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EAK16 (AEAEAKAKAEAEAKAK) and seven alternating polar/hydrophobic oligopeptides (derived from EAK16 by primary structure modifications) were tested for their resistance to free radical attack, in view of their possible use as biomimetic coatings of biomedical devices; in fact, oxidative radical stress, mainly due to hydroxyl radicals ((OH)-O-center dot), is a common physiological condition occurring under acute or chronic inflammatory response. (OH)-O-center dot radicals, mimicking an endogenous radical stress, were generated by gamma-radiolysis of water solutions. IR and Raman spectroscopies were used to investigate the damages induced by free radicals on the oligopeptides, in particular on their folding and inter-chain interactions. Some oligopeptides, i.e. EAK16 (pept1), pept3 (obtained by KO substitution), pept5 (AAbu substitution), pept7 (insertion at the N-terminus of the RGD sequence), and pept8 (RGD insertion and scrambling of the sequence) were not severely affected by the treatment and retained their prevalent structure (i.e. -helix for pept8 and -sheet for the others). The other peptides, i.e. pept2 (obtained by ED substitution), pept4 (ED and KO substitutions) and pept6 (AY substitution), showed significant conformational changes (i.e. increase in the -helix content) upon irradiation. In pept2 and pept4, modifications in hydrophobic and ionic interactions were observed; the latter were mainly ascribed to the decarboxylation of acidic amino acids. In pept6, the main target of the (OH)-O-center dot attack was Tyr, as deduced by the strong variations observed in Tyr bands both in IR and Raman spectra. This study may contribute to gain insight into the different biological performances of the EAK16-derived peptides and to identify the most adequate materials usable for bone regeneration.

Self-assembling peptides are a category of peptides which undergo spontaneous assembling into ordered nanostructures. These designed peptides have attracted huge interest in the field of nanotechnology for its potential application in areas such as biomedical nanotechnology, cell culturing, molecular electronics, and more. In the emerging field of tissue engineering, the development of synthetic materials promoting cell growth has led to the study of regularly alternating polar/non-polar amphiphilic oligopeptides, such as EAK16 (AEAEAKAK)2, also called LEGO peptides, which have been considered particularly promising. Self-assembling LEGO peptides have a preferential ?-sheet structure, are resistant to proteolytic cleavage, and are able to form an insoluble macroscopic membrane under physiological conditions. Their ability to create such stable structures derive from the hydrophobic interactions between the aliphatic groups of non-ionic residues and the complementary ionic bonds between acidic and basic amino acids. This stability can be enhanced by the pH regulation and the presence of monovalent ions. This chapter will be focused on some approaches useful for elucidating the influence of the sequence modifications and the interactions with a surface on the self-assembly capability of differently synthesised peptides. Eight different oligopeptides (from 16 to 19 residues), derived from EAK16, have been analysed by IR and Raman spectroscopies that are particularly useful for obtaining qualitative and quantitative information on the secondary structure of these peptides. Several modifications in the primary structure of peptides have been considered, namely acidic and/or basic substitutions (Glu -> Asp; Lys -> Orn), changes in the length of the aliphatic side chain of the spacer residues (Ala -> Abu or Ala -> Tyr), the addition of RGD (known to promote osteoblast adhesion), or scrambling of the sequence. As these peptides are widely used as biocompatible coatings of metallic implants, the peptide folding after adsorption on different surfaces, in particular on titanium oxide, will be also discussed. Finally, it will be shown that not all the oligopeptides examined can self-assemble into a homogeneous multilayer on metallic surfaces; however, most of the peptides take a prevailing ?-sheet structure which guarantees the best peptide-surface interaction. In particular, the interactions of polar, ionic and aromatic residues with surfaces will be discussed more in detail.

The design of composite scaffolds with slow degradation kinetics imposes the assessment of the time-course of degradation to predict the long-term in vitro behavior. In this work, the effect of hydroxyapatite (HA) particles on the hydrolytic degradation of poly e-caprolactone composite scaffold was investigated. The study of accelerated degradation mechanisms in alkaline medium enabled analysing comparable degradation profiles at different times. The accurate qualitative and quantitative study of morphology by scanning electron microscopy supported by image analysis demonstrated only a negligible effect on the structural porosity, to be ascribed to the addition of micrometric HA as a filler. Moreover, by comparing the Raman spectra with thermal analysis thermogravimetry and differential scanning calorimetry) the role of HA on the composite degradation mechanism was defined, by separately quantifying the contribution of HA particles in the bulk and on the surface, on the bone formation as a function of modifications induced in the pore morphology, as well as physical and chemical properties of the polymer matrix. Indeed, HA particles alter the poly e-caprolactone crystallinity inducing a ‘‘shielding’’ effect of the polymer matrix. Meanwhile, the slight reduction of pore size as a function of the increasing HA content and the improvement of the effective hydrophilicity of the scaffolds also influence the degradation by faster mechanisms. Finally, it has been proven that the presence of HA enhances the scaffold bioactivity and human osteoblast cell response, remarking the active role of bioactive signals on the promotion of the surface mineralization and, as a consequence, on the cell–material interaction

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(3-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 mm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are simultaneously requested.

This study aimed to characterize the chemical properties and bioactivity of an endodontic sealer (GuttaFlow Bioseal) based on polydimethylsiloxane (PDMS) and containing a calcium bioglass as a doping agent. Commercial PDMS-based cement free from calcium bioglass (GuttaFlow 2 and RoekoSeal) were characterized for comparison as well as GuttaFlow 2 doped with dicalcium phosphate dihydrate, hydroxyapatite, or a tricalcium silicate-based cement. IR and Raman analyses were performed on fresh materials as well as after aging tests in Hank’s Balanced Salt Solution (28 d, 37 °C). Under these conditions, the strengthening of the 970 cm−1 Raman band and the appearance of the IR components at 1455–1414, 1015, 868, and 600–559 cm−1 revealed the deposition of B-type carbonated apatite. The Raman I970/I638 and IR A1010/A1258 ratios (markers of apatite-forming ability) showed that bioactivity decreased along with the series: GuttaFlow Bioseal > GuttaFlow 2 > RoekoSeal. The PDMS matrix played a relevant role in bioactivity; in GuttaFlow 2, the crosslinking degree was favorable for Ca2+ adsorption/complexation and the formation of a thin calcium phosphate layer. In the less crosslinked RoekoSeal, such processes did not occur. The doped cements showed bioactivity higher than GuttaFlow 2, suggesting that the particles of the mineralizing agents are spontaneously exposed on the cement surface, although the hydrophobicity of the PDMS matrix slowed down apatite deposition. Relevant properties in the endodontic practice (i.e., setting time, radiopacity, apatite-forming ability) were related to material composition and the crosslinking degree.

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.

In the last decades, silk fibroin and wool keratin have been considered functional materials for biomedical applications. In this study, fabrics containing silk fibers from Bombyx mori and Tussah silk fibers from Antheraea pernyi, as well as wool keratin fabrics, were grafted with phosmer CL and phosmer M (commercial names, i.e., methacrylate monomers containing phosphate groups in the molecular side chain) with different weight gains. Both phosmers were recently proposed as flame retarding agents, and their chemical composition suggested a possible application in bone tissue engineering. IR and Raman spectroscopy were used to disclose the possible structural changes induced by grafting and identify the most reactive amino acids towards the phosmers. The same techniques were used to investigate the nucleation of a calcium phosphate phase on the surface of the samples (i.e., bioactivity) after ageing in simulated body fluid (SBF). The phosmers were found to polymerize onto the biopolymers efficiently, and tyrosine and serine underwent phosphorylation (monitored through the strengthening of the Raman band at 1600 cm−1 and the weakening of the Raman band at 1400 cm−1, respectively). In grafted wool keratin, cysteic acid and other oxidation products of disulphide bridges were detected together with sulphated residues. Only slight conformational changes were observed upon grafting, generally towards an enrichment in ordered domains, suggesting that the amorphous regions were more prone to react (and, sometimes, degrade). All samples were shown to be bioactive, with a weight gain of up to 8%. The most bioactive samples contained the highest phosmers amounts, i.e., the highest amounts of phosphate nucleating sites. The sulphate/sulphonate groups present in grafted wool samples appeared to increase bioactivity, as shown by the five-fold increase of the IR phosphate band at 1040 cm−1.

Cyanobacteria are oxygenic phototrophs that have an essential role in soil N2 fixation, fertility, and water retention. Cyanobacteria are also natural sources of bioactive metabolites beneficial to improve plant vigor and potentially active against fungal plant pathogens. Therefore, we studied the antifungal activity of water extract (WE) and phycobiliproteins (PBPs) from Anabaena minutissima strain BEA 0300B against the fungal plant pathogen Botrytis cinerea on tomato fruits and in vitro. The water extract and PBPs were characterized by using FT-IR and FT-Raman spectroscopies. Both water extract (5 mg/mL) and PBPs (ranged from 0.3 to 4.8 mg/mL) reduced disease incidence and disease severity on tomato fruits and mycelium growth and colony forming units in vitro. For mycelium growth, a linear PBP dose-response was found. Tomato fruits were also characterized by FT-IR and FT-Raman spectroscopies in order to evaluate structural modifications induced by pathogen and PBP treatment. PBPs preserved cutin and pectin structures by pathogen challenge. In conclusion, A. minutissima can be considered a potential tool for future large-scale experiments for plant disease control.

Cadophora luteo-olivacea represents a critical problem for kiwifruit in the post-harvest phase, mainly for its little note epidemiology. The study presented some results about the possibility of preserving kiwifruit from skin pitting symptoms using alternative methods to fungicides. By in vitro assays, antagonist mechanisms of action against pathogen isolates were tested. Trichoderma harzianum (Th1) showed the highest inhibitory activity against C. luteo-olivacea isolates by volatile, non-volatile, and by dual culture assay, displaying an inhibition respectively by 90%, 70.6%, and 78.8%, and with respect to Aureobasidium pullulans (L1 and L8) by 23.3% and 25.8%, 50% and 34.7%, and 22.5% and 23.6%, respectively. Further, the sensitivity on CFU and mycelial growth of C. luteo-olivacea isolates to fludioxonil, and CaCl2 was tested, displaying interesting EC50 values (0.36 and 0.92 g L−1, 22.5 g L−1, respectively). The effect of Brassica nigra defatted meal was tested as biofumigation assays and through FT-IR (Fourier-Transform Infrared) spectroscopy. The above-mentioned treatments were applied in vivo to evaluate their efficacy on kiwifruits. Our data demonstrated that alternative solutions could be considered to control postharvest pathogens such as C. luteo-olivacea.