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.

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.

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.

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.

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.

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.

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.

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.

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.

Strategies for the Covalent Anchoring of a BMP-2-Mimetic Peptide to PEEK Surface for Bone Tissue Engineering.

Researchers in the field of tissue engineering are always searching for new scaffolds for bone repair. Polyetheretherketone (PEEK) is a chemically inert polymer that is insoluble in conventional solvents. PEEK's great potential in tissue engineering applications arises from its ability to not induce adverse reactions when in contact with biological tissues and its mechanical properties, which are similar to those of human bone. These exceptional features are limited by the bio-inertness of PEEK, which causes poor osteogenesis on the implant surface. Here, we demonstrated that the covalent grafting of the sequence (48-69) mapped on the BMP-2 growth factor (GBMP1α) significantly enhances the mineralization and gene expression of human osteoblasts. Different chemical methods were employed for covalently grafting the peptide onto 3D-printed PEEK disks: (a) the reaction between PEEK carbonyls and amino-oxy groups inserted in the peptides' N-terminal sites (oxime chemistry) and (b) the photoactivation of azido groups present in the peptides' N-terminal sites, which produces nitrene radicals able to react with PEEK surface. The peptide-induced PEEK surface modification was assessed using X-ray photoelectron measurements, while the superficial properties of the functionalized material were analyzed by means of atomic force microscopy and force spectroscopy. Live and dead assays and SEM measurements showed greater cell cover on functionalized samples than the control, without any cytotoxicity induction. Moreover, functionalization improved the rate of cell proliferation and the amount of calcium deposits, as demonstrated by the AlamarBlue™ and alizarin red results, respectively. The effects of GBMP1α on h-osteoblast gene expression were assayed using quantitative real-time polymerase chain reaction.

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.

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.

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.

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.

Exploiting Conserved Quorum Sensing Signals in Streptococcus mutans and Streptococcus pneumoniae

Bacterial species of the Streptococcus genera are considered either commensal bacteria or potential pathogens, according to their metabolic evolution and production of quorum sensing (QS)-controlled virulence factors. S. mutans, in particular, has become one of the best-studied examples of bacteria that are able to get along or cheat commensal species, even of the same genera. S. mutans and S. pneumoniae share homolog QS pathways and a competence stimulating peptide (CSP) for regulating bacteriocin production. Intriguingly, the abundance of S. pneumoniae and S. mutans alternates in complex microbial communities, thus opening the role for the fratricide communication of homolog QS systems. Since the inhibition of the QS has been proposed in treating bacterial infections, in this study, we designed and synthesized analogs of S. pneumoniae CSP with precise residual modifications. We reported that S. pneumoniae CSP analogs reduced the expression of genes involved in the QS of S. mutans and biofilm formation without affecting bacterial growth. The CSP analogs inhibited bacteriocin production in S. mutans, as reported by co-cultures with commensal bacteria of the oral cavity. The peptide CSP1AA, bearing substitutions in the residues involved in QS receptor recognition and activation, reported the most significant quorum-quenching activities. Our findings provide new insights into specific chemical drivers in the CSP sequences controlling the interconnection between S. mutans and S. pneumoniae. We think that the results reported in this study open the way for new therapeutic interventions in controlling the virulence factors in complex microbial communities such as the oral microbiota.

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.

Mn-Containing Bioactive Glass-Ceramics: BMP-2-Mimetic Peptide Covalent Grafting Boosts Human-Osteoblast Proliferation and Mineral Deposition.

The addition of Mn in bioceramic formulation is gaining interest in the field of bone implants. Mn activates human osteoblast (h-osteoblast) integrins, enhancing cell proliferation with a dose-dependent effect, whereas Mn-enriched glasses induce inhibition of Gram-negative or Gram-positive bacteria and fungi. In an effort to further optimize Mn-containing scaffolds' beneficial interaction with h-osteoblasts, a selective and specific covalent functionalization with a bioactive peptide was carried out. The anchoring of a peptide, mapped on the BMP-2 wrist epitope, to the scaffold was performed by a reaction between an aldehyde group of the peptide and the aminic groups of silanized Mn-containing bioceramic. SEM-EDX, FT-IR, and Raman studies confirmed the presence of the peptide grafted onto the scaffold. In in vitro assays, a significant improvement in h-osteoblast proliferation, gene expression, and calcium salt deposition after 7 days was detected in the functionalized Mn-containing bioceramic compared to the controls.

Electrospun Chitosan Functionalized with C12, C14 or C16 Tails for Blood-Contacting Medical Devices.

Medical applications stimulate the need for materials with broad potential. Chitosan, the partially deacetylated derivative of chitin, offers many interesting characteristics, such as biocompatibility and chemical derivatization possibility. In the present study, porous scaffolds composed of electrospun interwoven nanometric fibers are produced using chitosan or chitosan functionalized with aliphatic chains of twelve, fourteen or sixteen methylene groups. The scaffolds were thoroughly characterized by SEM and XPS. The length of the aliphatic tail influenced the physico-chemical and dynamic mechanical properties of the functionalized chitosan. The electrospun membranes revealed no interaction of Gram+ or Gram- bacteria, resulting in neither antibacterial nor bactericidal, but constitutively sterile. The electrospun scaffolds demonstrated the absence of cytotoxicity, inflammation response, and eryptosis. These results open the door to their application for blood purification devices, hemodialysis membranes, and vascular grafts.