Pure titanium particle loaded nanocomposites: study on the polymer/filler interface and hMSC biocompatibility.

The integration of inorganic nanoparticles into polymer matrices allows for the modification of physical properties as well as the implementation of new features for unexplored application fields. Here, we propose the study of a new metal/polymer nanocomposite fabricated by dispersing pure Ti nanoparticles into a poly(methylmetacrilate) matrix via solvent casting process, to investigate its potential use as new biomaterial for biomedical applications. We demonstrated that Ti nanoparticles embedded in the poly(methylmetacrilate) matrix can act as reinforcing agent, not negatively influencing the biological response of human mesenchymal stem cell in terms of cytotoxicity and cell viability. As a function of relative amount and surface treatment, Ti nanoparticles may enhance mechanical strength of the composite-ranging from 31.1 ± 2.5 to 43.7 ± 0.7 MPa-also contributing to biological response in terms of adhesion and proliferation mechanisms. In particular, for 1 wt% Ti, treated Ti nanoparticles improve cell materials recognition, as confirmed by higher cell spreading-quantified in terms of cell area via image analysis-locally promoting stronger interactions at cell matrix interface. At this stage, these preliminary results suggest a promising use of pure Ti nanoparticles as filler in polymer composites for biomedical applications.

Design of bimodal PCL and PCL-HA nanocomposite scaffolds by two step depressurization during solid-state supercritical CO(2) foaming.

This communication reports the design and fabrication of porous scaffolds of poly(ε-caprolactone) (PCL) and PCL loaded with hydroxyapatite (HA) nanoparticles with bimodal pore size distributions by a two step depressurization solid-state supercritical CO(2) (scCO(2) ) foaming process. Results show that the pore structure features of the scaffolds are strongly affected by the thermal history of the starting polymeric materials and by the depressurization profile. In particular, PCL and PCL-HA nanocomposite scaffolds with bimodal and uniform pore size distributions are fabricated by quenching molten samples in liquid N(2) , solubilizing the scCO(2) at 37 °C and 20 MPa, and further releasing the blowing agent in two steps: (1) from 20 to 10 MPa at a slow depressurization rate, and (2) from 10 MPa to the ambient pressure at a fast depressurization rate. The biocompatibility of the bimodal scaffolds is finally evaluated by the in vitro culture of human mesenchymal stem cells (MSCs), in order to assess their potential for tissue engineering applications.

Thermo-mechanical, antimicrobial and biocompatible properties of PVC blends based on imidazolium ionic liquids.

The aim of this study was the development of antimicrobial polyvinylchloride (PVC) blends loaded with 0.1-10% (w/w) of the ILs 1-hexadecyl-3-methylimidazolium 1,3-dimethyl 5-sulfoisophthalate (HdmimDMSIP) and 1-octyloximethyl-3-methylimidazolium hexafluorophosphate (OOMmimPF6). The synthetized ILs were characterized by 1HNMR, MALDI-TOF, DSC and TGA. PVC/ILs films were obtained by solvent casting.Thermal and mechanical properties (tensile stress TS and elongation at break EB), morphology by SEM, surface wettability, antimicrobial activity, cytotoxicity and ILs release in sterile water from PVC/ILs film blends were determined. Results demonstrated that the presence of both ILs in PVC formulation slightly affected thermal and mechanical properties of blends. The loading of both ILs into PVC matrix made PVC/ILs films hydrophilic, especially at the highest concentration of HdmimDMSIP. The PVC/ILs blends displayed antibacterial activity up to ILs lowest concentrations (0.1-0.5%). The inhibition of Escherichia coli growth was lower than that showed toward Staphylococcus epidermidis. The addition of 10% ILs concentration resulted excessive as demonstrated by accumulation of ILs on film surfaces (SEM) and ILs high release from PVC/ILs blends during the first day of water immersion. Biocompatibility studies highlighted that the addition of low amounts of both ILs into PVC matrix is not cytotoxic for mouse fibroblast cells (L929), supporting their potential use for biomedical porposes.

Natural Formulations Provide Antioxidant Complement to Hyaluronic Acid-Based Topical Applications Used in Wound Healing.

Hyaluronic acid (HA) promotes wound healing, and, accordingly, formulations based on HA have been widely used in regenerative medicine. In addition, naturally derived compounds, e.g., plant-based extracts and vitamin E, have exhibited antioxidant activity. In this study, a formulation containing hyaluronic acid, vitamin E, raspberry extract, and green tea was developed for potential topical applications, targeting wound healing. Rheological analysis was performed along with antioxidant and biological studies. The rheological characterization showed that the HA-based formulation is a thixotropic platform and possesses higher mechanical properties than the control formulation. To evaluate the wound healing potential of the formulation, an in vitro "wound healing" assay was carried out using human derived fibroblasts (HDF) with a cell-free gap on the tissue culture dish. The formulation showed better wound healing ability than the control formulation.

Quantitative Study of Morphological Features of Stem Cells onto Photopatterned Azopolymer Films.

In the last decade, the use of photolithography for the fabrication of structured substrates with controlled morphological patterns that are able to interact with cells at micrometric and nanometric size scales is strongly growing. A promising simple and versatile microfabrication method is based on the physical mass transport induced by visible light in photosensitive azobenzene-containing polymers (or azopolymers). Such light-driven material transport produces a modulation of the surface of the azopolymer film, whose geometry is controlled by the intensity and the polarization distributions of the irradiated light. Herein, two anisotropic structured azopolymer films have been used as substrates to evaluate the effects of topological signals on the in vitro response of human mesenchymal stem cells (hMSCs). The light-induced substrate patterns consist of parallel microgrooves, which are produced in a spatially confined or over large-scale areas of the samples, respectively. The analysis of confocal optical images of the in vitro hMSC cells grown on the patterned films offered relevant information about cell morphology-i.e., nuclei deformation and actin filaments elongation-in relation to the geometry and the spatial extent of the structured area of substrates. The results, together with the possibility of simple, versatile, and cost-effective light-induced structuration of azopolymers, promise the successful use of these materials as anisotropic platforms to study the cell guidance mechanisms governing in vitro tissue formation.

Synthesis and characterization of divinyl-fumarate poly-ε-caprolactone for scaffolds with controlled architectures.

A vinyl-terminated polycaprolactone has been developed for tissue engineering applications using a one-step synthesis and functionalization method based on ring opening polymerization (ROP) of ε-Caprolactone, with hydroxyl ethyl vinyl ether (HEVE) acting both as the initiator of ROP and as photo-curable functional group. The proposed method employs a catalyst based on aluminium, instead of the most popular Tin(II) 2-ethylhexanoate, to reduce the cytotoxicity. Following the synthesis of the vinyl-terminated polycaprolactone, its reaction with fumaryl chloride (FuCl) results in a divinyl-fumarate polycaprolactone (VPCLF). The polymers obtained were thoroughly characterized using Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC) techniques. The polymer has been successfully employed, in combination with N-vinyl pyrrolidone (NVP), to fabricate films and computer-designed porous scaffolds by micro-stereolithography (µ-SL) with gyroid and diamond architectures. Characterization of the networks indicated the influence of NVP content on the network properties. Human mesenchymal stem cells adhered and spread onto VPCLF/NVP networks showing good biological properties and no cytotoxic effect. Copyright © 2016 John Wiley & Sons, Ltd.

Spontaneous arrangement of a tumor targeting hyaluronic acid shell on irinotecan loaded PLGA nanoparticles.

The arrangement of tumor targeting hyaluronic acid (HA) moieties on irinotecan (IRIN)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) has been directed by means of a gradient of lipophilicity between the oil and water phases of the emulsion used to produce the NPs. PLGA constitutes the NP bulk while HA is superficially exposed, with amphiphilic poloxamers acting as a bridge between PLGA and HA. Differential scanning calorimetry, zeta potential analyses and ELISA tests were employed to support the hypothesis of polymer assembly in NP formulations. The presence of flexible HA chains on NP surface enhances NP size stability over time due to an increased electrostatic repulsion between NPs and a higher degree of hydration of the device surface. IRIN in vitro release kinetics can be sustained up to 7-13 days. In vitro biologic studies indicated that HA-containing NPs were more toxic than bare PLGA NPs against CD44-overexpressing breast carcinoma cells (HS578T), therefore indicating their ability to target CD44 receptor.

The effect of matrix composition of 3D constructs on embryonic stem cell differentiation.

The use of embryonic stem (ES) cells as unlimited cell source in tissue engineering has ignited the hope of regenerating any kind of tissue in vitro. However, the role of the material in control and guidance of their development and commitment into complex and viable three-dimensional (3D) tissues is still poorly understood. In this work, we investigate the role of material composition and structure on promoting ES cells growth and differentiation, by culturing mouse ES cell-derived embryoid bodies (EBs) in various semi-interpenetrating polymer networks (SIPNs), made of collagen, fibronectin (FN) and laminin (LM). We show that both composition and strength of the supportive matrix play an important role in EBs development. High collagen concentrations inhibit EBs cavitation and hence the following EBs differentiation, by inhibiting apoptosis. The presence of FN in 3D collagen constructs strongly stimulates endothelial cell differentiation and vascularization. Conversely, LM increases the ability of ES cells to differentiate into beating cardiomyocytes. Our data suggest that matrix composition has an important role in EBs development and that it is possible to influence stem cell differentiation toward preferential pattern, by modulating the physical and biochemical properties of the scaffold.

New macroporous calcium phosphate glass ceramic for guided bone regeneration.

This work describes a method to obtain macroporous resorbable glass and glass ceramic scaffolds with controlled biodegradability for tissue engineering applications. The constructs consisted of glass and glass ceramics in the system P(2)O(5)-CaO-Na(2)O-TiO(2) and they were prepared by foaming a slurry of glass particles by addition of a H(2)O(2) solution, and subsequent sintering of the porous structures obtained. Different thermal treatments were applied to control the degree of devitrification of the glass. The resultant materials showed a porosity percentage between 40% and 55% with a wide variety of pores ranging from 20 to 500 microm in diameter as determined by SEM and Image Analysis. The resulting constructs were predominantly formed by a vitreous phase, although small amounts of calcium metaphosphate and pyrophosphates were detected by X-ray diffraction and Raman spectroscopy after the sintering process. The biological response was also evaluated by means of the MTT test, the material showed a non-cytotoxic effect.

Large defect-tailored composite scaffolds for in vivo bone regeneration.

The discovery of new strategies to repair large segmental bone defects is currently an open challenge for worldwide clinicians. In the treatment of critical-sized bone defects, an alternative strategy to traditional bone grafting is always more frequently the use of tailor-made scaffolds modelled on the final size and shape of the implant site. Here, poly-ε-caprolactone-based composite scaffolds including poly-L-lactic acid continuous fibres and hyaluronan derivates (i.e. HYAFF11®) have been investigated for the peculiar 3D architecture characterized by interconnected macroporous networks and tunable mechanical properties. Composite scaffolds were immersed in simulated body fluid solution in order to support in vivo tissue in-growth. Scaffolds loaded with autologous cells (bone marrow stromal cells) plus platelet-rich plasma and osteoconductive protein such bone morphogenetic protein-7 were also tested to evaluate eventual enhancement in bone regeneration. The morphological and mechanical properties of poly-L-lactic acid-reinforced composite scaffolds have been studied to identify the optimal scaffold design to match the implant-site requirements of sheep metatarsal defects. Dynamic mechanical tests allowed to underline the viscoelastic response of the scaffold - resulting in elastic moduli from 2.5 to 1.3 MPa, suitable to temporarily support the structural function of damaged bone tissue. In vivo preliminary investigations in a sheep model of metatarsus shaft defect also showed the attitude of the scaffold to promote osteogenesis, preferentially in association with bone marrow stromal cell and platelet-rich plasma, even if the highest amount of mature bone was reached in the case of scaffold loaded with human bone morphogenetic protein-7 released via hydrolytic degradation of HYAFF11® phases in the implant site.

Design of injectable organic-inorganic hybrid for bone tissue repair.

Injectable bone substitutes are rapidly gained success in tissue engineering applications for their less invasive surgical aspect. Here, the design and the characterization of a novel degradable paste of PCL reinforced with nanocrystals of hydroxyapatite have been presented aiming to mimic natural tissue. Nanohydroxyapatite has been successfully synthesized via sol-gel technique. Dynamic and steady state viscoelastic properties of the solutions and paste were investigated to control the kinetic of phase transition. Correspondingly, the morphology and composition were characterized via TEM, EDAX, and thermal analysis. Injection test underlines the completely ability of the paste of being injected without altering its features. Preliminary biological study showed that the composite paste is not cytotoxic. The synergistic rheological and biological properties, combined with the positive effect of chemical synthesis method indicate that the composite paste is very suitable as local bone substitute in low-load areas.

Effect of micro- and macroporosity of bone tissue three-dimensional-poly(epsilon-caprolactone) scaffold on human mesenchymal stem cells invasion, proliferation, and differentiation in vitro.

The design of porous scaffolds able to promote and guide cell proliferation, colonization, and biosynthesis in three dimensions is key determinant in bone tissue engineering (bTE). The aim of this study was to assess the role of the micro-architecture of poly(epsilon-caprolactone) scaffolds in affecting human mesenchymal stem cells' (hMSCs) spatial organization, proliferation, and osteogenic differentiation in vitro. Poly(epsilon-caprolactone) scaffolds for bTE and characterized by mono-modal and bi-modal pore size distributions were prepared by the combination of gas foaming and selective polymer extraction from co-continuous blends. The topological properties of the pore structure of the scaffolds were analyzed and the results correlated with the ability of hMSCs to proliferate, infiltrate, and differentiate in vitro in three dimensions. Results showed that the micro-architecture of the pore structure of the scaffolds plays a crucial role in defining cell seeding efficiency as well as hMSCs' three-dimensional colonization, proliferation, and osteogenic differentiation. Taken all together, our results indicated that process technologies able to allow a fine-tune of the topological properties of biodegradable porous scaffolds are essential for bTE strategies.

Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation.

The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27Kip1 and p16INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.