Publications by authors named "Cristina Lenardi"

53 Publications

Monolithic Three-Dimensional Functionally Graded Hydrogels for Bioinspired Soft Robots Fabrication.

Soft Robot 2021 Mar 2. Epub 2021 Mar 2.

CIMaINa, Department of Physics, Università degli Studi di Milano, Milan, Italy.

Bioinspired soft robotics aims at reproducing the complex hierarchy and architecture of biological tissues within artificial systems to achieve the typical motility and adaptability of living organisms. The development of suitable fabrication approaches to produce monolithic bodies provided with embedded variable morphological and mechanical properties, typically encountered in nature, is still a technological challenge. Here we report on a novel manufacturing approach to produce three-dimensional functionally graded hydrogels (3D-FGHs) provided with a controlled porosity gradient conferring them variable stiffness. 3D-FGHs are fabricated by means of a custom-designed liquid foam templating (LFT) technique, which relies on the inclusion of air bubbles generated by a blowing agent into the monomer-based template solution during ultraviolet-induced photopolymerization. The 3D-FGHs' apparent Young's modulus ranges from 0.37 MPa (bulky hydrogel region) to 0.09 MPa (highest porosity region). A fish-shaped soft swimmer is fabricated to demonstrate the feasibility of the LFT technique to produce bioinspired robots. Mobility tests show a significant improvement in terms of swimming speed when the robot is provided with a graded body. The proposed manufacturing approach constitutes an enabling solution for the development of macroscopic functionally graded hydrogel-based structures usable in biomimetic underwater soft robotics applications.
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http://dx.doi.org/10.1089/soro.2020.0137DOI Listing
March 2021

Nanostructure Determines the Wettability of Gold Surfaces by Ionic Liquid Ultrathin Films.

Front Chem 2021 5;9:619432. Epub 2021 Feb 5.

CIMaINa and Dipartimento di Fisica "Aldo Pontremoli", Università degli Studi di Milano, Milano, Italy.

Ionic liquids are employed in energy storage/harvesting devices, in catalysis and biomedical technologies, due to their tunable bulk and interfacial properties. In particular, the wettability and the structuring of the ionic liquids at the interface are of paramount importance for all those applications exploiting ionic liquids tribological properties, their double layer organization at electrified interfaces, and interfacial chemical reactions. Here we report an experimental investigation of the wettability and organization at the interface of an imidazolium-based ionic liquid ([Bmim][NTf2]) and gold surfaces, that are widely used as electrodes in energy devices, electronics, fluidics. In particular, we investigated the role of the nanostructure on the resulting interfacial interactions between [Bmim][NTf2] and atom-assembled or cluster-assembled gold thin films. Our results highlight the presence of the solid-like structured ionic liquid domains extending several tens of nanometres far from the gold interfaces, and characterized by different lateral extension, according to the wettability of the gold nanostructures by the IL liquid-phase.
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http://dx.doi.org/10.3389/fchem.2021.619432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892474PMC
February 2021

Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication.

Micromachines (Basel) 2021 Jan 18;12(1). Epub 2021 Jan 18.

CIMaINa and Dipartimento di Fisica, Università degli Studi di Milano, Via Celoria 16, 20133 Milano, Italy.

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.
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http://dx.doi.org/10.3390/mi12010094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829752PMC
January 2021

Hadron Therapy, Magnetic Nanoparticles and Hyperthermia: A Promising Combined Tool for Pancreatic Cancer Treatment.

Nanomaterials (Basel) 2020 Sep 25;10(10). Epub 2020 Sep 25.

Dipartimento di Fisica and INFN, Università degli Studi di Pavia, 27100 Pavia, Italy.

A combination of carbon ions/photons irradiation and hyperthermia as a novel therapeutic approach for the in-vitro treatment of pancreatic cancer BxPC3 cells is presented. The radiation doses used are 0-2 Gy for carbon ions and 0-7 Gy for 6 MV photons. Hyperthermia is realized via a standard heating bath, assisted by magnetic fluid hyperthermia (MFH) that utilizes magnetic nanoparticles (MNPs) exposed to an alternating magnetic field of amplitude 19.5 mTesla and frequency 109.8 kHz. Starting from 37 °C, the temperature is gradually increased and the sample is kept at 42 °C for 30 min. For MFH, MNPs with a mean diameter of 19 nm and specific absorption rate of 110 ± 30 W/go coated with a biocompatible ligand to ensure stability in physiological media are used. Irradiation diminishes the clonogenic survival at an extent that depends on the radiation type, and its decrease is amplified both by the MNPs cellular uptake and the hyperthermia protocol. Significant increases in DNA double-strand breaks at 6 h are observed in samples exposed to MNP uptake, treated with 0.75 Gy carbon-ion irradiation and hyperthermia. The proposed experimental protocol, based on the combination of hadron irradiation and hyperthermia, represents a first step towards an innovative clinical option for pancreatic cancer.
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http://dx.doi.org/10.3390/nano10101919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600442PMC
September 2020

Rational Design of a User-Friendly Aptamer/Peptide-Based Device for the Detection of .

Sensors (Basel) 2020 Sep 2;20(17). Epub 2020 Sep 2.

Department of Pharmaceutical Sciences, University of Milan, 20133 Milan, Italy.

The urgent need to develop a detection system for , one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.
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http://dx.doi.org/10.3390/s20174977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506613PMC
September 2020

Proteomic Analysis Reveals a Mitochondrial Remodeling of βTC3 Cells in Response to Nanotopography.

Front Cell Dev Biol 2020 29;8:508. Epub 2020 Jul 29.

Department of Veterinary Medicine, University of Milano, Milan, Italy.

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that β cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing β-cell differentiation and function in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of βTC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in βTC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different β-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs.
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http://dx.doi.org/10.3389/fcell.2020.00508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405422PMC
July 2020

Shaping Pancreatic β-Cell Differentiation and Functioning: The Influence of Mechanotransduction.

Cells 2020 02 11;9(2). Epub 2020 Feb 11.

Department of Excellence of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20134 Milan, Italy.

Embryonic and pluripotent stem cells hold great promise in generating β-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these β-like cells still fail to fully mirror the adult β-cell physiology. For their proper growth and functioning, β-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals We here review the current status of knowledge of biophysical cues provided by the niche where β-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in β-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that β-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the β-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of β-cell differentiation capacity in vitro.
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http://dx.doi.org/10.3390/cells9020413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072458PMC
February 2020

Nanoscale-Induced Formation of Silicide around Gold Nanoparticles Encapsulated in a-Si.

Langmuir 2020 Feb 22;36(4):939-947. Epub 2020 Jan 22.

Interdisciplinary Centre for Nanostructured Materials and Interfaces (CIMaINa) and Physics Department "Aldo Pontremoli" , University of Milan , Via Celoria 16 , Milan 20133 , Italy.

Decorating thin-film solar cells with plasmonic nanoparticles is being pursued in order to improve device efficiency through increased scattering and local field enhancement. Gold nanoparticles are particularly interesting due to their chemical inertness and plasmon resonance in the visible range of the spectrum. In this work, gold nanoparticles fabricated by a gas aggregation nanoparticle source and embedded in a-Si (a commercial solar cell material) are studied using X-ray photoelectron spectroscopy, transmission electron microscopy, electron energy-loss spectroscopy, and energy-dispersive X-ray spectroscopy. The formation of gold silicide around the nanoparticles is investigated, as it has important consequences for the optical and electronic properties of the structures. Different from previous studies, in which the silicide formation is observed for gold nanoparticles and thin films grown on top of crystalline silicon or silica, it is found that silicide formation is largely enhanced around the nanoparticles, owing to their increased surface/volume ratio. A detailed gold silicide formation mechanism is presented based on the results, and strategies for optimizing the design of plasmonically enhanced solar cells with gold nanoparticles encapsulated in a-Si are discussed.
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http://dx.doi.org/10.1021/acs.langmuir.9b02993DOI Listing
February 2020

Combined Effects of Electrical Stimulation and Protein Coatings on Myotube Formation in a Soft Porous Scaffold.

Ann Biomed Eng 2020 Feb 7;48(2):734-746. Epub 2019 Nov 7.

The BioRobotics Institute, Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, 56127, Pisa, Italy.

Compared to two-dimensional cell cultures, three-dimensional ones potentially allow recreating natural tissue environments with higher accuracy. The three-dimensional approach is being investigated in the field of tissue engineering targeting the reconstruction of various tissues, among which skeletal muscle. Skeletal muscle is an electroactive tissue which strongly relies upon interactions with the extracellular matrix for internal organization and mechanical function. Studying the optimization of myogenesis in vitro implies focusing on appropriate biomimetic stimuli, as biochemical and electrical ones. Here we present a three-dimensional polyurethane-based soft porous scaffold (porosity ~ 86%) with a Young's modulus in wet conditions close to the one of natural skeletal muscle tissue (~ 9 kPa). To study the effect of external stimuli on muscle cells, we functionalized the scaffold with extracellular matrix components (laminin and fibronectin) and observed an increase in myoblast proliferation over three days. Furthermore, the combination between laminin coating and electrical stimulation resulted in more spread and thicker myotubes compared to non-stimulated samples and samples receiving the single (non-combined) inputs. These results pave the way to the development of mature muscle tissue within three-dimensional soft scaffolds, through the combination of biochemical and electrical stimuli.
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http://dx.doi.org/10.1007/s10439-019-02397-9DOI Listing
February 2020

Mechanotransduction in neuronal cell development and functioning.

Biophys Rev 2019 Oct 15;11(5):701-720. Epub 2019 Oct 15.

Interdisciplinary Centre for Nanostructured Materials and Interfaces (C.I.Ma.I.Na.) and Department of Physics ``Aldo Pontremoli'', Università degli Studi di Milano, via Celoria 16, 20133, Milan, Italy.

Although many details remain still elusive, it became increasingly evident in recent years that mechanosensing of microenvironmental biophysical cues and subsequent mechanotransduction are strongly involved in the regulation of neuronal cell development and functioning. This review gives an overview about the current understanding of brain and neuronal cell mechanobiology and how it impacts on neurogenesis, neuronal migration, differentiation, and maturation. We will focus particularly on the events in the cell/microenvironment interface and the decisive extracellular matrix (ECM) parameters (i.e. rigidity and nanometric spatial organisation of adhesion sites) that modulate integrin adhesion complex-based mechanosensing and mechanotransductive signalling. It will also be outlined how biomaterial approaches mimicking essential ECM features help to understand these processes and how they can be used to control and guide neuronal cell behaviour by providing appropriate biophysical cues. In addition, principal biophysical methods will be highlighted that have been crucial for the study of neuronal mechanobiology.
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http://dx.doi.org/10.1007/s12551-019-00587-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815321PMC
October 2019

Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography.

ACS Biomater Sci Eng 2018 Dec 28;4(12):4062-4075. Epub 2018 Nov 28.

Interdisciplinary Centre for Nanostructured Materials and Interfaces (CIMaINa) and Department of Physics, Università degli Studi di Milano, Milano, Italy.

Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, because of the random architecture of these artificial networks. Important groundwork therefore resulted in the development of methods to confine neuronal adhesion at specific locations to match predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning. Here, we report the development and characterization of a novel approach that, by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere nonadhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on mechanotransductive processes, and can be used in their original state without any coating requirements. These micropatterned substrates were validated using (i) a neuron-like PC12 cell line and (ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated, in the case of the hippocampal neurons, up to the formation of mature, functionally active, and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional interdot communication between these subgroups of cells was observed.
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http://dx.doi.org/10.1021/acsbiomaterials.8b00916DOI Listing
December 2018

Author Correction: Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

Sci Rep 2018 Nov 27;8(1):17472. Epub 2018 Nov 27.

Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134, Milan, Italy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-35958-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255846PMC
November 2018

Magnetic Patterning by Electron Beam-Assisted Carbon Lithography.

ACS Appl Mater Interfaces 2018 Aug 1;10(32):27178-27187. Epub 2018 Aug 1.

Elettra-Sincrotrone Trieste , S.S. 14 km 163.5 in AREA Science Park , Basovizza, I-34149 Trieste , Italy.

We report on the proof of principle of a scalable method for writing the magnetic state by electron-stimulated molecular dissociative adsorption on ultrathin Co on Re(0001). Intense microfocused low-energy electron beams are used to promote the formation of surface carbides and graphitic carbon through the fragmentation of carbon monoxide. Upon annealing at the CO desorption temperature, carbon persists in the irradiated areas, whereas the clean surface is recovered elsewhere, giving origin to chemical patterns with nanometer-sharp edges. The accumulation of carbon is found to induce an in-plane to out-of-plane spin reorientation transition in Co, manifested by the appearance of striped magnetic domains. Irradiation at doses in excess of 1000 L of CO followed by ultrahigh vacuum annealing at 380 °C determines the formation of a graphitic overlayer in the irradiated areas, under which Co exhibits out-of-plane magnetic anisotropy. Domains with opposite magnetization are separated here by chiral Neél walls. Our fabrication protocol adds lateral control to spin reorientation transitions, permitting to tune the magnetic anisotropy within arbitrary regions of mesoscopic size. We envisage applications in the nano-engineering of graphene-spaced stacks exhibiting the desired magnetic state and properties.
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http://dx.doi.org/10.1021/acsami.8b07485DOI Listing
August 2018

Anisotropic cytocompatible electrospun scaffold for tendon tissue engineering elicits limited inflammatory response in vitro.

J Biomater Appl 2018 07;33(1):127-139

4 Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, UK.

Tendon tears are a relevant concern for today's national health systems because of their social impact and high recurrence rate. The current gold standard for fixing tendon tears is surgical repair; however, this strategy is not able to fully re-establish tendon integrity and functionality. Tissue engineering approaches aim at promoting tissue regeneration by delivering the opportune signals to the injured site combining biomaterials, cells and biochemical cues. Electrospinning is currently one of the most versatile polymer processing techniques that allows manufacturing of nano- and micro-fibres substrates. Such fibrous morphology is deemed to be an ideal substrate to convey topographical cues to cells. Here we evaluated the potential of polycaprolactone processed by means of electrospinning technology for tendon tissue engineering. Fibrous free-of-defects substrate with random and aligned fibres were successfully fabricated. Rat tenocytes were used to assess the cytocompatibility of the substrates for application as tendon tissue engineered devices. Tenocytes were able to proliferate and adapt to the substrates topography acquiring an elongated morphology, which is the precondition for oriented collagen deposition, when seeded on aligned fibres. Real time Polymerase Chain Reaction (Rt-PCR) also revealed the overall maintenance of tenocyte phenotype over 7 days culture. To verify suitability for in vivo implantation, the level of inflammatory cytokine genes expressed by THP-1 cells cultured in presence of electrospun polycaprolactone substrates was evaluated. Inflammatory response was limited. The novel preliminary in vitro work presented herein showing tenocytes compatibility and limited inflammatory cytokines synthesis suggests that electrospun polycaprolactone may be taken into consideration as substrate for tendon healing applications.
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http://dx.doi.org/10.1177/0885328218779846DOI Listing
July 2018

Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

Sci Rep 2018 07 2;8(1):9979. Epub 2018 Jul 2.

Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134, Milan, Italy.

Ex vivo expansion and differentiation of human pancreatic β-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which β-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates with tailored roughness to mimic the nanotopography of the extracellular matrix. We demonstrate that β-cells can perceive nanoscale features of the substrate and can convert these stimuli into mechanotransductive processes which promote long-term in vitro human islet culture, thus preserving β-cell differentiation and function. Proteomic and quantitative immunofluorescence analyses demonstrate that the process is driven by nanoscale topography, via remodelling of the actin cytoskeleton and nuclear architecture. These modifications activate a transcriptional program which stimulates an adaptive metabolic glucose response. Engineered cluster-assembled substrates coupled with proteomic approaches may provide a useful strategy for identifying novel molecular targets for treating diabetes mellitus and for enhancing tissue engineering in order to improve the efficacy of islet cell transplantation therapies.
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http://dx.doi.org/10.1038/s41598-018-28019-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028636PMC
July 2018

Decoration of RGD-mimetic porous scaffolds with engineered and devitalized extracellular matrix for adipose tissue regeneration.

Acta Biomater 2018 06 21;73:154-166. Epub 2018 Apr 21.

Department of Biomedicine, University Hospital of Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; Clinic of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital of Basel, Switzerland.

Fat grafting is emerging as a promising alternative to silicon implants in breast reconstruction surgery. Unfortunately, this approach does not provide a proper mechanical support and is affected by drawbacks such as tissue resorption and donor site morbidity. Synthetic scaffolds can offer a valuable alternative to address these challenges, but poorly recapitulate the biochemical stimuli needed for tissue regeneration. Here, we aim at combining the positive features of a structural, synthetic polymer to an engineered, devitalized extracellular matrix (ECM) to generate a hybrid construct that can provide a mix of structural and biological stimuli needed for adipose tissue regeneration. A RGD-mimetic synthetic scaffold OPAAF, designed for soft tissue engineering, was decorated with ECM deposited by human adipose stromal cells (hASCs). The adipoinductive potential of the hybrid ECM-OPAAF construct was validated in vitro, by culture with hASC in a perfusion bioreactor system, and in vivo, by subcutaneous implantation in nude mouse. Our findings demonstrate that the hybrid ECM-OPAAF provides proper mechanical support and adipoinductive stimuli, with potential applicability as off-the-shelf material for adipose tissue reconstruction.

Statement Of Significance: In this study we combined the functionalities of a synthetic polymer with those of an engineered and subsequently devitalized extracellular matrix (ECM) to generate a hybrid material for adipose tissue regeneration. The developed hybrid ECM-OPAAF was demonstrated to regulate human adipose stromal cells adipogenic commitment in vitro and adipose tissue infiltration in vivo. Our findings demonstrate that the hybrid ECM-OPAAF provide proper mechanical support and adipoinductive stimuli and represents a promising off-the-shelf material for adipose tissue reconstruction. We believe that our approach could offer an alternative strategy for adipose tissue reconstruction in case of mastectomy or congenital abnormalities, overcoming the current limitations of autologous fat based strategies such as volume resorption and donor site morbidity.
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http://dx.doi.org/10.1016/j.actbio.2018.04.039DOI Listing
June 2018

Exploring the potential of polyurethane-based soft foam as cell-free scaffold for soft tissue regeneration.

Acta Biomater 2018 06 12;73:141-153. Epub 2018 Apr 12.

Fondazione Filarete, Viale Ortles 22/4, 20139 Milan, Italy; CIMAINA, Dipartimento di Fisica, Università degli Studi di Milano, Via Celoria 16, 20133 Milan, Italy.

Reconstructive treatment after trauma and tumor resection would greatly benefit from an effective soft tissue regeneration. The use of cell-free scaffolds for adipose tissue regeneration in vivo is emerging as an attractive alternative to tissue-engineered constructs, since this approach avoids complications due to cell manipulation and lack of synchronous vascularization. In this study, we developed a biodegradable polyurethane-based scaffold for soft tissue regeneration, characterized by an exceptional combination between softness and resilience. Exploring the potential as a cell-free scaffold required profound understanding of the impact of its intrinsic physico-chemical properties on the biological performance in vivo. We investigated the effect of the scaffold's hydrophilic character, degradation kinetics, and internal morphology on (i) the local inflammatory response and activation of MGCs (foreign body response); (ii) its ability to promote rapid vascularisation, cell infiltration and migration through the scaffold over time; and (iii) the grade of maturation of the newly formed tissue into vascularized soft tissue in a murine model. The study revealed that soft tissue regeneration in vivo proceeded by gradual infiltration of undifferentiated mesenchymal cells though the periphery toward the center of the scaffold, where the rapid formation of a functional and well-formed vascular network supported cell viability overtime.

Statement Of Significance: Exploring the potential of polyurethane-based soft foam as cell-free scaffold for soft tissue regeneration. In this work, we address the unmet need for synthetic functional soft tissue substitutes that provide adequate biological and mechanical support to soft tissue. We developed a series of flexible cross-linked polyurethane copolymer scaffolds with remarkable fatigue-resistance and tunable physico-chemical properties for soft tissue regeneration in vivo. Accordingly, we could extend the potential of this class of biomaterials, which was so far confined for bone and osteochondral tissue regeneration, to other types of connective tissue.
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http://dx.doi.org/10.1016/j.actbio.2018.04.011DOI Listing
June 2018

Unravelling the nucleation mechanism of bimetallic nanoparticles with composition-tunable core-shell arrangement.

Nanoscale 2018 Apr;10(14):6684-6694

Laboratory of Solid-State Physics and Magnetism, KU Leuven, Celestijnenlaan 200D - Box 2414, 3001 Leuven, Belgium.

The structure and atomic ordering of Au-Ag nanoparticles grown in the gas phase are determined by a combination of HAADF-STEM, XPS and Refl-XAFS techniques as a function of composition. It is shown consistently from all the techniques that an inversion of chemical ordering takes place by going from Au-rich to Ag-rich compositions, with the minority element always occupying the nanoparticle core, and the majority element enriching the shell. With the aid of DFT calculations, this composition-tunable chemical arrangement is rationalized in terms of a four-step growth process in which the very first stage of cluster nucleation plays a crucial role. The four-step growth mechanism is based on mechanisms of a general character, likely to be applicable to a variety of binary systems besides Au-Ag.
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http://dx.doi.org/10.1039/c8nr01481gDOI Listing
April 2018

Proteomic Dissection of Nanotopography-Sensitive Mechanotransductive Signaling Hubs that Foster Neuronal Differentiation in PC12 Cells.

Front Cell Neurosci 2017 4;11:417. Epub 2018 Jan 4.

Department of Veterinary Medicine, Università degli Studi di Milano, Milan, Italy.

Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be "interpreted" by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD) allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calcium signaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive mechanotransductive signaling hubs participating in neuronal differentiation were dissected.
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http://dx.doi.org/10.3389/fncel.2017.00417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758595PMC
January 2018

Sol-gel TiO colloidal suspensions and nanostructured thin films: structural and biological assessments.

Nanotechnology 2018 Feb;29(5):055704

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.

The role of substrate topography in phenotype expression of in vitro cultured cells has been widely assessed. However, the production of the nanostructured interface via the deposition of sol-gel synthesized nanoparticles (NPs) has not yet been fully exploited. This is also evidenced by the limited number of studies correlating the morphological, structural and chemical properties of the grown thin films with those of the sol-gel 'brick' within the framework of the bottom-up approach. Our work intends to go beyond this drawback presenting an accurate investigation of sol-gel TiO NPs shaped as spheres and rods. They have been fully characterized by complementary analytical techniques both suspended in apolar solvents, by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) and after deposition on substrates (solid state configuration) by transmission electron microscopy (TEM) and powder x-ray diffraction (PXRD). In the case of suspended anisotropic rods, the experimental DLS data, analyzed by the Tirado-Garcia de la Torre model, present the following ranges of dimensions: 4-5 nm diameter (∅) and 11-15 nm length (L). These results are in good agreement with that obtained by the two solid state techniques, namely 3.8(9) nm ∅ and 13.8(2.5) nm L from TEM and 5.6(1) ∅ and 13.3(1) nm L from PXRD data. To prove the suitability of the supported sol-gel NPs for biological issues, spheres and rods have been separately deposited on coverslips. The cell response has been ascertained by evaluating the adhesion of the epithelial cell line Madin-Darby canine kidney. The cellular analysis showed that titania films promote cell adhesion as well clustering organization, which is a distinguishing feature of this type of cell line. Thus, the use of nanostructured substrates via sol-gel could be considered a good candidate for cell culture with the further advantages of likely scalability and interfaceability with many different materials usable as supports.
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http://dx.doi.org/10.1088/1361-6528/aa9ca0DOI Listing
February 2018

3D porous polyurethanes featured by different mechanical properties: Characterization and interaction with skeletal muscle cells.

J Mech Behav Biomed Mater 2017 11 14;75:147-159. Epub 2017 Jul 14.

Tensive s.r.l., Milan, Italy.

The fabrication of biomaterials for interaction with muscle cells has attracted significant interest in the last decades. However, 3D porous scaffolds featured by a relatively low stiffness (almost matching the natural muscle one) and highly stable in response to cyclic loadings are not available at present, in this context. This work describes 3D polyurethane-based porous scaffolds featured by different mechanical properties. Biomaterial stiffness was finely tuned by varying the cross-linking degree of the starting foam. Compression tests revealed, for the softest material formulation, stiffness values close to the ones possessed by natural skeletal muscles. The materials were also characterized in terms of local nanoindenting, rheometric properties and long-term stability through cyclic compressions, in a strain range reflecting the contraction extent of natural muscles. Preliminary in vitro tests revealed a preferential adhesion of C2C12 skeletal muscle cells over the softer, rougher and more porous structures. All the material formulations showed low cytotoxicity.
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http://dx.doi.org/10.1016/j.jmbbm.2017.07.018DOI Listing
November 2017

An Injectable System for Local and Sustained Release of Antimicrobial Agents in the Periodontal Pocket.

Macromol Biosci 2017 08 2;17(8). Epub 2017 May 2.

Tensive S.r.l., Via Timavo 34, 20124, Milano, Italy.

Periodontitis treatments usually require local administration of antimicrobial drugs with the aim to reduce the bacterial load inside the periodontal pocket. Effective pharmaceutical treatments may require sustained local drug release for several days in the site of interest. Currently available solutions are still not able to fulfill the clinical need for high-quality treatments, mainly in terms of release profiles and patients' comfort. This work aims to fill this gap through the development of an in situ gelling system, capable to achieve controlled and sustained release of antimicrobial agents for medium-to-long-term treatments. The system is composed of micrometer-sized β-cyclodextrin-based hydrogel (bCD-Jef-MPs), featured by a strong hydrophilic character, suspended in a synthetic block-co-polymer solution (Poloxamer 407), which is capable to undergo rapid thermally induced sol-gel phase transition at body temperature. The chemical structure of bCD-Jef-MPs was confirmed by cross-correlating data from Fourier transform infrared (FTIR) spectroscopy, swelling test, and degradation kinetics. The thermally induced sol-gel phase transition is demonstrated by rheometric tests. The effectiveness of the described system to achieve sustained release of antimicrobial agents is demonstrated in vitro, using chlorhexidine digluconate as a drug model. The results achieved in this work disclose the potential of the mentioned system in effectively treating periodontitis lesions.
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http://dx.doi.org/10.1002/mabi.201700103DOI Listing
August 2017

Electroactive Ionic Soft Actuators with Monolithically Integrated Gold Nanocomposite Electrodes.

Adv Mater 2017 Jun 18;29(23). Epub 2017 Apr 18.

Interdisciplinary Centre for Nanostructured Materials and Interfaces (CIMaINa), Physics Department, University of Milan, Via Celoria 16, 20133, Milan, Italy.

Electroactive ionic gel/metal nanocomposites are produced by implanting supersonically accelerated neutral gold nanoparticles into a novel chemically crosslinked ion conductive soft polymer. The ionic gel consists of chemically crosslinked poly(acrylic acid) and polyacrylonitrile networks, blended with halloysite nanoclays and imidazolium-based ionic liquid. The material exhibits mechanical properties similar to that of elastomers (Young's modulus ≈ 0.35 MPa) together with high ionic conductivity. The fabrication of thin (≈100 nm thick) nanostructured compliant electrodes by means of supersonic cluster beam implantation (SCBI) does not significantly alter the mechanical properties of the soft polymer and provides controlled electrical properties and large surface area for ions storage. SCBI is cost effective and suitable for the scaleup manufacturing of electroactive soft actuators. This study reports the high-strain electromechanical actuation performance of the novel ionic gel/metal nanocomposites in a low-voltage regime (from 0.1 to 5 V), with long-term stability up to 76 000 cycles with no electrode delamination or deterioration. The observed behavior is due to both the intrinsic features of the ionic gel (elasticity and ionic transport capability) and the electrical and morphological features of the electrodes, providing low specific resistance (<100 Ω cm ), high electrochemical capacitance (≈mF g ), and minimal mechanical stress at the polymer/metal composite interface upon deformation.
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http://dx.doi.org/10.1002/adma.201606109DOI Listing
June 2017

Quantitative Control of Protein and Cell Interaction with Nanostructured Surfaces by Cluster Assembling.

Acc Chem Res 2017 02 24;50(2):231-239. Epub 2017 Jan 24.

Centro Interdisciplinare Materiali e Interfacce Nanostrutturati (CIMAINA) e Dipartimento di Fisica, Università degli Studi di Milano , via Celoria 16, 20133 Milano, Italy.

The development of smart prosthetics, scaffolds, and biomaterials for tissue engineering and organ-on-a-chip devices heavily depends on the understanding and control of biotic/abiotic interfaces. In recent years, the nanometer scale emerged as the predominant dimension for processes impacting on protein adsorption and cellular responses on surfaces. In this context, the extracellular matrix (ECM) can be seen as the prototype for an intricate natural structure assembled by nanoscale building blocks forming highly variable nanoscale configurations, dictating cellular behavior and fate. How exactly the ECM nanotopography influences mechanotransduction, that is, the cellular capacity to convert information received from the ECM into appropriate responses, remains partially understood due to the complexity of the involved biological structures, limiting also the attempts to artificially reproduce the nanoscale complexity of the ECM. In this Account, we describe and discuss our strategies for the development of an efficient and large-scale bottom-up approach to fabricate surfaces with multiscale controlled disorder as substrates to study quantitatively the effect of nanoscale topography on biological entities. Our method is based on the use of supersonic cluster beam deposition (SCBD) to assemble, on a substrate, neutral clusters produced in the gas phase and accelerated by a supersonic expansion. The assembling of clusters in the ballistic deposition regime follows simple scaling laws, allowing the quantitative control of surface roughness and asperity layout over large areas. Due to their biocompatibility, we focused on transition metal oxide nanostructured surfaces assembled by titania and zirconia clusters. We demonstrated the engineering of structural and functional properties of the cluster-assembled surfaces with high relevance for interactions at the biotic/abiotic interface. We observed that isoelectric point and wettability, crucial parameters for the adhesion of biological entities on surfaces, are strongly influenced and controlled by the nanoscale roughness. By developing a high-throughput method (protein surface interaction microarray, PSIM), we characterized quantitatively the capacity of the nanostructured surfaces to adsorb proteins, showing that with increasing roughness the adsorption rises beyond what could be expected by the increase in specific area, paralleled by an almost linear decrease in protein binding affinity. We also determined that the spatial layout of the surface asperities effectively perceived by the cells mimics at the nanoscale the topographical ECM characteristics. The interaction with these features consequently regulates parameters significant for cell adhesion and mechanotransductive signaling, such as integrin clustering, focal adhesion maturation, and the correlated cellular mechanobiology, eventually impacting the cellular program and differentiation, as we specifically showed for neuronal cells.
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http://dx.doi.org/10.1021/acs.accounts.6b00433DOI Listing
February 2017

Calcium Stearate as an Effective Alternative to Poly(vinyl alcohol) in Poly-Lactic-co-Glycolic Acid Nanoparticles Synthesis.

Biomacromolecules 2017 02 18;18(2):452-460. Epub 2017 Jan 18.

Filarete Foundation, viale Ortles 22/4, 20139 Milano, Italy.

Poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are among the most studied systems for drug and gene targeting. So far, the synthesis of stable and uniform PLGA NPs has involved the use of a large excess of polyvinyl surfactants such as poly(vinyl alcohol) (PVA) and polyvinylpyrrolidone (PVP), whose removal requires multistep purification procedures of high ecological and economic impact. Hence the development of environment-friendly and cost-effective synthetic procedures for the synthesis of PLGA NPs would effectively boost their use in clinics. This work aims to address this issue by investigating more efficacious alternatives to the so far employed polyvinyl surfactants. More specifically, we developed an innovative synthetic process to achieve stable and uniformly distributed PLGA NPs that involves the use of calcium stearate (CSt), gaining benefits of its high biocompatibility and efficacy at low concentrations and avoiding consequently expensive purification steps. With the help of minimum quantities of polysorbate 60 and sorbitane monostearate, CSt-stabilized PLGA NPs with different sizes and structures were synthesized. The influence of CSt on the encapsulation efficiency of bioactive molecules has been also investigated. The effective encapsulation of both hydrophobic (curcumin) and hydrophilic (fibrinogen labeled with Alexa647) biomolecules into NPs was demonstrated by confocal microscopy, and their release quantified by spectrofluorimetric analyses. Finally, degradation and cytotoxicity studies showed that CSt stabilized NPs were stable under physiological conditions and with good biocompatibility, thus looking promising for further investigation as controlled release devices.
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http://dx.doi.org/10.1021/acs.biomac.6b01546DOI Listing
February 2017

Photocrosslinked poly(amidoamine) nanoparticles for central nervous system targeting.

Colloids Surf B Biointerfaces 2017 Mar 14;151:197-205. Epub 2016 Dec 14.

Fondazione Filarete, Viale Ortles 22/4, 20139 Milan, Italy. Electronic address:

This study presents an innovative method for the synthesis of polymeric nanoparticles (NPs) for central nervous system (CNS) targeting. The method is based on Ultraviolet light (UV)-induced crosslinking of diacrylamide-terminated oligomers of poly(amidoamine)s (PAAs), a widely used class of synthetic polymers in biomedical field research, especially in drug delivery thanks to their excellent biocompatibility and controlled biodegradability. Previous attempts aiming at preparing PAA-based NPs by self-assembly were challenged by lack of structural stability and consequently their early degradation and premature drug release. Here, the UV-induced crosslinked PAA NPs demonstrated to overcome main disadvantages of the self-assembled ones, as they showed improved stability and controlled release properties. Besides the remarkable efficiency to produce monodisperse and stable PAA NPs, the UV-induced crosslinking method is featured by great versatility and low environmental impact, since it does not require use of organic solvents and multiple purification steps. The capability of PAA NPs to encapsulate a fluorescently labelled model protein was experimentally demonstrated in this study. Cell culture experiments showed that PAA NPs were biocompatible and highly permeable across an in vitro blood-brain barrier model, thus highlighting their great potential as drug delivery vectors for CNS delivery.
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http://dx.doi.org/10.1016/j.colsurfb.2016.12.008DOI Listing
March 2017

Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways.

Front Cell Neurosci 2016 18;10:267. Epub 2016 Nov 18.

Dipartimento di Fisica, Centro Interdisciplinare Materiali e Interfacce Nanostrutturate, Università degli Studi di Milano Milan, Italy.

The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signaling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterized by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation.
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http://dx.doi.org/10.3389/fncel.2016.00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114288PMC
November 2016

An Efficient CuxO Photocathode for Hydrogen Production at Neutral pH: New Insights from Combined Spectroscopy and Electrochemistry.

ACS Appl Mater Interfaces 2016 Aug 9;8(33):21250-60. Epub 2016 Aug 9.

Department of Chemistry, University of Milan , Via Golgi 19, 20133 Milan, Italy.

Light-driven water splitting is one of the most promising approaches for using solar energy in light of more sustainable development. In this paper, a highly efficient p-type copper(II) oxide photocathode is studied. The material, prepared by thermal treatment of CuI nanoparticles, is initially partially reduced upon working conditions and soon reaches a stable form. Upon visible-light illumination, the material yields a photocurrent of 1.3 mA cm(-2) at a potential of 0.2 V vs a reversible hydrogen electrode at mild pH under illumination by AM 1.5 G and retains 30% of its photoactivity after 6 h. This represents an unprecedented result for a nonprotected Cu oxide photocathode at neutral pH. The photocurrent efficiency as a function of the applied potential was determined using scanning electrochemical microscopy. The material was characterized in terms of photoelectrochemical features; X-ray photoelectron spectroscopy, X-ray absorption near-edge structure, fixed-energy X-ray absorption voltammetry, and extended X-ray absorption fine structure analyses were carried out on pristine and used samples, which were used to explain the photoelectrochemical behavior. The optical features of the oxide are evidenced by direct reflectance spectroscopy and fluorescence spectroscopy, and Mott-Schottky analysis at different pH values explains the exceptional activity at neutral pH.
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http://dx.doi.org/10.1021/acsami.6b03345DOI Listing
August 2016

Biologically and mechanically driven design of an RGD-mimetic macroporous foam for adipose tissue engineering applications.

Biomaterials 2016 10 6;104:65-77. Epub 2016 Jul 6.

Filarete Foundation, Viale Ortles 22/4, 20139, Milano, Italy; CIMAINA, Dipartimento di Fisica, Università degli studi di Milano, Via Celoria 16, 20133, Milano, Italy.

Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. To address this need, we adopted a biologically and mechanically driven design to fabricate an RGD-mimetic poly(amidoamine) oligomer macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of in vivo vascularization and match of native tissue mechanical properties. Poly(amidoamine) oligomers were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. OPAAF's ability to support cell adhesion, proliferation and adipogenesis was assessed in vitro using epithelial, fibroblast and endothelial cells (MDCK, 3T3L1 and HUVEC respectively). In addition, in vivo subcutaneous implantation in murine model highlighted OPAAF potential to support both adipogenesis and vessels infiltration. Overall, the reported results support the use of OPAAF as a scaffold for engineered adipose tissue construct.
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http://dx.doi.org/10.1016/j.biomaterials.2016.07.004DOI Listing
October 2016

Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation.

J Nanobiotechnology 2016 Mar 9;14:18. Epub 2016 Mar 9.

CIMAINA, Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milan, 20133, Italy.

Background: Thanks to mechanotransductive components cells are competent to perceive nanoscale topographical features of their environment and to convert the immanent information into corresponding physiological responses. Due to its complex configuration, unraveling the role of the extracellular matrix is particularly challenging. Cell substrates with simplified topographical cues, fabricated by top-down micro- and nanofabrication approaches, have been useful in order to identify basic principles. However, the underlying molecular mechanisms of this conversion remain only partially understood.

Results: Here we present the results of a broad, systematic and quantitative approach aimed at understanding how the surface nanoscale information is converted into cell response providing a profound causal link between mechanotransductive events, proceeding from the cell/nanostructure interface to the nucleus. We produced nanostructured ZrO2 substrates with disordered yet controlled topographic features by the bottom-up technique supersonic cluster beam deposition, i.e. the assembling of zirconia nanoparticles from the gas phase on a flat substrate through a supersonic expansion. We used PC12 cells, a well-established model in the context of neuronal differentiation. We found that the cell/nanotopography interaction enforces a nanoscopic architecture of the adhesion regions that affects the focal adhesion dynamics and the cytoskeletal organization, which thereby modulates the general biomechanical properties by decreasing the rigidity of the cell. The mechanotransduction impacts furthermore on transcription factors relevant for neuronal differentiation (e.g. CREB), and eventually the protein expression profile. Detailed proteomic data validated the observed differentiation. In particular, the abundance of proteins that are involved in adhesome and/or cytoskeletal organization is striking, and their up- or downregulation is in line with their demonstrated functions in neuronal differentiation processes.

Conclusion: Our work provides a deep insight into the molecular mechanotransductive mechanisms that realize the conversion of the nanoscale topographical information of SCBD-fabricated surfaces into cellular responses, in this case neuronal differentiation. The results lay a profound cell biological foundation indicating the strong potential of these surfaces in promoting neuronal differentiation events which could be exploited for the development of prospective research and/or biomedical applications. These applications could be e.g. tools to study mechanotransductive processes, improved neural interfaces and circuits, or cell culture devices supporting neurogenic processes.
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http://dx.doi.org/10.1186/s12951-016-0171-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784317PMC
March 2016