Valentina Cirillo

Influence of Gelatin Cues in PCL Electrospun Membranes on Nerve Outgrowth

The design of functionalized polymers that can elicit specific biological responses and the development of methods to fabricate new devices that incorporate biological cues are of great interest to the biomedical community. The realization of nanostructured matrices that exhibit biological properties and that comprise fibers with diameters of similar scale to those of the natural extracellular matrix (ECM) would enable the provision of tailored materials for tissue engineering. Accordingly, the goal of this work is to create a biologically active functionalized electrospun matrix capable of guiding neurite growth for the regeneration of nerve tissue. In this study, nanoscale electrospun membranes made of poly ε-caprolactone enhanced with gelatin from calf skin were investigated to validate their biological response under in vitro culture of PC-12 nerve cells. Preliminary observations from SEM studies supported by image analysis highlighted the nanoscale texture of the scaffold with fiber diameters equal to 0.548 ± 0.140 μm. In addition, contact angle measurements confirmed the hydrophilic behavior of the membranes, ascribable to the gelatin content. We demonstrate that the balance of morphological and biochemical properties improves all the fundamental biological events of nerve regeneration, enhancing cell adhesion, proliferation, and differentiation in comparison with PCL nanofibrous scaffolds, as well as supporting the neurite outgrowth.

hMSC interaction with PCL and PCL/gelatin platforms: A comparative study on films and electrospun membranes:

Polycaprolactone (PCL) and PCL/gelatin membranes and films were fabricated by electrospinning and solvent casting. A systematic analysis of the morphology evolution, as degradation occurred, was made to separate the contribution of fiber nanotexture and gelatin biochemical signal on cell adhesion and proliferation. Field emission scanning electron microscope was used to assess the contribution of platform architecture on the gelatin degradation by the morphological changes that occurred at different times. The evaluation of human mesenchymal stem cells’ biocompatibility confirmed the role of architecture and chemical composition on cell response. The nanostructured surfaces positively affected the cell recognition by increasing the surface area. The gelatin embedded in the PCL matrix of the nanofibers improved the cell/material interaction and provided support to the proliferation. The PCL/gelatin electrospun membranes showed an increase in mineralization when conditioned in osteogenic medium; this system has promise for long-term in vitro investigations of bone regeneration.

Tuning Size Scale and Crystallinity of PCL Electrospun Fibres via Solvent Permittivity to Address hMSC Response

The effect of solvent permittivity on the fibre morphology of PCL electrospun membranes for tissue engineering applications is studied. Morphological results indicate that polar solvents with higher permittivity are able to promote the formation of sub-micrometric fibres, while apolar solvents yield microfibres with an average fibre diameter of 2.86 ± 0.31 µm. Polymer/solvent interactions and electrospinning process parameters influence the mechanism of fibre and bead formation. It is shown that the dielectric properties of solvents influence the fibre size scale and crystallinity and directly contribute to the biological response of stem cells. Solvent permittivity is a key factor in controlling the morphological and physical properties of electrospun fibre meshes.

In vitro mineralization and bone osteogenesis in poly(ε-caprolactone)/gelatin nanofibers†

The implementation of bio-inspired strategies in developing scaffolds for the reconstruction of oral, craniofacial and bone skeletal tissues after injury or resection remains a challenge. Currently, advanced scaffolds comprising nanofibers endowed with biochemical/biophysical signaling capability offer great advantages in bone regeneration, because of their faithful mimesis of the characteristic size scales encountered in the fibrous network of the native extracellular matrix (ECM). In this study, we investigate the biological potential of nanofibers made of polycaprolactone and gelatin on guiding the regenerative mechanisms of bone. Contact angle measurements and environmental SEM investigations indicate a weak linkage of gelatin molecules to PCL chains, facilitating an efficient adhesion signal to cells up to 3 days of culture. In vitro studies performed on human mesenchymal stem cells (hMSC) until 3 weeks in culture medium with osteogenic supplementation, clearly showing the effectiveness of PCL/Gelatin electrospun scaffolds in promoting bone osteogenesis and mineralization. The increase of alkaline phosphatase activity (ALP) and gene expression of bone-related molecules (bone sialoprotein, osteopontin and osteocalcin), indicated by immunodetection and upregulation level of mRNA, confirm that proposed nanofibers promote the osteogenic differentiation of hMSC, preferentially in osteogenic medium. Moreover, the evidence of newly formed collagen fibers synthesis by SIRCOL and their mineralization evaluated by Alizarin Red staining and EDS mapping of the elements Ca, P and Mg corroborate the idea that native osteoid matrix is ultimately deposited. All these data suggest that PCL and gelatin electrospun nanofibers have great potential as osteogenesis promoting scaffolds for successful application in bone surgery.

Influence of electrospun fiber mesh size on hMSC oxygen metabolism in 3D collagen matrices: Experimental and theoretical evidences

The traditional paradigm of tissue engineering of regenerating in vitro tissue or organs, through the combination of an artificial matrix and a cellular population has progressively changed direction. The most recent concept is the realization of a fully functional biohybrid, where both, the artificial and the biotic phase, concur in the formation of the novel organic matter. In this direction, interest is growing in approaches taking advantage of the control at micro‐ and nano‐scale of cell material interaction based on the realization of elementary tassels of cells and materials which constitute the beginning point for the expansion of 3D more complex structures. Since a spontaneous assembly of all these components is expected, however, it becomes more fundamental than ever to define the features influencing cellular behavior, either they were material functional properties, or material architecture. In this work, it has been investigated the direct effect of electrospun fiber sizes on oxygen metabolism of h‐MSC cells, when any other culture parameter was kept constant. To this aim, thin PCL electrospun membranes, with micro‐ and nano‐scale texturing, were layered between two collagen slices up to create a sandwich structure (µC‐PCL‐C and nC‐PCL‐C). Cells were seeded on membranes, and the oxygen consumption was determined by a phosphorescence quenching technique. Results indicate a strong effect of the architecture of scaffolds on cell metabolism, also revealed by the increasing of HIF1‐α gene expression in nC‐PCL‐C. These findings offer new insights into the role of materials in specific cell activities, also implying the existence of very interesting criteria for the control of tissue growth through the tuning of scaffold architecture. Biotechnol. Bioeng. 2011; 108:1965–1976.

Polymer-based platforms by electric field-assisted techniques for tissue engineering and cancer therapy

A large variety of processes and tools has been investigated to acquire better knowledge on the natural evolution of healthy or pathological tissues in 3D scaffolds to discover new solutions for tissue engineering and cancer therapy. Among them, electrodynamic techniques allow revisiting old scaffold manufacturing approach by utilizing electrostatic forces as the driving force to assemble fibers and/or particles from an electrically charged solution. By carefully selecting materials and processing conditions, they allow to fine control of characteristic shapes and sizes from micro to sub-micrometric scale and incorporate biopolymers/molecules (e.g., proteins, growth factors) for time- and space-controlled release for use in drug delivery and passive/active targeting. This review focuses on current advances to design micro or nanostructured polymer platforms by electrodynamic techniques, to be used as innovative scaffolds for tissue engineering or as 3D models for preclinical in vitro studies of in vivo tumor growth.

Bicomponent electrospun scaffolds to design extracellular matrix tissue analogs

In the last decade, bicomponent fibers have been proposed to fabricate bio-inspired systems for tissue repair, regenerative medicine, medical healthcare and clinical applications. In comparison with monocomponent fibers, key advantage concerns their ability of self-adapting to the physiological conditions through an extended pattern of signals – morphological, chemical and physical ones – confined at the single fiber level. Hydrophobic/hydrophilic phases may be variously organized by tuneable processing modes (i.e., blending, core/shell, interweaving) thus offering different benefits in terms of biological activity, fluid sorption and molecular transport properties (first generation). The possibility to efficiently graft cell-adhesive proteins and peptide sequences onto the fiber surface mediated by spacers or impregnating hydrogels allows to trigger cell late activities by a controlled and sustained release in vitro of specific biomolecules (i.e., morphogens, growth factors). Here, we introduce an overview of current approaches based on bicomponent fiber use as extra cellular matrix analogs with cell-instructive functions and hierarchal organization of living tissues.

5-azacytidine mediated hMSC behaviour on electrospun scaffolds for skeletal muscle regeneration

Incomplete regeneration after trauma or muscular dysfunction is a common problem in muscle replacement therapies. Recent approaches in tissue engineering allow for the replication of skeletal muscle structure and function in vitro and in vivo by molecular therapies and implantable scaffolds which properly address muscle cells toward myotube differentiation and maturation. Here, we investigate the in vitro response of human mesenchymal stem cells (hMSC) on electrospun fibers made of polycaprolactone (PCL) in the presence of 5-azacytidine (5-AZA) to evaluate how fibrous network may influence the therapeutic effect of drug during in vitro myogenesis. Biological studies demonstrate the ability of hMSCs to differentiate in mature myofibers in supplemented (myogenic) and, preferentially, in 5-AZA-enriched culture. PCL electrospun fibers amplify the 5-AZA capability to induce a low proliferation rate in hMSC, thus promoting hMSC differentiation (MTT assay). Qualitative (Azan Mallory stain, immunofluorescence assay, SEM analyses) and quantitative (ELISA test) assays confirm the synergistic contribution of PCL electrospun fibers and 5-AZA on in vitro myotubes formation and maturation. This result is also confirmed by the expression of muscle-specific proteins related to the myogenic mechanisms in the presence of other muscle inductive signals (i.e., oxytocin, Tweak). Hence, we suggest the use of PCL electrospun fibers as interesting preclinical model to explore the effect of drugs and chemotherapeutics administration after damaged muscle resection.

Electro fluido dynamic techniques to design instructive biomaterials for tissue engineering and drug delivery

A large variety of processes and tools is continuously investigated to discover new solutions to design instructive materials with controlled chemical, physical and biological properties for tissue engineering and drug delivery. Among them, electro fluido dynamic techniques (EFDTs) are emerging as an interesting strategy, based on highly flexible and low-cost processes, to revisit old biomaterial’s manufacturing approach by utilizing electrostatic forces as the driving force for the fabrication of 3D architectures with controlled physical and chemical functionalities to guide in vitro and in vivo cell activities. By a rational selection of polymer solution properties and process conditions, EFDTs allow to produce fibres and/or particles at micro and/or nanometric size scale which may be variously assembled by tailored experimental setups, thus giving the chance to generate a plethora of different 3D devices able to incorporate biopolymers (i.e., proteins, polysaccharides) or active molecules (e.g., drugs)...

Degradation and early in vitro activity of healthy hepatocytes onto bicomponent electrospun fibers

ABSTRACT Bicomponent electrospun fibers (BEFs) have been designed as building blocks of various biomedical devices, i.e., scaffolds, conduits, grafts, for the repair/regeneration of in vitro and in vivo tissues. With respect to monocomponent fibers, BEF may exert diversified patterns of signals—morphological, chemical or physical—confined at the single fiber level, conferring them a unique ability of self-adapting to local microenvironment and influencing cell/fiber interaction. Herein, an investigation of basic relationships between degradation properties of BEF and in vitro response of healthy hepatocytes to prove their use as in vitro model to predict in vivo liver regeneration is proposed. GRAPHICAL ABSTRACT