPS) to confirm the peptide enrichment. The biological results showed that both peptides were able to increase cell proliferation at 3 and 21 days. D2HVP functionalized PEEK resulted in an enhanced proliferation across all time points investigated with higher calcium deposition and more elongated cell morphology.

Proprotein convertases regulate trafficking and maturation of key proteins within the secretory pathway.

Proprotein Convertases (PCs) are serine endoproteases that regulate the homeostasis of protein substrates in the cell. The PCs family counts 9 members-PC1/3, PC2, PC4, PACE4, PC5/6, PC7, Furin, SKI-1/S1P, and PCSK9. The first seven PCs are known as Basic Proprotein Convertases due to their propensity to cleave after polybasic clusters. SKI-1/S1P requires the additional presence of hydrophobic residues for processing, whereas PCSK9 is catalytically dead after autoactivation and exerts its functions using mechanisms alternative to direct cleavage. All PCs traffic through the canonical secretory pathway, reaching different compartments where the various substrates reside. Despite PCs members do not share the same subcellular localization, most of the cellular organelles count one or more Proprotein Convertases, including ER, Golgi stack, endosomes, secretory granules, and plasma membranes. The widespread expression of these enzymes at the systemic level speaks for their importance in the homeostasis of a large number of biological functions. Among others, PCs cleave precursors of hormones and growth factors and activate receptors and transcription factors. Notably, dysregulation of the enzymatic activity of Proprotein Convertases is associated to major human pathologies, such as cardiovascular diseases, cancer, diabetes, infections, inflammation, autoimmunity diseases, and Parkinson. In the current COVID-19 pandemic, Furin has further attracted the attention as a key player for conferring high pathogenicity to SARS-CoV-2. Here, we review the Proprotein Convertases family and their most important substrates along the secretory pathway. Knowledge about the complex functions of PCs is important to identify potential drug strategies targeting this class of enzymes.

Selective Grafting of Protease-Resistant Adhesive Peptides on Titanium Surfaces.

In orthopedic, dental, and maxillofacial fields, joint prostheses, plates, and screws are widely used in the treatment of problems related to bone tissue. However, the use of these prosthetic systems is not free from complications: the fibrotic encapsulation of endosseous implants often prevents optimal integration of the prostheses with the surrounding bone. To overcome these issues, biomimetic titanium implants have been developed where synthetic peptides have been selectively grafted on titanium surfaces via Schiff base formation. We used the retro-inverted sequence (DHVPX) from [351-359] human Vitronectin and its dimer (D2HVP). Both protease-resistant peptides showed increased human osteoblast adhesion and proliferation, an augmented number of focal adhesions, and cellular spreading with respect to the control. D2HVP-grafted samples significantly enhance Secreted Phosphoprotein 1, Integrin Binding Sialoprotein, and Vitronectin gene expression vs. control. An estimation of peptide surface density was determined by Two-photon microscopy analysis on a silanized glass model surface labeled with a fluorescent analog.

DAR 16-II Primes Endothelial Cells for Angiogenesis Improving Bone Ingrowth in 3D-Printed BCP Scaffolds and Regeneration of Critically Sized Bone Defects.

Bone is a highly vascularized tissue and relies on the angiogenesis and response of cells in the immediate environmental niche at the defect site for regeneration. Hence, the ability to control angiogenesis and cellular responses during osteogenesis has important implications in tissue-engineered strategies. Self-assembling ionic-complementary peptides have received much interest as they mimic the natural extracellular matrix. Three-dimensional (3D)-printed biphasic calcium phosphate (BCP) scaffolds coated with self-assembling DAR 16-II peptide provide a support template with the ability to recruit and enhance the adhesion of cells. In vitro studies demonstrated prompt the adhesion of both human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC), favoring endothelial cell activation toward an angiogenic phenotype. The SEM-EDS and protein micro bicinchoninic acid (BCA) assays demonstrated the efficacy of the coating. Whole proteomic analysis of DAR 16-II-treated HUVECs demonstrated the upregulation of proteins involved in cell adhesion (HABP2), migration (AMOTL1), cytoskeletal re-arrangement (SHC1, TMOD2), immuno-modulation (AMBP, MIF), and morphogenesis (COL4A1). In vivo studies using DAR-16-II-coated scaffolds provided an architectural template, promoting cell colonization, osteogenesis, and angiogenesis. In conclusion, DAR 16-II acts as a proactive angiogenic factor when adsorbed onto BCP scaffolds and provides a simple and effective functionalization step to facilitate the translation of tailored 3D-printed BCP scaffolds for clinical applications.

Development of Two-Layer Hybrid Scaffolds Based on Oxidized Polyvinyl Alcohol and Bioactivated Chitosan Sponges for Tissue Engineering Purposes.

Oxidized polyvinyl alcohol (OxPVA) is a new polymer for the fabrication of nerve conduits (NCs). Looking for OxPVA device optimization and coupling it with a natural sheath may boost bioactivity. Thus, OxPVA/chitosan sponges (ChS) as hybrid scaffolds were investigated to predict in the vivo behaviour of two-layered NCs. To encourage interaction with cells, ChS were functionalized with the self-assembling-peptide (SAP) EAK, without/with the laminin-derived sequences -IKVAV/-YIGSR. Thus, ChS and the hybrid scaffolds were characterized for mechanical properties, ultrastructure (Scanning Electron Microscopy, SEM), bioactivity, and biocompatibility. Regarding mechanical analysis, the peptide-free ChS showed the highest values of compressive modulus and maximum stress. However, among +EAK groups, ChS+EAK showed a significantly higher maximum stress than that found for ChS+EAK-IKVAV and ChS+EAK-YIGSR. Considering ultrastructure, microporous interconnections were tighter in both the OxPVA/ChS and +EAK groups than in the others; all the scaffolds induced SH-SY5Y cells' adhesion/proliferation, with significant differences from day 7 and a higher total cell number for OxPVA/ChS+EAK scaffolds, in accordance with SEM. The scaffolds elicited only a slight inflammation after 14 days of subcutaneous implantation in Balb/c mice, proving biocompatibility. ChS porosity, EAK 3D features and neuro-friendly attitude (shared with IKVAV/YIGSR motifs) may confer to OxPVA certain bioactivity, laying the basis for future appealing NCs.

Biofunctionalization of bioactive ceramic scaffolds to increase the cell response for bone regeneration.

Biofunctionalization was investigated for polymers and metals considering their scarce integration ability. On the contrary few studies dealt with ceramic biofunctionalization because the bioactive and bioresorbable surfaces of ceramics are able to positively interact with biological environment. In this study the cell-response improvement on biofunctionalized wollastonite and diopside-based scaffolds was demonstrated. The ceramics were first obtained by heat treatment of a silicone embedding reactive oxide fillers and then biofunctionalized with adhesive peptides mapped on vitronectin. The most promisingin vitroresults, in terms of h-osteoblast proliferation and bone-related gene expression, were reached anchoring selectively a peptide stable toward proteolytic degradation induced by serum-enriched medium. Inin vivoassays the anchoring of this protease-stable adhesive peptide was combined with self-assembling peptides, for increasing cell viability and angiogenesis. The results demonstrated external and internal cell colonization of biofunctionalized scaffolds with formation of new blood vessels (neoangiogenesis) and stimulation of ectopic mineralization.

SERS Investigation on Oligopeptides Used as Biomimetic Coatings for Medical Devices.

The surface-enhanced Raman scattering (SERS) spectra of three amphiphilic oligopeptides derived from EAK16 (AEAEAKAK)2 were examined to study systematic amino acid substitution effects on the corresponding interaction with Ag colloidal nanoparticles. Such self-assembling molecular systems, known as "molecular Lego", are of particular interest for their uses in tissue engineering and as biomimetic coatings for medical devices because they can form insoluble macroscopic membranes under physiological conditions. Spectra were collected for both native and gamma-irradiated samples. Quantum mechanical data on two of the examined oligopeptides were also obtained to clarify the assignment of the prominent significative bands observed in the spectra. In general, the peptide-nanoparticles interaction occurs through the COO- groups, with the amide bond and the aliphatic chain close to the colloid surface. After gamma irradiation, mimicking a free oxidative radical attack, the SERS spectra of the biomaterials show that COO- groups still provide the main peptide-nanoparticle interactions. However, the spatial arrangement of the peptides is different, exhibiting a systematic decrease in the distance between aliphatic chains and colloid nanoparticles.

Breast Cancer Cell Cultures on Electrospun Poly(ε-Caprolactone) as a Potential Tool for Preclinical Studies on Anticancer Treatments.

During anticancer drug development, most compounds selected by in vitro screening are ineffective in in vivo studies and clinical trials due to the unreliability of two-dimensional (2D) in vitro cultures that are unable to mimic the cancer microenvironment. Herein, HCC1954 cell cultures on electrospun polycaprolactone (PCL) were characterized by morphological analysis, cell viability assays, histochemical staining, immunofluorescence, and RT-PCR. Our data showed that electrospun PCL allows the in vitro formation of cultures characterized by mucopolysaccharide production and increased cancer stem cell population. Moreover, PCL-based cultures were less sensitive to doxorubicin and electroporation/bleomycin than those grown on polystyrene plates. Collectively, our data indicate that PCL-based cultures may be promising tools for preclinical studies.

Breast cancer cells grown on hyaluronic acid-based scaffolds as 3D in vitro model for electroporation.

Nowadays, electroporation (EP) represents a promising method for the intracellular delivery of anticancer drugs. To setting up the process, the EP efficiency is usually evaluated by using cell suspension and adherent cell cultures that are not representative of the in vivo conditions. Indeed, cells are surrounded by extracellular matrix (ECM) whose composition and physical characteristics are different for each tissue. So, various three-dimensional (3D) in vitro models, such as spheroids and hydrogel-based cultures, have been proposed to mimic the tumour microenvironment. Herein, a 3D breast cancer in vitro model has been proposed. HCC1954 cells were seeded on crosslinked and lyophilized matrices composed of hyaluronic acid (HA) and ionic complementary self-assembling peptides (SAPs) already known to provide a fibrous structure mimicking collagen network. Herein, SAPs were functionalized with laminin derived IKVAV adhesion motif. Cultures were characterized by spheroids surrounded by ECM produced by cancer cells as demonstrated by collagen1a1 and laminin B1 transcripts. EP was carried out on both 2D and 3D cultures: a sequence of 8 voltage pulses at 5 kHz with different amplitude was applied using a plate electrode. Cell sensitivity to EP seemed to be modulated by the presence of ECM and the different cell organization. Indeed, cells cultured on HA-IKVAV were more sensitive than those treated in 2D and HA cultures, in terms of both cell membrane permeabilization and viability. Collectively, our results suggest that HA-IKVAV cultures may represent an interesting model for EP studies. Further studies will be needed to elucidate the influence of ECM composition on EP efficiency.

The Efficiency of Gene Electrotransfer in Breast-Cancer Cell Lines Cultured on a Novel Collagen-Free 3D Scaffold.

Gene Electro-Transfer (GET) is a powerful method of DNA delivery with great potential for medical applications. Although GET has been extensively studied in vitro and in vivo, the optimal parameters remain controversial. 2D cell cultures have been widely used to investigate GET protocols, but have intrinsic limitations, whereas 3D cultures may represent a more reliable model thanks to the capacity of reproducing the tumor architecture. Here we applied two GET protocols, using a plate or linear electrode, on 3D-cultured HCC1954 and MDA-MB231 breast cancer cell lines grown on a novel collagen-free 3D scaffold and compared results with conventional 2D cultures. To evaluate the electrotransfer efficiency, we used the plasmid pEGFP-C3 encoding the enhanced green fluorescent protein (EGFP) reporter gene. The novel 3D scaffold promoted extracellular matrix deposition, which particularly influences cell behavior in both in vitro cell cultures and in vivo tumor tissue. While the transfection efficiency was similar in the 2D-cultures, we observed significant differences in the 3D-model. The transfection efficiency in the 3D vs 2D model was 44% versus 15% (p < 0.01) and 24% versus 17% (p < 0.01) in HCC1954 and MDA-MB231 cell cultures, respectively. These findings suggest that the novel 3D scaffold allows reproducing, at least partially, the peculiar morphology of the original tumor tissues, thus allowing us to detect meaningful differences between the two cell lines. Following GET with plate electrodes, cell viability was higher in 3D-cultured HCC1954 (66%) and MDA-MB231 (96%) cell lines compared to their 2D counterpart (53% and 63%, respectively, p < 0.001). Based on these results, we propose the novel 3D scaffold as a reliable support for the preparation of cell cultures in GET studies. It may increase the reliability of in vitro assays and allow the optimization of GET parameters of in vivo protocols.

EAK Hydrogels Cross-Linked by Disulfide Bonds: Cys Number and Position Are Matched to Performances.

Hydrogels produced by self-assembling peptides are intrinsically biocompatible and thus appropriate for many biomedical purposes. Their application field may be even made wider by reducing the softness and improving the hydrogel mechanical properties through cross-linking treatments. To this aim, modifications of EAK16-II sequence by including Cys residues in its sequence were here investigated in order to obtain hydrogels cross-linkable through a disulfide bridge. Two sequences, namely, C-EAK and C-EAK-C, that contain Cys residues at the N-terminus or at both ends were characterized. Fiber-forming abilities and biological and dynamic mechanical properties were explored before and after the oxidative treatment. In particular, the oxidized version of C-EAK presents a good cell viability and sustains osteoblast proliferation. Furthermore, molecular dynamics (MD) simulations on monomeric and assembled forms of the peptides were performed. MD simulations explained how a specific Cys functionalization was better than the other one. In particular, the results suggested that EAK16-II functionalization with a single Cys residue, instead of two, together with biocompatible cross-linking may be considered an intriguing strategy to obtain a support with better dynamic mechanical properties and biological performances.

Cysteine-Modified Self-Assembling Peptides on Gold: The Role of the Head and Tail.

Molecular self-assembly consists of the spontaneous aggregation of molecules into a well-defined structure guided by noncovalent bonds. The self-assembly strategy is ubiquitous in nature and recently has been proposed as a nature-mimetic strategy in polymer science and biomaterial engineering. In this context, we aim at designing and testing innovative but simple chemical strategies to efficiently modify surfaces by exploiting minor modifications in the bioactive molecule functionalities, for example, introducing cysteine (Cys) as a terminal residue in self-assembling peptides (SAPs). In this work, we report the attenuated total reflection-Fourier transform infrared spectroscopy, synchrotron radiation-induced X-ray photoelectron spectroscopy, near-edge X-ray absorption fine structure spectroscopy, and time-of-flight secondary ion mass spectrometry investigation of self-assembled layers of oligopeptides anchored onto gold surfaces through cysteine residues, opportunely inserted in an SAP (EAK16-II) main chain in three different positions: at the amine end group, at the carboxyl end group, and at both terminal groups (i.e., a bidentate SAP). This study, which allowed us to individuate in the bidentate SAP the best candidate for the controlled production of ordered SAP layers on the gold substrate surface, is envisaged to open wide perspectives for efficient chemical modification of surfaces with biomolecules, leading to obtaining innovative bioactive materials for applications in the field of tissue engineering.

A Novel 3D Scaffold for Cell Growth to Asses Electroporation Efficacy.

Tumor electroporation (EP) refers to the permeabilization of the cell membrane by means of short electric pulses thus allowing the potentiation of chemotherapeutic drugs. Standard plate adhesion 2D cell cultures can simulate the in vivo environment only partially due to lack of cell-cell interaction and extracellular matrix (ECM). In this study, we assessed a novel 3D scaffold for cell cultures based on hyaluronic acid and ionic-complementary self-assembling peptides (SAPs), by studying the growth patterns of two different breast carcinoma cell lines (HCC1569 and MDA-MB231). This 3D scaffold modulates cell shape and induces extracellular matrix deposit around cells. In the MDA-MB 231 cell line, it allows three-dimensional growth of structures known as spheroids, while in HCC1569 it achieves a cell organization similar to that observed in vivo. Interestingly, we were able to visualize the electroporation effect on the cells seeded in the new scaffold by means of standard propidium iodide assay and fluorescence microscopy. Thanks to the presence of cell-cell and cell-ECM interactions, the new 3D scaffold may represent a more reliable support for EP studies than 2D cancer cell cultures and may be used to test new EP-delivered drugs and novel EP protocols.

3D Synthetic Peptide-based Architectures for the Engineering of the Enteric Nervous System.

Damage of enteric neurons and partial or total loss of selective neuronal populations are reported in intestinal disorders including inflammatory bowel diseases and necrotizing enterocolitis. To develop three-dimensional scaffolds for enteric neurons we propose the decoration of ionic-complementary self-assembling peptide (SAP) hydrogels, namely EAK or EAbuK, with bioactive motives. Our results showed the ability of EAK in supporting neuronal cell attachment and neurite development. Therefore, EAK was covalently conjugated to: RGD, (GRGDSP)4K (fibronectin), FRHRNRKGY (h-vitronectin, named HVP), IKVAV (laminin), and type 1 Insulin-like Growth Factor (IGF-1). Chemoselective ligation was applied for the SAP conjugation with IGF-1 and the other longer sequences. Freshly isolated murine enteric neurons attached and grew on all functionalized EAK but IGF-1. Cell-cell contact was evident on hydrogels enriched with (GRGDSP)4K and HVP. Moreover (GRGDSP)4K significantly increased mRNA expression of neurotrophin-3 and nerve growth factor, two trophic factors supporting neuronal survival and differentiation, whereas IKVAV decoration specifically increased mRNA expression of acetylcholinesterase and choline acetyltransferase, genes involved in synaptic communication between cholinergic neurons. Thus, decorated hydrogels are proposed as injectable scaffolds to support in loco survival of enteric neurons, foster synaptic communication, or drive the differentiation of neuronal subtypes.

Cell-seeded 3D scaffolds as in vitro models for electroporation.

Electroporation of cells is usually studied using cell suspensions or monolayer cultures. 3D scaffolds for cell culture have been recently designed in order to reproduce in vitro the complex and multifactorial environment experimented in vivo by cells. In fact, it is well known that 2D cell cultures are not able to simulate the complex interactions between the cells and their extracellular matrix (ECM). Recently, some examples of 3D models, like spheroids, have been investigated also in the electroporation field. Spheroids have been proposed in electrochemotherapy (ECT) studies to mimic tumor in vivo conditions: they are easy-to-handle 3D models but their sensitivity to electric field pulses depends from their diameter and, more interestingly, despite being relevant for intercellular junctions, they are not so much so for cell-ECM interactions. In this work, we propose a 3D macroscopic myxoid matrix for cell culture that would mimic the in vivo environment of myxoid stroma tumors. The myxoid stroma consists of abundant basic substances with large amounts of glycosaminoglycans (hyaluronic acid) and proteoglycans, poor collagen fibers and no elastin content. In the proposed approach, tumor cells seeded on 3D scaffolds mimic of myxoid stroma can establish both cell-cell and cell-ECM 3D interactions. The MCF7 cells (human breast adenocarcinoma cell line) were seeded in complete culture medium. Cell cultures were incubated at 37 °C for either 24 h, 3 days or 7 day. Some samples were used to assess cell vitality using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) test and others for electroporation tests and for histopathological analysis. The electroporation has been verified by the fluorescent dye Propidium cellular uptake. The proposed myxoid stroma scaffold induces cell proliferation and shows fibrous structures produced by cells, the concentration of which increases with culture time. The proposed matrix will be used for further investigations as a new scaffold for cell culture. Tumor cells grown into these new scaffolds will be used to evaluate electroporation including the stroma effect.

Biocompatible Materials Based on Self-Assembling Peptides on Ti25Nb10Zr Alloy: Molecular Structure and Organization Investigated by Synchrotron Radiation Induced Techniques.

In this work, we applied advanced Synchrotron Radiation (SR) induced techniques to the study of the chemisorption of the Self Assembling Peptide EAbuK16, i.e., H-Abu-Glu-Abu-Glu-Abu-Lys-Abu-Lys-Abu-Glu-Abu-Glu-Abu-Lys-Abu-Lys-NH2 that is able to spontaneously aggregate in anti-parallel ß-sheet conformation, onto annealed Ti25Nb10Zr alloy surfaces. This synthetic amphiphilic oligopeptide is a good candidate to mimic extracellular matrix for bone prosthesis, since its ß-sheets stack onto each other in a multilayer oriented nanostructure with internal pores of 5-200 nm size. To prepare the biomimetic material, Ti25Nb10Zr discs were treated with aqueous solutions of EAbuK16 at different pH values. Here we present the results achieved by performing SR-induced X-ray Photoelectron Spectroscopy (SR-XPS), angle-dependent Near Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy, FESEM and AFM imaging on Ti25Nb10Zr discs after incubation with self-assembling peptide solution at five different pH values, selected deliberately to investigate the best conditions for peptide immobilization.