Ricardo A. Pires

Controlling Cancer Cell Fate Using Localized Biocatalytic Self-Assembly of an Aromatic Carbohydrate Amphiphile

We report on a simple carbohydrate amphiphile able to self-assemble into nanofibers upon enzymatic dephosphorylation. The self-assembly can be triggered by alkaline phosphatase (ALP) in solution or in situ by the ALP produced by osteosarcoma cell line, SaOs2. In the latter case, assembly and localized gelation occurs mainly on the cell surface. The gelation of the pericellular environment induces a reduction of the SaOs2 metabolic activity at an initial stage (≤7 h) that results in cell death at longer exposure periods (≥24 h). We show that this effect depends on the phosphatase concentration, and thus, it is cell-selective with prechondrocytes ATDC5 (that express ∼15-20 times lower ALP activity compared to SaOs2) not being affected at concentrations ≤1 mM. These results demonstrate that simple carbohydrate derivatives can be used in an antiosteosarcoma strategy with limited impact on the surrounding healthy cells/tissues.

Immobilization of bioactive factor-loaded liposomes on the surface of electrospun nanofibers targeting tissue engineering

Electrospun nanofiber meshes (NFM), due to their morphology and fibrous structure, are extensively proposed as biomedical devices, for tissue engineering on scaffolds and also as drug delivery systems. Liposomes are nanoparticles prepared from a biologically derived material (phospholipid), which are already in clinical use as a drug release device. Liposomes may be combined with biomaterial scaffolds to promote a local and sustained delivery of loaded bioactive agents. The main objective of the present study is to evaluate the efficacy of dexamethasone (Dex)-loaded liposomes immobilized on the surface of electrospun polycaprolactone (PCL) NFM for promoting the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). The in vitro release profile demonstrates a sustained release of Dex over 21 days, after an initial burst release over 12 h. Biological assays show that Dex-loaded liposomes immobilized on the surface of electrospun PCL NFMs do not exhibit any cytotoxic effect, being able to successfully promote the osteogenic differentiation of hBMSCs. We herein validate the concept of using liposomes immobilized on the surface of a nanostructured fibrous system to be used as an advanced cell carrier device with autonomous release of growth/differentiation factors relevant for tissue engineering and regenerative medicine strategies.

Bioresorbable ureteral stents from natural origin polymers

In this work, stents were produced from natural origin polysaccharides. Alginate, gellan gum, and a blend of these with gelatin were used to produce hollow tube (stents) following a combination of templated gelation and critical point carbon dioxide drying. Morphological analysis of the surface of the stents was carried out by scanning electron microscopy. Indwelling time, encrustation, and stability of the stents in artificial urine solution was carried out up to 60 days of immersion. In vitro studies carried out with simulated urine demonstrated that the tubes present a high fluid uptake ability, about 1000%. Despite this, the materials are able to maintain their shape and do not present an extensive swelling behavior. The bioresorption profile was observed to be highly dependent on the composition of the stent and it can be tuned. Complete dissolution of the materials may occur between 14 and 60 days. Additionally, no encrustation was observed within the tested timeframe. The ability to resist bacterial adherence was evaluated with Gram-positive Staphylococcus aureus and two Gram-negatives Escherichia coli DH5 alpha and Klebsiella oxytoca. For K. oxytoca, no differences were observed in comparison with a commercial stent (Biosoft(®) duo, Porges), although, for S. aureus all tested compositions had a higher inhibition of bacterial adhesion compared to the commercial stents. In case of E. coli, the addition of gelatin to the formulations reduced the bacterial adhesion in a highly significant manner compared to the commercial stents. The stents produced by the developed technology fulfill the requirements for ureteral stents and will contribute in the development of biocompatible and bioresorbable urinary stents.

Carboxymethylation of ulvan and chitosan and their use as polymeric components of bone cements.

Ulvan, extracted from the green algae Ulva lactuca, and chitosan, extracted from Loligo forbesis squid-pen, were carboxymethylated, yielding polysaccharides with an average degree of substitution of ∼98% (carboxymethyl ulvan, CMU) and ∼87% (carboxymethyl chitosan, N,O-CMC). The carboxymethylation was confirmed by Fourier transform infrared spectroscopy and quantified by conductimetric titration and 1H nuclear magnetic resonance. The average molecular weight increased with the carboxymethylation (chitosan, Mn 145→296 kDa and Mw 227→416 kDa; ulvan, Mn 139→261 kDa and Mw 368→640 kDa), indicating successful chemical modifications. Mixtures of the modified polysaccharides were tested in the formulation of polyacrylic acid-free glass-ionomer bone cements. Mechanical and in vitro bioactivity tests indicate that the inclusion of CMU in the cement formulation, i.e. 0.50:0.50 N,O-CMC:CMU, enhances its mechanical performance (compressive strength 52.4±8.0 MPa and modulus 2.3±0.3 GPa), generates non-cytotoxic cements and induces the diffusion of Ca and/or P-based moieties from the surface to the bulk of the cements.

Interactions between exogenous FGF-2 and sulfonic groups: in situ characterization and impact on the morphology of human adipose-derived stem cells.

FGF-2 is often used as a supplement to stem cells culture medium aiming at preserving their self-renewal capacity and plasticity through the passages. However, little is known on the influence of the underlying substrate in these interactions. In this study, we have used mixed self-assembled monolayers with different ratios of -SO3H and -OH tail groups to investigate the influence of substrate properties (e.g., charge) on the FGF-2 adsorption and activity. QCM-D data demonstrated that, in the presence of -OH groups, the quantity of the adsorbed FGF-2 is proportional to the percentage of surface -SO3H groups. The bioactivity of the adsorbed FGF-2 follows the same tendency as demonstrated by its interactions with anti-FGF-2. Surprisingly, the adlayer of FGF-2 formed on the surface containing only SO3H-tailed SAMs was similar to the surface with 25% of -SO3H groups, demonstrating that FGF-2 adsorption is not solely driven by electrostatic interactions. We related these results with changes in the morphology of adipose-derived stem cells (ASCs) cultured on the same surfaces.

The study of a commercial dental resin by 1H stray-field magnetic resonance imaging

Abstract A dentine/enamel resin containing methacrylate monomers, included in a new generation adhesive system, was used to evaluate the potentialities of recent nuclear magnetic resonance imaging (MRI) techniques to obtain spatially-resolved information on photo-polymerization reaction and subsequent polymerization shrinkage. 1 H stray-field (STRAFI)-MRI one-dimensional images (1D profiles) of visible-light cured resins were obtained in the presence of oxygen from the atmosphere, and the variation of magnetization with irradiation time was recorded for each resin slice. The polymerization shrinkage was obtained from 1D profiles. The spatial distribution of the unreacted methyl methacrylate groups was obtained from 3D STRAFI experiments. In particular, the thickness of the surface remaining unpolymerised was measured.

Aluminum-free glass-ionomer bone cements with enhanced bioactivity and biodegradability

Al-free glasses of general composition 0.340SiO2:0.300ZnO:(0.250-a-b)CaO:aSrO:bMgO:0.050Na2O:0.060P2O5 (a, b=0.000 or 0.125) were synthesized by melt quenching and their ability to form glass-ionomer cements was evaluated using poly(acrylic acid) and water. We evaluated the influence of the poly(acrylic acid) molecular weight and glass particle size in the cement mechanical performance. Higher compressive strength (25±5 MPa) and higher compressive elastic modulus (492±17 MPa) were achieved with a poly(acrylic acid) of 50 kDa and glass particle sizes between 63 and 125 μm. Cements prepared with glass formulation a=0.125 and b=0.000 were analyzed after immersion in simulated body fluid; they presented a surface morphology consistent with a calcium phosphate coating and a Ca/P ratio of 1.55 (similar to calcium-deficient hydroxyapatite). Addition of starch to the cement formulation induced partial degradability after 8 weeks of immersion in phosphate buffer saline containing α-amylase. Micro-computed tomography analysis revealed that the inclusion of starch increased the cement porosity from 35% to 42%. We were able to produce partially degradable Al-free glass-ionomer bone cements with mechanical performance, bioactivity and biodegradability suitable to be applied on non-load bearing sites and with the appropriate physical characteristics for osteointegration upon partial degradation. Zn release studies (concentrations between 413 μM and 887 μM) evidenced the necessity to tune the cement formulations to reduce the Zn concentration in the surrounding environment.

Sulfonic groups induce formation of filopodia in mesenchymal stem cells

Glycosaminoglycans (GAGs) are an integral part of the extracellular matrix and glycocalix, i.e. the closest cellular environment. They are abundant in –OH groups and their bioactivity is also associated with the presence of negatively charged –SO3H functionalities. Therefore, we have investigated and discussed the influence of these functional units on mesenchymal stem cells (MSCs) behaviour using single component and mixed self-assembled monolayers of alkanethiols with –SO3H and –OH end groups. In the absence of serum, MSCs attachment, spreading, cytoskeleton organisation and motility were significantly influenced by the surface chemistry. We found that the sulfonic groups induce star-like cell shape with very intense actin staining and a high density net of filopodia that enlarge from the base of lamellipodia structures. Moreover, this response is concentration dependent and is apparent only for very short culture time in the presence of serum.

Reinforcement of poly-L-lactic acid electrospun membranes with strontium borosilicate bioactive glasses for bone tissue engineering

UNLABELLED Herein, for the first time, we combined poly-l-lactic acid (PLLA) with a strontium borosilicate bioactive glass (BBG-Sr) using electrospinning to fabricate a composite bioactive PLLA membrane loaded with 10% (w/w) of BBG-Sr glass particles (PLLA-BBG-Sr). The composites were characterised by scanning electron microscopy (SEM) and microcomputer tomography (μ-CT), and the results showed that we successfully fabricated smooth and uniform fibres (1-3μm in width) with a homogeneous distribution of BBG-Sr microparticles (<45μm). Degradation studies (in phosphate buffered saline) demonstrated that the incorporation of BBG-Sr glass particles into the PLLA membranes increased their degradability and water uptake with a continuous release of cations. The addition of BBG-Sr glass particles enhanced the membranes mechanical properties (69% higher Young modulus and 36% higher tensile strength). Furthermore, cellular in vitro evaluation using bone marrow-derived mesenchymal stem cells (BM-MSCs) demonstrated that PLLA-BBG-Sr membranes promoted the osteogenic differentiation of the cells as demonstrated by increased alkaline phosphatase activity and up-regulated osteogenic gene expression (Alpl, Sp7 and Bglap) in relation to PLLA alone. These results strongly suggest that the composite PLLA membranes reinforced with the BBG-Sr glass particles have potential as an effective biomaterial capable of promoting bone regeneration. STATEMENT OF SIGNIFICANCE PLLA membranes were reinforced with 10% (w/w) of strontium-bioactive borosilicate glass microparticles, and their capacity to induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was evaluated. These membranes presented an increased: degradability, water uptake, Young modulus and tensile strength. We also demonstrated that these membranes are non-cytotoxic and promote the attachment of BM-MSCs. The addition of the glass microparticles into the PLLA membranes promoted the increase of ALP activity (under osteogenic conditions), as well as the BM-MSCs osteogenic differentiation as shown by the upregulation of Alpl, Sp7 and Bglap gene expression. Overall, we demonstrated that the reinforcement of PLLA with glass microparticles results in a biomaterial with the appropriate properties for the regeneration of bone tissue.

The role of alumina in aluminoborosilicate glasses for use in glass–ionomer cements

Aluminoborosilicate glasses of general composition (0.14 − x/2.2)SiO2:(0.07 − x/4.2)B2O3:(0.09 − x/3.3)Na2O:0.40CaO:xAl2O3 (0.00 ≤ x ≤ 0.30) were synthesised using standard melt quenching methods and analysed by 11B, 23Na, 27Al and 29Si solid-state nuclear magnetic resonance (NMR) spectroscopy. Calculations based on electroneutrality considerations were used to check the consistency of the NMR assignments in both glasses and cements. Glass–ionomer cement (GIC) formation was tested with all the prepared glass compositions, but only the samples with higher concentrations of Al2O3, x ≥ 0.20, produced sustainable cements. The compressive strengths (CS) of the cements varied from 66.7 to 74.2 MPa, although the differences were not statistically significant. The leaching of aluminium from the glass into the cement matrix varied between 6 and 12 mol%. It is proposed that aluminium plays a key role in glass–ionomer cements, not only through leaching of Al3+ ions from the glass into the cement matrix during formation, but also through reinforcement of the resulting cement by the residual glass particles which have high alumina content and confer greater mechanical strength.