Annj Zamuner

Electrospun Scaffolds for Osteoblast Cells: Peptide-Induced Concentration-Dependent Improvements of Polycaprolactone

The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype.

A peptide nucleic acid label-free biosensor for Mycobacterium tuberculosis DNA detection via azimuthally controlled grating-coupled SPR

Grating coupled surface plasmon resonance phenomena under azimuthal control of incident light (φ ≠ 0° GC-SPR) have recently been exploited for the development of biosensing solutions with a sensitivity similar to that of classic prism-coupled SPR sensors, with the advantage of higher miniaturization potential. Here we combined the use of φ ≠ 0° GC-SPR with the use of peptide nucleic acid (PNA) probes and a strategy for maximizing the signal-to-noise ratio for the sensitive detection of Mycobacterium tuberculosis (MT) DNA. We focused our attention on the optimization of the PNA-based sensing layer by controlling the sensing surface composition with the PNA-based probe and a poly(ethylene oxide) (PEO)-based antifouling layer. We tested the sensor response first in the presence of complementary and non-complementary oligonucleotides, and then we applied our strategy for the detection of PCR amplified samples, using the fluorescence-based microarray technology as the control. With the φ ≠ 0° GC-SPR set-up adopted, a limit of detection (LOD 0.26 pM) more than one order of magnitude lower than that obtained by the fluorescence method (LOD 8.9 pM) was observed using a complementary oligonucleotide target. Also when PCR amplicons were analysed on SPR grating surfaces, lower DNA concentrations were detectable with the SPR readout as compared to the fluorescence one, and with an experimental protocol that does not include the need to use expensive fluorophore molecules. The whole approach, involving the sensor fabrication, the sensing surface control and DNA detection, has demonstrated that φ ≠ 0° GC-SPR is a good starting point for a sensitive, versatile and scalable biosensing technique that will be further investigated in future experiments.

Driving h‐osteoblast adhesion and proliferation on titania: peptide hydrogels decorated with growth factors and adhesive conjugates

Hydrogels from self‐assembling ionic complementary peptides have been receiving much interest from the scientific community as mimetics of the extracellular matrix that can offer three‐dimensional support for cell growth or become vehicles for the delivery of stem cells or drugs. These scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In order to develop a novel bioactive titanium implant, we propose the introduction of a layer of ionic‐complementary self‐assembling peptides (EAbuK) on Ti whose surface has been previously sandblasted and acid etched. The peptide layer is anchored to the metal by covalent functionalization of titania with self‐assembling sequences. The peptide layer has also been enriched by the insulin‐like growth factor‐1 incorporated to the layer and/or a conjugate obtained by chemoselective ligation between EAbuK and a sequence of 25 residues containing four GRGDSP motifs per chain. X‐ray photoelectron spectroscopy studies confirmed a change in the surface composition in agreement with the proposed decorations. An evaluation of the contact angle showed a substantial change in wettability induced by the peptide layer. The human osteoblast adhesion and proliferation assays showed an increase in adhesion for the surfaces enriched with conjugate at a concentration of 3.8 × 10−7 m and an enhanced proliferation for samples enriched with insulin‐like growth factor‐1 at the highest concentration tested (2.1 × 10−5 m). Copyright

Design of Decorated Self-Assembling Peptide Hydrogels as Architecture for Mesenchymal Stem Cells

Hydrogels from self-assembling ionic complementary peptides have been receiving a lot of interest from the scientific community as mimetic of the extracellular matrix that can offer three-dimensional supports for cell growth or can become vehicles for the delivery of stem cells, drugs or bioactive proteins. In order to develop a 3D “architecture” for mesenchymal stem cells, we propose the introduction in the hydrogel of conjugates obtained by chemoselective ligation between a ionic-complementary self-assembling peptide (called EAK) and three different bioactive molecules: an adhesive sequence with 4 Glycine-Arginine-Glycine-Aspartic Acid-Serine-Proline (GRGDSP) motifs per chain, an adhesive peptide mapped on h-Vitronectin and the growth factor Insulin-like Growth Factor-1 (IGF-1). The mesenchymal stem cell adhesion assays showed a significant increase in adhesion and proliferation for the hydrogels decorated with each of the synthesized conjugates; moreover, such functionalized 3D hydrogels support cell spreading and elongation, validating the use of this class of self-assembly peptides-based material as very promising 3D model scaffolds for cell cultures, at variance of the less realistic 2D ones. Furthermore, small amplitude oscillatory shear tests showed that the presence of IGF-1-conjugate did not alter significantly the viscoelastic properties of the hydrogels even though differences were observed in the nanoscale structure of the scaffolds obtained by changing their composition, ranging from long, well-defined fibers for conjugates with adhesion sequences to the compact and dense film for the IGF-1-conjugate.

Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides

Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

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.

Natural Scaffolds for Regenerative Medicine: Direct Determination of Detergents Entrapped in Decellularized Heart Valves

The increasing urgency for replacement of pathological heart valves is a major stimulus for research on alternatives to glutaraldehyde-treated grafts. New xenogeneic acellular heart valve substitutes that can be repopulated by host cells are currently under investigation. Anionic surfactants, including bile acids, have been widely used to eliminate the resident cell components chiefly responsible for the immunogenicity of the tissue, even if detergent toxicity might present limitations to the survival and/or functional expression of the repopulating cells. To date, the determination of residual detergent has been carried out almost exclusively on the washings following cell removal procedures. Here, a novel HPLC-based procedure is proposed for the direct quantification of detergent (cholate, deoxycholate, and taurodeoxycholate) residues entrapped in the scaffold of decellularized porcine aortic and pulmonary valves. The method was demonstrated to be sensitive, reproducible, and extendable to different types of detergent. This assessment also revealed that cell-depleted heart valve scaffolds prepared according to procedures currently considered for clinical use might contain significant amount of surfactant.

Self-assembling peptide hydrogels immobilized on silicon surfaces.

Abstract The hydrogels of self-assembling ionic complementary peptides have collected in the scientific community increasing consensus as mimetics of the extracellular matrix that can offer 3D supports for cell growth or be vehicles for the delivery of stem cells or drugs. Such scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In this context, our research deals with the introduction of a layer of self-assembling peptides on a silicon surface by covalent anchoring and subsequent physisorption. In this work, we present a spectroscopic investigation of the proposed bioactive scaffolds, carried out by surface-sensitive spectroscopic techniques such as XPS (X-ray photoelectron spectroscopy) and RAIRS (Reflection Absorption Infrared Spectroscopy) and by state-of-the-art synchrotron radiation methodologies such as angle dependent NEXAFS (Near Edge X-ray Absorption Fine Structure). XPS studies confirmed the change in the surface composition in agreement with the proposed enrichments, and led to assess the self-assembling peptide chemical stability. NEXAFS spectra, collected in angular dependent mode at the N K-edge, allowed to investigate the self-assembling behavior of the macromolecules, as well as to determine their molecular orientation on the substrate. Furthermore, Infrared Spectroscopy measurements demonstrated that the peptide maintains its secondary structure (β-sheet anti-parallel) after deposition on the silicon surface. The complementary information acquired by means of XPS, NEXAFS and RAIRS lead to hypothesize a “layer-by-layer” arrangement of the immobilized peptides, giving rise to an ordered 3D nanostructure.

Covalent grafting of Ti surfaces with peptide hydrogel decorated with growth factors and self-assembling adhesive sequences

Hydrogels from self‐assembling ionic complementary peptides have been receiving much interest from the scientific community as mimetics of the extracellular matrix that can offer three‐dimensional support for cell growth or become vehicles for the delivery of stem cells or drugs. These scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In order to develop a novel bioactive titanium implant, we propose the introduction of a layer of ionic‐complementary self‐assembling peptides (EAbuK) on Ti whose surface has been previously sandblasted and acid etched. The peptide layer is anchored to the metal by covalent functionalization of titania with self‐assembling sequences. The peptide layer has also been enriched by the insulin‐like growth factor‐1 incorporated to the layer and/or a conjugate obtained by chemoselective ligation between EAbuK and a sequence of 25 residues containing four GRGDSP motifs per chain. X‐ray photoelectron spectroscopy studies confirmed a change in the surface composition in agreement with the proposed decorations. An evaluation of the contact angle showed a substantial change in wettability induced by the peptide layer. The human osteoblast adhesion and proliferation assays showed an increase in adhesion for the surfaces enriched with conjugate at a concentration of 3.8 × 10−7 m and an enhanced proliferation for samples enriched with insulin‐like growth factor‐1 at the highest concentration tested (2.1 × 10−5 m). Copyright

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

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