Aureliana Sousa

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

The use of human Mesenchymal Stem Cells (hMSC) as therapeutic agents for advanced clinical therapies relies on their in vitro expansion. Over the last years, several efforts have been made to optimize hMSC culture protocols, namely by mimicking the cell physiological microenvironment, which strongly relies on signals provided by the extracellular matrix (ECM). ECM glycosaminoglycans, such as heparan-sulfate, sequester adhesive proteins and soluble growth factors at the cell membrane, orchestrating signaling pathways that control cell proliferation. Surfaces exposing the synthetic polypeptide poly(L-lysine, L-leucine) (pKL) have previously been shown to bind heparin from human plasma in a selective and concentration-dependent manner. To evaluate its effect on hMSC expansion, pKL was immobilized onto self-assembled monolayers (SAMs). The pKL-SAMs were able to bind heparin, fibronectin and other serum proteins, as demonstrated by quartz crystal microbalance with dissipation (QCM-D) studies. hMSC adhesion and proliferation were significantly increased in pKL-SAMs compared to controls, most probably related to increased heparin and fibronectin binding to pKL surfaces. This proof-of-concept study highlights the potential of pKL surfaces to improve hMSC in vitro expansion possible through selective heparin/serum protein binding at the cell-material interface.

Polycaprolactone (PCL) is widely used in tissue engineering due to its interesting properties, namely biocompatibility, biodegradability, elastic nature, availability, cost efficacy, and the approval of health authorities such as the American Food and Drug Administration (FDA). The PCL degradation rate is not the most adequate for specific applications such as skin regeneration due to the hydrophobic nature of bulk PCL. However, PCL electrospun fiber meshes, due to their low diameters resulting in high surface area, are expected to exhibit a fast degradation rate. In this work, in vitro and in vivo degradation studies were performed over 90 days to evaluate the potential of electrospun PCL as a wound dressing. Enzymatic and hydrolytic degradation studies in vitro, performed in a static medium, demonstrated the influence of lipase, which promoted a rate of degradation of 97% for PCL meshes. In an in vivo scenario, the degradation was slower, although the samples were not rejected, and were well-integrated in the surrounding tissues inside the subcutaneous pockets specifically created.

Healing of intestinal chronic wounds remains a major challenge as current therapies are ineffective in promoting proper regeneration of the damaged intestinal wall. An innovative concept, based on a bioinspired multifunctional alginate-melanin hybrid 3D scaffold, to target both inflammatory and regenerative processes, is proposed herein. Hydrogel-entrapped melanin nanoparticles demonstrated free-radical scavenging activity, supported by the neutralization of free-radicals in solution (90%), and the in vitro capture of reactive oxygen species (ROS) produced by stimulated macrophages in an inflammatory-mimicking environment. Notably, scaffolds could be reused (at least 3 times), while maintaining these properties. The extracellular matrix (ECM)-inspired biomaterial, containing protease-sensitive and integrin-binding domains, exhibited remarkable ability for cell colonisation. Human intestinal fibroblasts and epithelial cells (Caco-2) co-seeded on lyophilized scaffolds were able to invade/colonize the construct and produce endogenous ECM, key for neo-tissue formation and re-epithelialization. Scaffolds presented tuneable mechanical properties and could be used both in hydrated and freeze-dried states, maintaining their performance upon rehydration, which are attractive features for clinical application. Collectively, our results highlight the potential of biofunctionalized alginate-melanin hybrid 3D scaffolds as multi-therapeutic patches for modulating inflammation and tissue regeneration in chronic intestinal wounds, which address a major but still unmet clinical need. The proposed multi-therapeutic strategy may potentially be extended to the treatment of other chronic wounds.

A calcium phosphate (CaP)-based scaffold used as synthetic bone grafts, which smartly combines precise dimensions, controlled porosity and therapeutic functions, presents benefits beyond those offered by conventional practices, although its fabrication is still a challenge. The sintering step normally required to improve the strength of the ceramic scaffolds precludes the addition of any biomolecules or functional particles before this stage. This study presents a proof of concept of multifunctional CaP-based scaffolds, fabricated by additive manufacturing from an innovative ink composition, with potential for bone regeneration, cancer treatment by local magnetic hyperthermia and drug delivery platforms. Highly loaded inks comprising iron-doped hydroxyapatite and β-tricalcium phosphate powders suspended in a chitosan-based solution, in the presence of levofloxacin (LEV) as model drug and magnetic nanoparticles (MNP), were developed. The sintering step was removed from the production process, and the integrity of the printed scaffolds was assured by the polymerization capacity of the ink composite, using genipin as a crosslinking agent. The effects of MNP and LEV on the inks’ rheological properties, as well as on the mechanical and structural behaviour of non-doped and iron-doped scaffolds, were evaluated. Magnetic and magneto-thermal response, drug delivery and biological performance, such as cell proliferation in the absence and presence of an applied magnetic field, were also assessed. The addition of a constant amount of MNP in the iron-doped and non-doped CaP-based inks enhances their magnetic response and induction heating, with these effects more pronounced for the iron-doped CaP-based ink. These results suggest a synergistic effect between the iron-doped CaP-based powders and the MNP due to ferro/ferrimagnetic interactions. Furthermore, the iron presence enhances human mesenchymal stem cell metabolic activity and proliferation.

Antimicrobial resistance (AMR) has become a global concern as many bacterial species have developed resistance to commonly prescribed antibiotics, making them ineffective to treatments. One type of antibiotics, gallium(III) compounds, stands out as possible candidates due to their unique “Trojan horse” mechanism to tackle bacterial growth, by substituting iron(III) in the metabolic cycles of bacteria. In this study, we tested three polysaccharides (carboxymethyl cellulose (CMC), alginate, and pectin) as the binding and delivery agent for gallium on three bacteria (Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) with a potential bioresponsive delivery mode. Two types of analysis on bacterial growth (minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)) were carried out while iron(III)-loaded polysaccharide samples were also tested for comparison. The results suggested that gallium showed an improved inhibitory activity on bacterial growth, in particular gallium(III)-loaded carboxymethyl cellulose (Ga-CMC) sample showing an inhibiting effect on growth for all three tested bacteria. At the MIC for all three bacteria, Ga-CMC showed no cytotoxicity effect on human dermal neonatal fibroblasts (HDNF). Therefore, these bioresponsive gallium(III) polysaccharide compounds show significant potential to be developed as the next-generation antibacterial agents with controlled release capability.