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.
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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.
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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
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 (
, IC
= 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
high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound
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,
represents an interesting antiviral candidate.
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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
and
.
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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.
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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)
K (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)
K and HVP. Moreover (GRGDSP)
K 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.
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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.
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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.
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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
M and 10
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.
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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.
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