Edmondo Battista

Cells preferentially grow on rough substrates

Substrate nanotopography affects cell adhesion and proliferation and is fundamental to the rational design of bio-adhesives, to tissue engineering and to the development of assays for in-vitro screening. Cell behavior on rough substrates is still elusive, and the results presented in the open literature remain controversial. Here, the proliferation of cells on electrochemically etched silicon substrates with different roughness and nearly similar surface energy was studied over three days with confocal and atomic force microscopy. The surface profile of the substrates is a self-affine fractal with a roughness R(a) growing with the etching time from approximately 2 to 100 nm and a fractal dimension D ranging between about 2 (nominally flat surface) and 2.6. For four cell types, the number of adhering cells and their proliferation rates exhibited a maximum on moderately rough (R(a) approximately 10-45 nm) nearly Brownian (D approximately 2.5) substrates. The observed cell behavior was satisfactorily interpreted within the theory of adhesion to randomly rough solids. These findings demonstrated the importance of nanogeometry in cell stable adhesion and growth, suggesting that moderately rough substrates with large fractal dimension could selectively boost cell proliferation.

A unique three-dimensional SCID-polymeric scaffold (SCID-synth-hu) model for in vivo expansion of human primary multiple myeloma cells.

Multiple myeloma (MM) cells home to the bone marrow (BM) and adhere to extracellular matrix (ECM) proteins and to bone marrow stromal cells (BMSCs). The close cross talk between MM cells and cells of the non-tumor compartment within the BM has a key role in supporting tumor growth, survival and development of drug resistance. This biological scenario has led to a growing interest in novel drugs, targeting MM cells and/or interfering with their human BM milieu (HuBMM).1, 2 Based on this, appropriate in vivo models that recapitulate the complex interactions occurring between MM and its HuBMM are required for preclinical evaluation of new anti-MM agents. To date, the in vivo study of MM pathobiology and the validation of therapeutic anti-MM agents has been carried out using a variety of models of murine MM or human MM xenografts in immunocompromised mice.3 These models, however, do not replicate the HuBMM. The development of the SCID (severe combined immunodeficiency)-hu model has been an important advance, as it was the first model to recapitulate a HuBMM in mice.4, 5, 6 However, although the SCID-hu system remains a highly relevant model for preclinical investigation, it does have important limitations: (i) restricted availability of human fetal bone chips; (ii) the allogeneic nature of the fetal BM milieu versus MM cells; and (iii) the significant heterogeneity of implanted human bone chips, collected from different individuals at different gestational age, that may produce experimental variability.

Surface investigation on biomimetic materials to control cell adhesion: the case of RGD conjugation on PCL.

The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(epsilon-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates.

Fluorescence Lifetimes and Quantum Yields of Rhodamine Derivatives: New Insights from Theory and Experiment

Although lifetimes and quantum yields of widely used fluorophores are often largely characterized, a systematic approach providing a rationale of their photophysical behavior on a quantitative basis is still a challenging goal. Here we combine methods rooted in the time-dependent density functional theory and fluorescence lifetime imaging microscopy to accurately determine and analyze fluorescence signatures (lifetime, quantum yield, and band peaks) of several commonly used rhodamine and pyronin dyes. We show that the radiative lifetime of rhodamines can be correlated to the charge transfer from the phenyl toward the xanthene moiety occurring upon the S(0) ← S(1) de-excitation, and to the xanthene/phenyl relative orientation assumed in the S(1) minimum structure, which in turn is variable upon the amino and the phenyl substituents. These findings encourage the synergy of experiment and theory as unique tool to design finely tuned fluorescent probes, such those conceived for modern optical sensors.

Supramolecular Spectrally Encoded Microgels with Double Strand Probes for Absolute and Direct miRNA Fluorescence Detection at High Sensitivity

We present novel microgels as a particle-based suspension array for direct and absolute microRNA (miRNA) detection. The microgels feature a flexible molecular architecture, antifouling properties, and enhanced sensitivity with a large dynamic range of detection. Specifically, they possess a core-shell molecular architecture with two different fluorescent dyes for multiplex spectral analyses and are endowed with a fluorescent probe for miRNA detection. Encoding and detection fluorescence signals are distinguishable by nonoverlapping emission spectra. Tunable fluorescence probe conjugation and emission confinement on single microgels allow for ultrasensitive miRNA detection. Indeed, the suspension array has high selectivity and sensitivity with absolute quantification, a detection limit of 10(-15) M, a dynamic range from 10(-9) to 10(-15) M, and higher accuracy than qRT-PCR. The antifouling properties of the microgels also permit the direct measurement of miRNAs in serum, without sample pretreatment or target amplification. A multiplexed assay has been tested for a set of miRNAs chosen as cancer biomarkers.

Evolutionary screening and adsorption behavior of engineered M13 bacteriophage and derived dodecapeptide for selective decoration of gold interfaces.

There is a growing interest in identifying biomacromolecules such as proteins and peptides to functionalize metallic surfaces through noncovalent binding. One method for functionalizing materials without fundamentally changing their inherent structure is using biorecognition moieties. Here, we proved a general route to select a biomolecule adhesive motif for surface functionalization by comprehensively screening phage displayed peptides. In particular, we selected a genetically engineered M13 bacteriophage and a linear dodecapeptide derived from its pIII domain for recognizing gold surfaces in a specific and selective manner. In the phage context, we demonstrated the adhesive motif was capable to adsorb on gold in a preferential way with a morphological and viscoelastic signature of the adsorbed layer as evidenced by QCM-D and AFM investigations. Out of the phage context, the linear dodecapeptide is reproducibly found to adhere to the gold surface, and by quantitative SPR measurements, high affinity constants (K(eq)~10(6)M(-1), binding energy ~-8 kcal/mol) were determined. We proved that the interactions occurring at gold interface were mainly hydrophobic as a consequence of high frequency of hydrophobic residues in the peptide sequence. Moreover, by CD, molecular dynamics and steered molecular dynamics, we demonstrated that the molecular flexibility only played a minor role in the peptide adsorption. Such noncovalent but specific modification of inorganic surfaces through high affinity biomolecule adsorption represents a general strategy to modulate the functionality of multipurpose metallic surfaces.

Selective modulation of cell response on engineered fractal silicon substrates

A plethora of work has been dedicated to the analysis of cell behavior on substrates with ordered topographical features. However, the natural cell microenvironment is characterized by biomechanical cues organized over multiple scales. Here, randomly rough, self-affinefractal surfaces are generated out of silicon,where roughness Ra and fractal dimension Df are independently controlled. The proliferation rates, the formation of adhesion structures, and the morphology of 3T3 murine fibroblasts are monitored over six different substrates. The proliferation rate is maximized on surfaces with moderate roughness (Ra ~ 40 nm) and large fractal dimension (Df ~ 2.4); whereas adhesion structures are wider and more stable on substrates with higher roughness (Ra ~ 50 nm) and lower fractal dimension (Df ~ 2.2). Higher proliferation occurson substrates exhibiting densely packed and sharp peaks, whereas more regular ridges favor adhesion. These results suggest that randomly roughtopographies can selectively modulate cell behavior.

Thermoresponsive PNIPAAm hydrogel scaffolds with encapsulated AuNPs show high analyte-trapping ability and tailored plasmonic properties for high sensing efficiency

The fabrication of a scaffold able to control the positioning of AuNPs and to trap and concentrate target molecules inside them is a promising idea for a large variety of sensing applications. In this work, we designed and fabricated a scaffold of already-prepared 20 nm AuNPs encapsulated in a PNIPAAm hydrogel and utilizing surface enhanced Raman spectroscopy (SERS), we used it as a sensor with remarkably low limits of detection. In fact, as the target is trapped inside the hydrogel, the following takes place: (a) the concentration of the target increases dramatically and (b) the localization of the AuNPs and thus of the hotspots (areas with extremely high SERS enhancement factors) work synergistically, improving the sensing ability of the scaffold. The SERS enhancement ability of our scaffolds was checked with adenine, 2-naphthalenethiol and melamine molecules; the trapping efficiency was investigated for the melamine and a partition coefficient of k = 5 × 105 was found. Finally, by focusing on a single PNIPAAm hydrogel with encapsulated AuNPs, we managed to detect 10-6 M or rather 108 molecules of melamine trapped inside the scaffold.

Ligand engagement on material surfaces is discriminated by cell mechanosensoring

Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.

Microstructure, degradation and in vitro MG63 cells interactions of a new poly(ε-caprolactone), zein, and hydroxyapatite composite for bone tissue engineering

Novel biodegradable biomaterials were investigated for potential application in bone tissue engineering. The biomaterials were prepared by blending poly(ε-caprolactone) and thermoplastic zein, a corn protein, with or without the incorporation of hydroxyapatite particles. The biomaterials were characterized in vitro to assess the degradation in phosphate buffer saline for 56 days by monitoring weight change, morphology, wettability, and tensile properties. The interaction between the biomaterials and MG63 was evaluated by proliferation, morphological characterization, and osteogenic differentiation assays up to 28 days in in vitro cultures. The incorporation of thermoplastic zein within poly(ε-caprolactone) enhanced the hydrophilicity and degradability, while minor effects were observed after the inclusion of the hydroxyapatite particles. Compared to the neat poly(ε-caprolactone), the multiphase poly(ε-caprolactone)/thermoplastic zein–hydroxyapatite composite improved the osteogenic differentiation of MG63 cells and is being considered a candidate material for bone tissue engineering applications.