Multilayer Electrospun Scaffolds of Opposite-Charged Chitosans.

Chitosan (CS) is a polysaccharide obtainable by the deacetylation of chitin, which is highly available in nature and is consequently low-cost. Chitosan is already used in the biomedical field (e.g., guides for nerve reconstruction) and has been proposed as a biomaterial for tissue regeneration in different body districts, including bone tissue. The interest in chitosan as a biomaterial stems from its ease of functionalization due to the presence of reactive groups, its antibacterial properties, its ease of processing to obtain porous matrices, and its inherent similarity to polysaccharides that constitute the human extracellular matrix, such as hyaluronic acid (HA). Here, chitosan was made to react with succinic anhydride to develop a negatively charged chitosan (SCS) that better mimics HA. FT-IR and NMR analyses confirmed the presence of the carboxylic groups in the modified polymer. Four different electrospun matrices were prepared: CS, SCS, a layer-by-layer matrix (LBL), and a matrix with both CS and SCS simultaneously electrospun (HYB). All the matrices containing SCS showed increased human osteoblast proliferation, mineralization, and gene expression, with the best results obtained with HYB compared to the control (CS). Moreover, the antibacterial potential of CS was preserved in all the SCS-containing matrices, and the pure SCS matrix demonstrated a significant reduction in bacterial proliferation of both S. aureus and E. coli.

Screening of Small-Molecule Libraries Using SARS-CoV-2-Derived Sequences Identifies Novel Furin Inhibitors.

SARS-CoV-2 is the pathogen responsible for the most recent global pandemic, which has claimed hundreds of thousands of victims worldwide. Despite remarkable efforts to develop an effective vaccine, concerns have been raised about the actual protection against novel variants. Thus, researchers are eager to identify alternative strategies to fight against this pathogen. Like other opportunistic entities, a key step in the SARS-CoV-2 lifecycle is the maturation of the envelope glycoprotein at the RARR685↓ motif by the cellular enzyme Furin. Inhibition of this cleavage greatly affects viral propagation, thus representing an ideal drug target to contain infection. Importantly, no Furin-escape variants have ever been detected, suggesting that the pathogen cannot replace this protease by any means. Here, we designed a novel fluorogenic SARS-CoV-2-derived substrate to screen commercially available and custom-made libraries of small molecules for the identification of new Furin inhibitors. We found that a peptide substrate mimicking the cleavage site of the envelope glycoprotein of the Omicron variant (QTQTKSHRRAR-AMC) is a superior tool for screening Furin activity when compared to the commercially available Pyr-RTKR-AMC substrate. Using this setting, we identified promising novel compounds able to modulate Furin activity in vitro and suitable for interfering with SARS-CoV-2 maturation. In particular, we showed that 3-((5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic acid (P3, IC50 = 35 µM) may represent an attractive chemical scaffold for the development of more effective antiviral drugs via a mechanism of action that possibly implies the targeting of Furin secondary sites (exosites) rather than its canonical catalytic pocket. Overall, a SARS-CoV-2-derived peptide was investigated as a new substrate for in vitro high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound P3 as a promising hit with an innovative chemical scaffold. Given the key role of Furin in infection and the lack of any Food and Drug Administration (FDA)-approved Furin inhibitor, P3 represents an interesting antiviral candidate.

SARS-CoV-2 S Mutations: A Lesson from the Viral World to Understand How Human Furin Works.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the etiological agent responsible for the worldwide pandemic and has now claimed millions of lives. The virus combines several unusual characteristics and an extraordinary ability to spread among humans. In particular, the dependence of the maturation of the envelope glycoprotein S from Furin enables the invasion and replication of the virus virtually within the entire body, since this cellular protease is ubiquitously expressed. Here, we analyzed the naturally occurring variation of the amino acids sequence around the cleavage site of S. We found that the virus grossly mutates preferentially at P positions, resulting in single residue replacements that associate with gain-of-function phenotypes in specific conditions. Interestingly, some combinations of amino acids are absent, despite the evidence supporting some cleavability of the respective synthetic surrogates. In any case, the polybasic signature is maintained and, as a consequence, Furin dependence is preserved. Thus, no escape variants to Furin are observed in the population. Overall, the SARS-CoV-2 system per se represents an outstanding example of the evolution of substrate-enzyme interaction, demonstrating a fast-tracked optimization of a protein stretch towards the Furin catalytic pocket. Ultimately, these data disclose important information for the development of drugs targeting Furin and Furin-dependent pathogens.

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.

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.

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.

Bio-Functionalized Chitosan for Bone Tissue Engineering.

Hybrid biomaterials allow for the improvement of the biological properties of materials and have been successfully used for implantology in medical applications. The covalent and selective functionalization of materials with bioactive peptides provides favorable results in tissue engineering by supporting cell attachment to the biomaterial through biochemical cues and interaction with membrane receptors. Since the functionalization with bioactive peptides may alter the chemical and physical properties of the biomaterials, in this study we characterized the biological responses of differently functionalized chitosan analogs. Chitosan analogs were produced through the reaction of GRGDSPK (RGD) or FRHRNRKGY (HVP) sequences, both carrying an aldehyde-terminal group, to chitosan. The bio-functionalized polysaccharides, pure or "diluted" with chitosan, were chemically characterized in depth and evaluated for their antimicrobial activities and biocompatibility toward human primary osteoblast cells. The results obtained indicate that the bio-functionalization of chitosan increases human-osteoblast adhesion (p < 0.005) and proliferation (p < 0.005) as compared with chitosan. Overall, the 1:1 mixture of HVP functionalized-chitosan:chitosan is the best compromise between preserving the antibacterial properties of the material and supporting osteoblast differentiation and calcium deposition (p < 0.005 vs. RGD). In conclusion, our results reported that a selected concentration of HVP supported the biomimetic potential of functionalized chitosan better than RGD and preserved the antibacterial properties of chitosan.

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 synthetic BMP-2 mimicking peptide induces glioblastoma stem cell differentiation.

BACKGROUND: Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments. METHODS: The BMP-2 mimicking peptide, named GBMP1a, was synthesized on solid-phase by Fmoc chemistry. Structural characterization and prediction of receptor binding were obtained by Circular Dicroism (CD) and NRM analyses. Activation of BMP signalling was evaluated by a luciferase reporter assay and western blot. Pro-differentiating effects of GBMP1a were verified by immunostaining and neurosphere assay in primary glioblastoma cultures. RESULTS: CD and NMR showed that GBMP1a correctly folds into expected tridimensional structures and predicted its binding to BMPR-IA to the same epitope as in the native complex. Reporter analysis disclosed that GBMP1a is able to activate BMP signalling in GBM cells. Moreover, BMP-signalling activation was specifically dependent on smad1/5/8 phosphorylation. Finally, we confirmed that GBMP1a treatment is sufficient to enhance osteogenic differentiation of Mesenchymal Stem Cells and to induce astroglial differentiation of glioma stem cells (GSCs) in vitro. CONCLUSIONS: GBMP1a was demonstrated to be a good inducer of GSC differentiation, thus being considered a potential anti-cancer tool to be further developed for GBM treatment. GENERAL SIGNIFICANCE: These data highlight the role of BMP-mimicking peptides as potential anti-cancer agents against GBM and stimulate the further development of GBMP1a-based structures in order to enhance its stability and activity.

Facile and selective covalent grafting of an RGD-peptide to electrospun scaffolds improves HUVEC adhesion.

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly-ε-caprolactone or poly(L-lactic acid-co-ɛ-caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly-Arg-Gly-Asp-Ser-Pro motifs per chain and a p-azido-Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly-ε-caprolactone embedded with adhesive peptides were produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24 h with a dose-dependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results.

Osteogenic properties of a short BMP-2 chimera peptide.

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2.

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.

Bottlenose dolphin (Tursiops truncatus) immortalized fibroblasts on novel 3D in vitro collagen-free scaffolds.

Dolphins, as apex predators, can be considered relevant sentinels of the health of marine ecosystems. The creation of 3D cell models to assess in vitro cell-to-cell and cell-to-matrix interactions in environmental-mimicking conditions, is of considerable interest. However, to date the establishment of cetacean 3D culture systems has not yet been accomplished. Thus, in this study, different 3D systems of bottlenose dolphin (Tursiops truncatus) skin fibroblasts have been analyzed. Particularly, novel scaffolds based on hyaluronic acid and ionic-complementary self-assembling peptides such as RGD-EAbuK and EAbuK-IKVAV have been compared to Matrigel. Histological and fluorescent staining, electron microscopy (TEM) analyses and viability assays have been performed and RT-PCR has been used to detect extracellular matrix (ECM) components produced by cells. Results showed that Matrigel induced cells to form aggregates with lower viability and no ECM production compared to the novel scaffolds. Moreover, scaffolds allowed dispersed cells to produce a collagenous ECM containing collagen1a1, laminin B1 and elastin. The HA-EAbuK-IKVAV scaffold resulted in the most suitable 3D model in terms of cell quantity and viability. The development of this innovative approach is the first step towards the possibility to create 3D in vitro models for this protected species.

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.

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.

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.

Proteolytically Resistant Bioactive Peptide-Grafted Sr/Mg-Doped Hardystonite Foams: Comparison of Two Covalent Functionalization Strategies.

Hardystonite-based (HT) bioceramic foams were easily obtained via thermal treatment of silicone resins and reactive oxide fillers in air. By using a commercial silicone, incorporating strontium oxide and magnesium oxide precursors (as well as CaO and ZnO), and treating it at 1100 °C, a complex solid solution (Ca1.4Sr0.6Zn0.85Mg0.15Si2O7) that has superior biocompatibility and bioactivity properties compared to pure hardystonite (Ca2ZnSi2O7) can be obtained. Proteolytic-resistant adhesive peptide mapped on vitronectin (D2HVP), was selectively grafted to Sr/Mg-doped HT foams using two different strategies. Unfortunately, the first method (via protected peptide) was unsuitable for acid-sensitive materials such as Sr/Mg-doped HT, resulting in the release of cytotoxic levels of Zinc over time, with consequent negative cellular response. To overcome this unexpected result, a novel functionalization strategy requiring aqueous solution and mild conditions was designed. Sr/Mg-doped HT functionalized with this second strategy (via aldehyde peptide) showed a dramatic increase in human osteoblast proliferation at 6 days compared to only silanized or non-functionalized samples. Furthermore, we demonstrated that the functionalization treatment does not induce any cytotoxicity. Functionalized foams enhanced mRNA-specific transcript levels coding IBSP, VTN, RUNX2, and SPP1 at 2 days post-seeding. In conclusion, the second functionalization strategy proved to be appropriate for this specific biomaterial and was effective at enhancing the material's bioactivity.

Finite Element Evaluation of the Electric Field Distribution in a Non-Homogeneous Environment.

Finite element analysis is used in this study to investigate the effect of media inhomogeneity on the electric field distribution in a sample composed of cells and their extracellular matrix. The sample is supposed to be subjected to very high pulsed electric field. Numerically computed electric field distribution and transmembrane potential at the cell membrane in electroporation conditions are considered in order to study cell behavior at different degrees of inhomogeneity. The different inhomogeneity grade is locally obtained using a representative model of fixed volume with cell-cell distance varying in the range of 1-283 um. The conductivity of the extracellular medium was varied between plain collagen and a gel-like myxoid matrix through combinations of the two, i.e., collagen and myxoid. An increase in the transmembrane potential was shown in the case of higher aggregate. The results obtained in this study show the effect of the presence of the cell aggregates and collagen on the transmembrane potential. In particular, by increasing the cell aggregation in the two cases, the transmembrane potential increased. Finally, the simulation results were compared to experimental data obtained by culturing HCC1954 cells in a hyaluronic acid-based scaffold. The experimental validation confirmed the behavior of the transmembrane potential in presence of the collagen: an increase in electroporation at a lower electric field intensity was found for the cells cultured in the scaffolds where there is the formation of collagen areas.

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.

Development and preclinical evaluation of bioactive nerve conduits for peripheral nerve regeneration: A comparative study.

In severe peripheral nerve injuries, nerve conduits (NCs) are good alternatives to autografts/allografts; however, the results the available devices guarantee for are still not fully satisfactory. Herein, differently bioactivated NCs based on the new polymer oxidized polyvinyl alcohol (OxPVA) are compared in a rat model of sciatic nerve neurotmesis (gap: 5 mm; end point: 6 weeks). Thirty Sprague Dawley rats are randomized to 6 groups: Reverse Autograft (RA); Reaxon®; OxPVA; OxPVA + EAK (self-assembling peptide, mechanical incorporation); OxPVA + EAK-YIGSR (mechanical incorporation); OxPVA + Nerve Growth Factor (NGF) (adsorption). Preliminarily, all OxPVA-based devices are comparable with Reaxon® in Sciatic Functional Index score and gait analysis; moreover, all conduits sustain nerve regeneration (S100, ß-tubulin) without showing substantial inflammation (CD3, F4/80) evidences. Following morphometric analyses, OxPVA confirms its potential in PNI repair (comparable with Reaxon®) whereas OxPVA + EAK-YIGSR stands out for its myelinated axons total number and density, revealing promising in injury recovery and for future application in clinical practice.