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4. Functional and Smart Materials by Electrospinning for Advanced Applications.

Author

Raisch, M., Genovese, D., Fornaia, G., Zaccheroni, N., Schmidt, S. B., Focarete, M. L., Sommer, M., and Gualandi, C.

Subjects
SMART materials, ELECTROSPINNING, COMPOSITE materials, CHEMICAL synthesis, FIBERS
Abstract

The processing of advanced and functional polymers with electrospinning brings enormous potentialities to either improve or extend their properties. A representative example is the field of mechanochromic materials, potentially exploitable for imaging mechanical damages and stress/strain distribution. An effective stress-sensing material must respond to low deformation with a detectable color change that should be quickly reversible upon force unloading. In the present study we processed a spiropyran main chain polymer by electrospinning and fibers with a proper weaving were included in a PDMS elastomeric matrix to produce composite materials. Measurements of fiber birefringence demonstrated that, after electrospinning, polymer chains were successfully vitrified in a highly oriented conformation. Stress strain tests, coupled with a real-time detection of color, showed that fibers displayed a clear color change after only 5% of deformation. When these highly sensitive mechanochromic nanofibers were incorporated in a PDMS matrix, either anisotropic or isotropic mechanochromic behavior was achieved, depending on fiber alignment. The unique mechanochromic properties of the proposed composites, i.e. reversibility, sensitivity and directionality, derive by a smart combination of chemical synthesis, processing and composite design, respectively, and make them ideal to act as real-time stress/strain-sensing materials. [ABSTRACT FROM AUTHOR]

Published
2019
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5. Structure-morphology correlation in electrospun fibers of semicrystalline polymers by simultaneous synchrotron SAXS-WAXD

Author

Gazzano, M., Gualandi, C., Zucchelli, A., Sui, T., Korsunsky, A. M., Reinhard, C., Focarete, M. L., Gazzano, M, Gualandi, C., Zucchelli, A., Sui, T., Korsunsky, A.M., Reinhard, C., and Focarete, M.L.

Subjects
chemistry.chemical_classification, Materials science, Morphology (linguistics), Electrospinning, Polymers and Plastics, Small-angle X-ray scattering, Organic Chemistry, Polyacrylonitrile, Polymer, Microstructure, Crystal phase, Crystallinity, chemistry.chemical_compound, chemistry, Synchrotron SAXS-WAXD, Materials Chemistry, Lamellar structure, Composite material
Abstract

Simultaneous SAXS-WAXD measurements are carried out to gain information about the microstructure and the molecular orientation developed by polymeric chains during the electrospinning process. Three semicrystalline polymers were studied, namely polyacrylonitrile, Nylon 6,6, and poly(ethylene oxide), as non-woven mats with either randomly arranged or aligned electrospun fibers. Mat thermal and morphological properties are investigated, together with their structural details in order to derive their hierarchical structure from the macro to the nano-scale. SAXS patterns have an elliptical shape with the main axis along the equator direction. No reflections are noticeable along the meridional direction, suggesting that the investigated electrospun fibers have a fibrillar structure with no trace of lamellar morphology. Combining the values of the unit cell and of the crystal size it can be concluded that in the fibers the ordered domains are organized into a bundle of fibrils due to the lateral aggregation of roughly 10 unit cells and the regular ordering of about either 50 (Nylon 6,6 and polyacrylonitrile) or 25 (poly(ethylene oxide)) cells in the chain direction.

Published
2015

9. Atmospheric pressure non-thermal plasma for the production of composite materials

Author

Bloise, Nora, primary, Sampaolesi, Maurilio, additional, Visai, Livia, additional, Colombo, V., additional, Gherardi, M., additional, Focarete, M. L., additional, Gualandi, C., additional, Laurita, R., additional, Liguori, A., additional, Mauro, Nicolo, additional, Manfredi, Amedea, additional, Ferruti, Paolo, additional, and Ranucci, Elisabetta, additional

Published
2015
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11. Polyvinylidene Difluoride-Polyethyleneoxide Blends for Electrospun Separators in Li-Ion Batteries.

Author

Monaca, A. La, De Giorgio, F., Focarete, M. L., Fabiani, D., Zaccaria, M., and Arbizzani, C.

Subjects
LITHIUM-ion batteries, POLYVINYLIDENE fluoride
Abstract

Polyvinylidenedifluoride (PVdF) and polyethyleneoxide (PEO) are blended and electrospun in order to obtain membranes suitable as Li-ion battery separators. The separators are characterized, and their properties investigated and compared with those of PVdF and commercial separators. The PVdF-PEO based separators ensure increased conductivities, greater electrolyte uptake and higher porosities than commercial polyolefines, all factors that improve cell performance. They are also safer than PVdF separators thanks to lower shutdown temperature, even if their mechanical properties are not yet comparable with those of the latter. [ABSTRACT FROM AUTHOR]

Published
2017
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13. Atmospheric plasma surface modification of electrospun poly(L-lactic acid): Effect on mat properties and cell culturing

Author

Alessandri, M., primary, Calza, L., additional, Colombo, V., additional, Dolci, L. S., additional, Fiorani, A., additional, Focarete, M. L., additional, Ghedini, E., additional, Gherardi, M., additional, Gualandi, C., additional, Laurita, R., additional, Liguori, A., additional, Quiroga, S. D., additional, and Sanibondi, P., additional

Published
2013
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14. Structural Study of Poly(<SCP>l</SCP>-lactic acid) Spherulites

Author

Gazzano, M., Focarete, M. L., Riekel, C., and Scandola, M.

Abstract

Spherulites of poly(l-lactic acid) (PLLA) and of its blends with atactic poly(3-hydroxybutyrate) (a-PHB, from 10 to 75 wt %) were investigated by microfocus X-ray diffraction using synchrotron radiation. Radial scans in 5 μm steps with 3 μm beam diameter were performed. In PLLA, tens of identical diffraction images were collected. The unit cell a-axis was radially oriented, and the other axes lacked any specific orientation. In contrast, all PLLA/a-PHB blends showed a periodic change of diffraction pattern with increasing distance from the spherulite center. In all cases, the a-axis lay along the radius, while the b- and c-axes rotated about a with a defined periodicity. The unit cell twisting frequency increased with a-PHB content and closely matched the band spacing observed by polarized optical microscopy, which changed from 250 to 60 μm when the amount of a-PHB increased from 10 to 75 wt %. Concomitantly, a gradual broadening of all X-ray reflections was observed.

Published
2004

15. Copolymers of ω-Pentadecalactone and Trimethylene Carbonate from Lipase Catalysis:  Influence of Microstructure on Solid-State Properties

Author

Focarete, M. L., Gazzano, M., Scandola, M., Kumar, A., and Gross, R. A.

Abstract

Unusual copolymers were prepared by Candida antarctica Lipase B (Novozyme-435) catalyzed copolymerization of ω-pentadecalactone (PDL) with trimethylene carbonate (TMC). Atypical solid-state properties were revealed by thermogravimetric analysis, differential scanning calorimetry (DSC), and X-ray diffraction analyses. Thermal degradation of poly(PDL−TMC) occurs in two steps:  the first well above the degradation range of poly(TMC) and the other at a temperature (430 °C) comparable with that of poly(PDL) decomposition. Thermal stability of PDL−TMC copolymers increases with randomization of comonomer distribution. All poly(PDL−TMC) investigated are highly crystalline, even those with equimolar comonomer content and close-to-random distribution. This result indicates that PDL and TMC units cocrystallize. The copolymers show two melting processes:  the higher one at about 90 °C, i.e., close to poly(PDL) melting, and the other 30 °C lower. The relative intensity of the two phenomena changes with copolymer microstructure. The X-ray diffraction patterns of poly(PDL−TMC) are practically identical to that of poly(PDL) in the range 10° &lt; 2θ &lt; 80°. Significant differences appear in the low-angle region below 2θ = 10°, indicating in some copolymers the presence of crystals with higher periodicity than poly(PDL) along the chain direction. Comparison of DSC and X-ray results with copolymer microstructure shows that two crystal phases can develop in poly(PDL−TMC):  poly(PDL) crystals and&sbd;whenever a significant amount of heterodiads are present&sbd;another lower melting crystal phase, characterized by a larger fiber axis periodicity and possibly associated with crystallization of alternate PDL−TMC sequences.

Published
2002
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16. Miscibility and Mechanical Properties of Blends of (<SCP>l</SCP>)-Lactide Copolymers with Atactic Poly(3-hydroxybutyrate)

Author

Focarete, M. L., Scandola, M., Dobrzynski, P., and Kowalczuk, M.

Abstract

Poly(l-lactide-co-glycolide) (PLLAGA) copolymers and poly(dl-lactide) (PDLLA) were synthesized using the low toxicity compound zirconium(IV) acetylacetonate. Blends with synthetic atactic poly(3-hydroxybutyrate) (a-PHB) were prepared in film form by solvent casting. The solid-state properties of the plain polymers and of the blends were investigated by thermogravimetric analysis coupled with mass spectrometry (TGA−MS), differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD) and stress−strain measurements. The a-PHB/PDLLA blends are miscible over the whole composition range. On the other hand, blends of a-PHB with PLLAGA copolymers containing 5 and 16 mol % of GA units show partial miscibility. The solubility limit of a-PHB in PLLAGA is around 20 wt % in both cases. In the blends, the a-PHB in excess to this value segregates as a pure phase. The mechanical properties of blend films of either PDLLA or PLLAGA containing up to 50 wt % of a-PHB can be modulated by changing blend composition. The tenacity progressively increases by the addition of a-PHB not only in the miscible amorphous a-PHB/PDLLA system but also in the partially miscible a-PHB/PLLAGA blends.

Published
2002
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17. Bacterial Poly(3-hydroxybutyrate):  An Optical Microscopy and Microfocus X-ray Diffraction Study

Author

Gazzano, M., Focarete, M. L., Riekel, C., and Scandola, M.

Abstract

Two-dimensional spatially resolved microfocus X-ray diffraction has been used to investigate spherulites of pure bacterial poly(3-hydroxybutyrate) (PHB) and of a blend of natural and synthetic atactic PHB (a-PHB) crystallized at a relatively high temperature (Tc = 140 °C). Both samples investigated contained practically two-dimensional spherulites, characterized by wide extinction bands (band spacing > 80 μm). The X-ray diffraction patterns confirmed that the unit cell a-axis is oriented along the spherulite radius in PHB and that the same is true for the a-PHB containing blend. Comparison of the matrix of diffraction patterns with the polarized optical micrograph of the scanned sample area indicated a very clear correlation between pattern changes and banding, yielding a straightforward picture of the structural variations within the spherulite.

Published
2000

18. Further Evidence of Crystallinity-Induced Biodegradation of Synthetic Atactic Poly(3-hydroxybutyrate) by PHB-Depolymerase A from Pseudomonas lemoignei. Blends of Atactic Poly(3-hydroxybutyrate) with Crystalline Polyesters

Author

Focarete, M. L., Ceccorulli, G., Scandola, M., and Kowalczuk, M.

Abstract

Blends of atactic poly[(R,S)-3-hydroxybutyrate], a-PHB, with poly(ε-caprolactone), PCL, and with poly(l-lactic acid), PLLA, were obtained in the form of compression-molded films. The phase behavior of the blends was different:  a-PHB and PCL were totally immiscible, whereas a-PHB/PLLA blends were miscible over the whole composition range. Biodegradation experiments were carried out on the blends and on the plain polymers in a buffered solution of PHB-depolymerase A from Pseudomonas lemoignei (Tris−HCl, pH = 8, T = 37 °C). None of the pure blend components (a-PHB, PCL, PLLA) showed any weight loss upon enzyme exposure. Conversely, both a-PHB/PCL and a-PHB/PLLA blends biodegraded. Analysis of the biodegradation products and of blend composition changes during biodegradation demonstrated that in both blends only the a-PHB component undergoes enzymatic hydrolysis. The results support the hypothesis that the crystalline polyester blended with a-PHB promotes a-PHB enzymatic hydrolysis by providing stable binding sites to the enzyme. The dependence of biodegradation rate on blend composition is different in (immiscible) a-PHB/PCL and (miscible) a-PHB/PLLA blends and can be explained in terms of different phase behavior and morphology.

Published
1998

19. Bioassimilation of Atactic Poly[(R,S)-3-hydroxybutyrate] Oligomers by Selected Bacterial Strains

Author

Focarete, M. L., Scandola, M., Jendrossek, D., Adamus, G., Sikorska, W., and Kowalczuk, M.

Abstract

Water-soluble a-PHB oligomers (from dimer to dodecamer) were synthesized by anionic oligomerization of (R,S)-β-butyrolactone and characterized by electrospray ionization tandem mass spectrometry (ESI-MSn). The oligomers were analogous to the degradation products remaining after enzymatic hydrolysis of synthetic high molecular weight atactic poly[(R,S)-3-hydroxybutyrate] by extracellular PHB depolymerases. Selected bacterial strains were used to test bioassimilability of a-PHB oligomers. It was found that not only two PHB-degrading bacteria (Alcaligenes faecalis T1 and Comamonas sp.) but also a non-PHB-degrading bacterium (Ralstonia eutropha H16) could grow on a-PHB oligomers as sole source of carbon and energy. Utilization of a-PHB oligomers by the three bacterial strains (total oligomer consumption and molecular weight distribution changes) was investigated by ESI-MS. Total oligomer consumption (tested after 30 h of bacterial growth) followed the same trend as the observed bacterial growth (A. faecalis > Comamonas sp. > R. eutropha). Mineralization of a-PHB oligomers demonstrates total biodegradability of synthetic high molecular weight atactic poly[(R,S)-3-hydroxybutyrate].

Published
1999

20. Crystallinity-Induced Biodegradation of Novel [(R,S)-β-Butyrolactone]-b-pivalolactone Copolymers

Author

Scandola, M., Focarete, M. L., Gazzano, M., Matuszowicz, A., Sikorska, W., Adamus, G., Kurcok, P., Kowalczuk, M., and Jedlinski, Z.

Abstract

Novel model block copolymers of (R,S)-β-butyrolactone with pivalolactone (PVL) are prepared in order to define the effect of crystalline domains provided by poly(pivalolactone) on the biodegradability of atactic poly(β-butyrolactone), a-PHB. The “living” a-PHB is synthesized from racemic β-butyrolactone, in the presence of potassium alkoxide/18-crown-6 complex, and such a living polymer is applied for polymerization of PVL, yielding block copolymers, a-PHB-b-PPVL, of tailored molecular weight and composition. The copolymers contain an amorphous phase with Tg = 5 °C, associated with the a-PHB block, and a high melting crystalline phase, whose amount increases with PPVL content. Films of copolymers containing 9 (PVL9), 17 (PVL17), and 23 mol % of PPVL (PVL23) are exposed to PHB-depolymerase A from Pseudomonas lemoignei (37 °C, Tris−HCl buffer pH = 8). While plain a-PHB does not biodegrade, the biodegradation rate of a-PHB-b-PPVL copolymers increases (PVL9 ≪ PVL17 &lt; PVL23) along with the increase of crystalline PPVL domains. The biodegradation rate of PVL23 is similar to that of natural (crystalline) PHB. On the basis of a comparison of a-PHB-b-PPVL composition changes (by 1H NMR) with weight loss during biodegradation experiments, it is concluded that in the copolymers studied only the a-PHB block is attacked by the enzyme and that the crystalline block of nonbiodegradable PPVL efficiently promotes enzymatic attack to a-PHB, by providing a binding support to the enzyme.

Published
1997

21. Polymer Blends of Natural Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and a Synthetic Atactic Poly(3-hydroxybutyrate). Characterization and Biodegradation Studies

Author

Scandola, M., Focarete, M. L., Adamus, G., Sikorska, W., Baranowska, I., Swierczek, S., Gnatowski, M., Kowalczuk, M., and Jedlinski, Z.

Abstract

Blends of synthetic atactic poly(3-hydroxybutyrate) (a-PHB) with a natural bacterial isotactic copolymer of 3-hydroxybutyrate with 3-hydroxyvalerate (PHBV) containing 10 mol % of 3HV units were prepared using a simple casting procedure. In the range of compositions explored (10−50% a-PHB), blends of bacterial PHBV and synthetic atactic a-PHB were miscible in the melt and solidified with spherulitic morphology. The influence of a-PHB content on the thermal and mechanical properties of the blends was evaluated. The degree of crystallinity decreased with increasing content of a-PHB in the film samples, and the elongation at break for a sample containing 50% of a-PHB was 30-fold that of pure PHBV. Degradation experiments, both hydrolytic (pH = 7.4, T = 70 °C) and enzymatic (PHB-depolymerase A from Pseudomonas lemoignei, Tris-HCl buffer (pH = 8), T = 37 °C), were performed for both polymers and polymer blends. The rate of enzymatic degradation of the blends was higher than that of PHBV and increased with a-PHB content in the blends studied, whereas pure a-PHB did not biodegrade under these conditions. 3-Hydroxybutyric acid and its dimer were identified by HPLC as biodegradation products of both pure PHBV and its blends with a-PHB. Higher oligomers up to heptamer were detected as degradation products of the blends by APCI-MS and ESI-MS.

Published
1997

22. Simple Kinetic Model for the Heterogeneous Enzymatic Hydrolysis of Natural Poly(3-hydroxybutyrate)

Author

Scandola, M., Focarete, M. L., and Frisoni, G.

Abstract

The kinetics of the enzymatic degradation of bacterial poly(3-hydroxybutyrate) (PHB) is studied using PHB-depolymerase A from Pseudomonas lemoignei (Tris−HCl buffer, pH = 8, T = 37 °C). Biodegradation experiments are performed on PHB in the form of both compression-molded films and powder suspension. From WAXS and DSC measurements the two substrates show the same crystalline fraction. The rate of hydrolysis of PHB films is determined by gravimetry and also through spectrophotometric quantification of the hydrolysis products at λ = 210 nm. For the suspension of PHB particles, a turbidimetric determination of the biodegradation rate is applied. A simple two-step kinetic model is proposed, which predicts that the hydrolysis rate per unit substrate surface area reaches a plateau at high enzyme concentrations. The model satisfactorily describes the enzymatic degradation results of PHB film and PHB powder suspension, provided that the remarkable changes of exposed area caused by enzymatic attack to the latter substrate are taken into account. Analysis of the enzymatic degradation results yields analogous hydrolysis rate constants for film (1.48 μg cm-2 min-1) and powder suspension (1.42 μg cm-2 min-1).

Published
1998

24. Collagen functionalisation by plasma coupled to chemical grafting

Author

Cipolla, L., Russo, L., Lupo, C., Zanini, S., Riccardi, C., Panseri, S., Russo, A., Marcacci, M., Andrea Fiorani, Gualandi, C., Focarete, M. L., Nicotra, F., L. Cipolla, L. Russo, C. Cupo, S. Zanini, C. Riccardi, S. Pansieri, A. Russo, M. Marcacci, A. Fiorani, C. Gualandi, M.L. Focarete, and F. Nicotra

Subjects
Electrospinning, BIOMATERIAL SURFACE MODIFICATIONS, TISSUE ENGINEERING, COLLAGEN
Abstract

The promising trends in biotechnology and tissue engineering are based on the development of advanced materials with biomimetic features in order to recreate the native environment promoting the appropriate cell behavior for tissue regeneration. Cell therapy together with novel functionalized biomaterials represent a very promising approach in regenerative medicine for cartilage regeneration. Articular cartilage exhibits a well-ordered organization with an extracellular matrix arranged as a network of collagen fibers and proteoglycans that allow for cell adhesion, mechanical support, transduction of chemical and mechanical signals from the surrounding tissue to the cells. Electrospun materials are considered highly promising scaffolds for cartilage tissue engineering given their specific fibrous morphology that resembles the fibrous component of tissue extracellular matrix. Many synthetic and natural polymers have been successfully electrospun to obtain scaffolds. Among natural polymers, collagen is universally applied as biomaterial in regenerative medicine because of its unique biocompatibility, and structural property. Robust techniques for surface “biodecoration” are currently required and the appropriate surface functionalization still remains a critical variable for the optimal performance of a wide range of biomaterials. Covalent bonding of bioactive molecules to material surface is a valid strategy in order to allow a sufficiently strong and specific affinity of biomolecules with the surface itself; in addition covalent bonding may permit site-directed immobilization and preservation of specific conformation and exposition to control biological responses. Plasma processes allow to tune surface properties of materials with negligible effect on their bulk. The need of improving cell/surface interaction has decisively introduced plasma techniques in the field of biomaterials; In addition, a wide range of compounds can be chosen as a monomer for plasma polymerization, providing a great diversity of possible surface modifications with different functional groups, including amine, anhydride, epoxide, carboxylic acid, cyano, halide, hydroxyl, furfuryl, and perfluoroalkyl.

25. Study on the polarization process for piezoelectric nanofibrous layers

Author

G. Selleri, M. E. Gino, L. Gasperini, M. Zanoni, C. Gualandi, M. L. Focarete, D. Fabiani, Selleri, G., Gino, M. E., Gasperini, L., Zanoni, M., Gualandi, C., Focarete, M. L., and Fabiani, D.

Subjects
piezolectric, nanofibers, energy harversting, sensing, polymers
Published
2021

26. Electrospun filters for organic pollutants removal

Author

Anitha Senthamizhan, Tamer Uyar, Brabu Balusamy, Focarete, M. L., Gualandi, C., and Ramakrishna, S.

Subjects
Pollutant, Contact time, 02 engineering and technology, Pesticide, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, 0104 chemical sciences, law.invention, Adsorption, Functional importance, Hazardous waste, law, Environmental chemistry, Environmental science, Composite membrane, 0210 nano-technology, Filtration
Abstract

Chapter 6 Increasing demand for access to clean and safe water around the globe emphasizes the development of new technologies for removing environmental pollutants. Especially, organic pollutants including dyes, volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), pesticides, herbicides, and antibiotics prominently affect environmental health due to their hazardous nature. In the past several decades, advancements in electrospun fibrous membranes have resulted as an efficient filtering platform for removal of various pollutants in water, air, and soil. Electrospun nanofibers are efficient filters complementing their unique feature of accommodating a variety of functional molecules. The choice of material and the effect of experimental condition including pH, contact time, and adsorbent dosage on pollutant removal efficiency have been extensively reviewed previously. Our chapter focuses on recent progress in the developments of the electrospun functional nanofibrous composite membrane for various organic pollutants removal.

Published
2018

28. Fabrication and characterization of electrospun scaffolds for tendon reconstruction

Author

A. Sensini, M. L. Focarete, C. Gualandi, A. Zucchelli, G. Tozzi, L. Boyle, G. Reilly, L. Cristofolini, Sensini, A., Focarete, M. L., Gualandi, C., Zucchelli, A., Tozzi, G., Boyle, L., Reilly, G., and Cristofolini, L.

Subjects
technology, industry, and agriculture, Electrospinning, tendon tissue engineering, x-ray tomography, cell viability
Abstract

Tendon injuries represent an unsolved clinical need. Electrospun bundles using blends of poly-L-lactic acid (PLLA) and collagen (Coll) in different percentages were prepared by an ‘ad hoc’ crosslinking system to increase the mechanical properties and reduce the loss of collagen after ageing in a physiological environment. The mechanical properties and cell proliferation were tested. High-resolution x- ray tomography was also performed to investigate the morphologyof the bundles.

Published
2017

30. Carbon on poly(ε-caprolactone) (PCL) ink-jet printed sensor for monitoring cell cultures of myoblasts

Author

Mariagrazia Marziano, Sarah Tonello, Maurizio Memo, Maurizio Toselli, Michela Borghetti, Marialaura Serzanti, Emilio Sardini, Maria Letizia Focarete, Stefano Pandini, Daniela Uberti, Patrizia Dell'Era, Mauro Serpelloni, Chiara Gualandi, Andrea Merlettini, Massimo Messori, Nicola Lopomo, Marziano, M., Tonello, S., Serzanti, M., Borghetti, M., Lopomo, N. F., Serpelloni, M., Pandini, S., Merlettini, A., Gualandi, C., Focarete, M. L., Messori, M., Toselli, M., Uberti, D., Memo, M., Dell’Era, P., and Sardini, E.

Subjects
Scaffold, Materials science, Biocompatibility, Biomedical Engineering, Bioengineering, 02 engineering and technology, Substrate (printing), 010402 general chemistry, 01 natural sciences, Cell monitoring, Electrochemical Cell-substrate Impedance Spectroscopy (ECIS), Ink-jet printed sensors, chemistry.chemical_compound, Composite material, Cell adhesion, Inkwell, Ink-jet printed sensor, Adhesion, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Dielectric spectroscopy, chemistry, 0210 nano-technology, Caprolactone, Biomedical engineering
Abstract

Nowadays techniques for sensitive non-invasive, real-time monitoring of cell differentiation and maturation are highly demanded. In light of this, the development of electrochemical printed sensors impedance-based could represent a promising tool. In the present work, we developed 2D ink-jet printed sensors for myoblasts adhesion monitoring, using carbon-based ink on a substrate consisting in non-woven electrospun mats made in crosslinked poly(ε-caprolactone) (PCL). First of all, sensors printability was optimized and the biocompatibility tested. In order to determine the possibility to employ the prepared systems as scaffolds for dynamic cellular cultures, the mechanical response of the PCL scaffold was evaluated through the application of cyclic deformation tests. After that, electrical characterization of ink and substrate was performed, followed by electrochemical impedance-based measurements to evaluate myoblasts adhesion. Biocompatibility assessment showed good results for both carbon and PCL. Mechanical tests findings suggested that a training of 50 cycles and a proper value of strain should be applied before the cell seeding, in order to ensure a subsequent controlled strain amplitude. The sensorized scaffold allowed us to correlate cell adhesion with an increase of impedance module, in agreement with biocompatibility testing. Thus, this first preliminary testing suggested that this non-invasive impedance spectroscopy-based measurement system can be used for sensitive monitoring of cells adhesion, in static and moreover, as suggested from mechanical characterization, in dynamic conditions.

Published
2017

34. Hierarchical electrospun tendon-ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions.

Author

Sensini A, Cristofolini L, Zucchelli A, Focarete ML, Gualandi C, DE Mori A, Kao AP, Roldo M, Blunn G, and Tozzi G

Subjects
Biocompatible Materials chemistry, Biomimetic Materials, Cells, Cultured, Collagen physiology, Fibroblasts cytology, Fibroblasts ultrastructure, Humans, Ligaments pathology, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Nanofibers chemistry, Polyesters chemistry, Regenerative Medicine, Tendons physiology, Tomography, X-Ray, Fibroblasts physiology, Tissue Engineering methods, Tissue Scaffolds chemistry
Abstract

The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high-resolution X-ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L-lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads. LAY DESCRIPTION: To enhance the regeneration of injured tendons and ligaments cells need to growth on dedicated structures (scaffolds) with mechanical properties and a fibrous morphology similar to the natural tissue. In particular, the morphological organisation of scaffolds is fundamental in leading cells to colonise them, regenerating the collagen extracellular matrix. Electrospinning is a promising technique to produce fibres with a similar to the human collagen fibres, suitable to design complex scaffolds able to guide cells in the reconstruction of the natural tissue. Moreover, it is well established that the cyclic stretching of these scaffolds inside dedicated systems called bioreactors, can speed up cells growth and their shape modification. The aim of the present study was to investigate how hierarchically structured electrospun scaffolds, made of resorbable material such as poly(L-lactic acid) and collagen, could induce morphological changes in human fibroblasts, while cultured during static and dynamic conditions. These scaffolds were composed by an external electrospun membrane that grouped inside it a ring-shaped bundle, made of axially aligned nanofibres, resembling the morphological arrangement of tendon and ligament tissue. After 7 days of parallel cultures, the scaffolds were investigated using the following imaging techniques: (i) high-resolution X-ray tomography (XCT); (ii) scanning electron microscopy (SEM); (iii) fluorescence microscopy and (iv) histology. The results showed that fibroblasts were able to grow on the external nanofibrous sheath of the static scaffolds, by elongating themselves along their circumference. The dynamic cultures revealed instead a preferential axial orientation of fibroblasts grown on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds allowed an axial distribution of the fibroblasts along the nanofibres direction. This study has demonstrated that the electrospun hierarchically structured scaffolds investigated can modify the fibroblasts morphology both in static and dynamic conditions, in relation with the direction of the applied loads., (© 2019 The Authors Journal of Microscopy © 2019 Royal Microscopical Society.)

Published
2020
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35. High-resolution x-ray tomographic morphological characterisation of electrospun nanofibrous bundles for tendon and ligament regeneration and replacement.

Author

Sensini A, Cristofolini L, Focarete ML, Belcari J, Zucchelli A, Kao A, and Tozzi G

Subjects
Caprolactam analogs & derivatives, Collagen, Ligaments, Microscopy, Electron, Scanning, Polyesters, Polymers, Regeneration, Tendons, Biocompatible Materials, Materials Testing, Nanofibers, Tissue Scaffolds, X-Ray Microtomography methods
Abstract

Repair of ligaments and tendons requires scaffolds mimicking the spatial organisation of collagen in the natural tissue. Electrospinning is a promising technique to produce nanofibres of both resorbable and biostable polymers with desired structural and morphological features. The aim of this study was to perform high-resolution x-ray tomography (XCT) scans of bundles of Nylon6.6, pure PLLA and PLLA-Collagen blends, where the nanofibres were meant to have a predominant direction. Characterisation was carried out via a dedicated methodology to firmly hold the specimen during the scan and a workflow to quantify the directionality of the nanofibres in the bundle. XCT scans with 0.4 and 1.0 μm voxel size were successfully collected for all bundle compositions. Better image quality was achieved for those bundles formed by thicker nanofibres (i.e. 0.59 μm for pure PLLA), whereas partial volume effect was more pronounced for thinner nanofibres (i.e. 0.26 μm for Nylon6.6). As expected, the nanofibres had a predominant orientation along the axis of the bundles (more than 20% of the nanofibres within 3° and more than 60% within 18° from the bundle axis), with a Gaussian-like dispersion in the other directions. The directionality assessment was validated by comparison against a similar analysis performed on SEM images: the XCT analysis overestimated the amount of nanofibres very close to the bundle axis, especially for the materials with thinnest nanofibres, but adequately identified the amount of nanofibres within 12°. LAY DESCRIPTION: Repair of ligaments and tendons requires dedicated materials (scaffolds) mimicking the spatial organisation of the collagen (the main material composing such natural tissue). Electrospinning is a promising technique that allows production of fibres with nanometric dimension using high voltage to stretch very tiny drops of polymeric solutions. Electrospinning allows processing both polymers that can be resorbed by the host tissue, and nonresorbable ones, to obtain the desired structural and morphological features by arranging the nanofibres in bundles. The aim of this study was to perform high-resolution x-ray computed tomography (XCT) scans of bundles, where the nanofibres were meant to have a predominant direction. The investigation included bundles of different compositions: a biostable polymer (Nylon) and bioresorbable ones (pure Poly-L-lactic acid (PLLA) and PLLA-Collagen blends). The electrospun bundles were produced using a validated method (Sensini et al 2017: https://doi.org/10.1088/1758-5090/aa6204). To this end, we developed a dedicated methodology to scan such small specimens, and a workflow to quantify the directionality of the nanofibres in the bundle. For all the compositions, XCT scans with extremely high resolution (i.e. down to 0.4 μm) were successfully collected. As expected, better images were obtained for those bundles where the nanofibres were thicker than the scanning resolution (i.e. 0.59 μm for pure PLLA). The images of the thinnest nanofibres (i.e. 0.26 μm for Nylon) were poorer because the fibre diameter was smaller than the resolution (partial volume effect). The nanofibres had a predominant orientation along the axis of the bundles (more than 60% of the nanofibres were within 18° from the bundle axis). The nanofibres had a Gaussian-like dispersion in the other directions. As this is the first time that XCT is used to quantify the directionality of this kind of bundles, the directionality assessment was further validated by comparison against a similar analysis performed on SEM images. Overall, this study has demonstrated the usefulness and reliability of using high-resolution x-ray computed tomography (XCT) scans to investigate the morphology of polymeric scaffolds made of electrospun nanofibres., (© 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.)

Published
2018
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36. Influence of biological matrix and artificial electrospun scaffolds on proliferation, differentiation and trophic factor synthesis of rat embryonic stem cells.

Author

Alessandri M, Lizzo G, Gualandi C, Mangano C, Giuliani A, Focarete ML, and Calzà L

Subjects
Animals, Apoptosis, Brain-Derived Neurotrophic Factor genetics, Brain-Derived Neurotrophic Factor metabolism, Caspase 3 metabolism, Cell Culture Techniques, Cell Survival, Cells, Cultured, Culture Media, Electrochemical Techniques, Extracellular Matrix metabolism, Gene Expression, Lactic Acid chemistry, Nerve Growth Factor metabolism, Octamer Transcription Factor-3 genetics, Octamer Transcription Factor-3 metabolism, Polyesters, Polymers chemistry, Rats, Vascular Endothelial Growth Factor A genetics, Vascular Endothelial Growth Factor A metabolism, Cell Differentiation, Cell Proliferation, Embryonic Stem Cells physiology, Paracrine Communication, Tissue Scaffolds chemistry
Abstract

Two-dimensional vs three-dimensional culture conditions, such as the presence of extracellular matrix components, could deeply influence the cell fate and properties. In this paper we investigated proliferation, differentiation, survival, apoptosis, growth and neurotrophic factor synthesis of rat embryonic stem cells (RESCs) cultured in 2D and 3D conditions generated using Cultrex® Basement Membrane Extract (BME) and in poly-(L-lactic acid) (PLLA) electrospun sub-micrometric fibres. It is demonstrated that, in the absence of other instructive stimuli, growth, differentiation and paracrine activity of RESCs are directly affected by the different microenvironment provided by the scaffold. In particular, RESCs grown on an electrospun PLLA scaffolds coated or not with BME have a higher proliferation rate, higher production of bioactive nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) compared to standard 2D conditions, lasting for at least 2 weeks. Due to the high mechanical flexibility of PLLA electrospun scaffolds, the PLLA/stem cell culture system offers an interesting potential for implantable neural repair devices., (© 2013.)

Published
2014
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37. A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids.

Author

Handrick R, Reinhardt S, Focarete ML, Scandola M, Adamus G, Kowalczuk M, and Jendrossek D

Subjects
Amino Acid Sequence, Carboxylic Ester Hydrolases isolation & purification, Electrophoresis, Polyacrylamide Gel, Hot Temperature, Hydrogen-Ion Concentration, Lipase chemistry, Molecular Sequence Data, Protein Binding, Substrate Specificity, Temperature, Time Factors, Bacterial Proteins, Carboxylic Ester Hydrolases chemistry, Gram-Negative Anaerobic Straight, Curved, and Helical Rods enzymology, Hydrolases chemistry, Polyesters chemistry, Polymers chemistry
Abstract

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.

Published
2001
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