María Concepción Serrano

Deep Eutectic Solvents in Polymerizations: A Greener Alternative to Conventional Syntheses

The use of deep eutectic solvents (DESs) that act as all-in-one solvent-template-reactant systems offers an interesting green alternative to conventional syntheses in materials science. This Review aims to provide a comprehensive overview to emphasize the similarities and discrepancies between DES-assisted and conventional syntheses and rationalize certain green features that are common for the three DES-assisted syntheses described herein: one case of radical polymerization and two cases of polycondensations. For instance, DESs contain the precursor itself and some additional components that either provide certain functionality (e.g., drug delivery and controlled release, or electrical conductivity) to the resulting materials or direct their formation with a particular structure (e.g., hierarchical-type). Moreover, DESs provide a reaction medium, so polymerizations are ultimately carried out in a solventless fashion. This means that DES-assisted syntheses match green chemistry principles 2 and 5 because of the economy of reagents and solvents, whereas the functionality incorporated by the second component allows the need for any post-synthesis derivatization to be minimized or even fully avoided (principle 8). DESs also provide new precursors that favor more efficient polymerization (principle 6) by decreasing the energy input required for reaction progress. Finally, the use of mild reaction conditions in combination with the compositional versatility of DESs, which allows low-toxic components to be selected, is also of interest from the viewpoint of green chemistry because it opens up the way to design biocompatible and/or eco-friendly synthetic methods (principle 3).

Recent Insights Into the Biomedical Applications of Shape‐memory Polymers

Shape-memory polymers (SMP) are versatile stimuli-responsive materials that can switch, upon stimulation, from a temporary to a permanent shape. This advanced functionality makes SMP suitable and promising materials for diverse technological applications, including the fabrication of smart biomedical devices. In this paper, advances in the design of SMP are discussed, with emphasis on materials investigated for medical applications. Future directions necessary to bring SMP closer to their clinical application are also highlighted.

3D free-standing porous scaffolds made of graphene oxide as substrates for neural cell growth

The absence of efficient therapies for the treatment of lesions affecting the central nervous system encourages scientists to explore new materials in an attempt to enhance neural tissue regeneration while preventing inhibitory fibroglial scars. In recent years, the superlative properties of graphene-based materials have provided a strong incentive for their application in biomedicine. Nonetheless, a few attempts to date have envisioned the use of graphene for the fabrication of three-dimensional (3D) substrates for neural repair, but none of these involve graphene oxide (GOx) despite some attractive features such as higher hydrophilicity and versatility of functionalization. In this paper, we report novel, free-standing, porous and flexible 3D GOx-based scaffolds, produced by the biocompatible freeze-casting procedure named ISISA, with potential utility in neural tissue regeneration. The resulting materials were thoroughly characterized by Fourier-transform infrared, Raman, and X-ray photoelectron spectroscopies and scanning electron microscopy, as well as flexibility testing. Embryonic neural progenitor cells were then used to investigate adhesion, morphology, viability, and neuronal/glial differentiation. Highly viable and interconnected neural networks were formed on these 3D scaffolds, containing both neurons and glial cells and rich in dendrites, axons and synaptic connections, and the results are in agreement with those obtained in initial studies performed with two-dimensional GOx films. These results encourage further investigation in vivo on the use of these scaffolds as guide substrates to promote the repair of neural injuries.

Characterization of Porcine Circulating Progenitor Cells: Toward a Functional Endothelium

The lack of available healthy vessels, significant patient morbidity, and high costs hinders the successful clinical utilization of autologous endothelial cells (ECs). Herein we assess the feasibility of using endothelial progenitor cells (EPC) found in circulating blood to engineer a functional endothelium on poly(1,8-octanediol-co-citrate) (POC), a hemocompatible and biodegradable elastomer used in vascular tissue engineering. EPCs were isolated from porcine blood and biochemically differentiated into porcine endothelial (PE)-like cells in vitro. Once differentiated, EC phenotype and function on POC were assessed according to the presence of the EC-specific markers von Willebrand factor, platelet EC adhesion molecule, and vascular endothelial cadherin; metabolism of acetylated low-density lipoprotein; secretion of the anti-thrombogenic factors nitric oxide and prostacyclin; and inhibition of platelet adhesion and clotting processes in vitro. The effects of PE-like cells on porcine aortic smooth muscle cells (PASMCs) were also investigated via co-culture. PE-like cells on POC had phenotype, function, and clotting responses similar to those of primary aortic ECs. The presence of PE-like cells resulted in a 71 +/- 20% decrease in PASMC proliferation; a 52 +/- 2% decrease in the protein:deoxyribonucleic acid ratio; and an elongated, spindle-shaped morphology indicative of a shift from the proliferative to the contractile phenotype. These data suggest that EPCs and POC can provide the basis for a functional tissue-engineered endothelium.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

Deep eutectic solvent-assisted synthesis of biodegradable polyesters with antibacterial properties.

Bacterial infection related to the implantation of medical devices represents a serious clinical complication, with dramatic consequences for many patients. In past decades, numerous attempts have been made to develop materials with antibacterial and/or antifouling properties by the incorporation of antibiotic and/or antiseptic compounds. In this context, deep eutectic solvents (DESs) are acquiring increasing interest not only as efficient carriers of active principle ingredients (APIs) but also as assistant platforms for the synthesis of a wide repertoire of polymer-related materials. Herein, we have successfully prepared biodegradable poly(octanediol-co-citrate) polyesters with acquired antibacterial properties by the DES-assisted incorporation of quaternary ammonium or phosphonium salts into the polymer network. In the resulting polymers, the presence of these salts (i.e., choline chloride, tetraethylammonium bromide, hexadecyltrimethylammonium bromide, and methyltriphenylphosphonium bromide) inhibits bacterial growth in the early postimplantation steps, as tested in cultures of Escherichia coli on solid agar plates. Later, positive polymer cytocompatibility is expected to support cell colonization, as anticipated from in vitro preliminary studies with L929 fibroblasts. Finally, the attractive elastic properties of these polyesters permit matching those of soft tissues such as skin. For all of these reasons, we envisage the utility of some of these antibacterial, biocompatible, and biodegradable polyesters as potential candidates for the preparation of antimicrobial wound dressings. These results further emphasize the enormous versatility of DES-assisted synthesis for the incorporation, in the synthesis step, of a wide palette of APIs into polymeric networks suitable for biomedical applications.

Chondroitin sulphate-based 3D scaffolds containing MWCNTs for nervous tissue repair

Nervous tissue lesions are an important social concern due to their increasing prevalence and their high sanitary costs. Their treatment still remains a challenge because of the reduced ability of nervous tissue to regenerate, its intrinsic structural and functional complexity and the rapid formation of fibroglial scars inhibiting neural repair. Herein, we show that 3D porous scaffolds made of chondroitin sulphate (CS), a major regulatory component of the nervous tissue, and multi-walled carbon nanotubes (MWCNTs) are selective substrates for the formation of a viable and neuron-enriched network with a transitory low glial content. Scaffolds have been fabricated by using the ice segregation-induced self-assembly technique and cultured with embryonic neural progenitor cells. Cell adhesion, morphology, viability, neuron/glial differentiation, calcium signaling dynamics, and mitochondrial activity have been studied over time on the scaffolds and compared to appropriate 2D control substrates. Our results indicate the formation of viable cultures enriched in neuron cells for up to 20 days, with ability to display calcium transients and active mitochondria, even in the absence of poly-D-lysine coating. A synergistic neural-permissive signaling from both the scaffold structure and its components (i.e., MWCNTs and CS) is suggested as the major responsible factor for these findings. We anticipate that these scaffolds may serve nerve regeneration if implanted in the acute phase after injury, as it is during the first stages of graft implantation when the most critical sequence of phenomena takes place to drive either nervous regeneration or fibroglial scar formation. The temporary glial inhibition found may be, indeed, beneficial for promoting the formation of neuron-enriched circuits at early phases while guaranteeing posterior glial integration to support longer-term neuron survival and activity.

Subacute Tissue Response to 3D Graphene Oxide Scaffolds Implanted in the Injured Rat Spinal Cord

The increasing prevalence and high sanitary costs of lesions affecting the central nervous system (CNS) at the spinal cord are encouraging experts in different fields to explore new avenues for neural repair. In this context, graphene and its derivatives are attracting significant attention, although their toxicity and performance in the CNS in vivo remains unclear. Here, the subacute tissue response to 3D flexible and porous scaffolds composed of partially reduced graphene oxide is investigated when implanted in the injured rat spinal cord. The interest of these structures as potentially useful platforms for CNS regeneration mainly relies on their mechanical compliance with neural tissues, adequate biocompatibility with neural cells in vitro and versatility to carry topographical and biological guidance cues. Early tissue responses are thoroughly investigated locally (spinal cord at C6 level) and in the major organs (i.e., kidney, liver, lung, and spleen). The absence of local and systemic toxic responses, along with the positive signs found at the lesion site (e.g., filler effect, soft interface for no additional scaring, preservation of cell populations at the perilesional area, presence of M2 macrophages), encourages further investigation of these materials as promising components of more efficient material-based platforms for CNS repair.

Modulating the cytocompatibility of tridimensional carbon nanotube-based scaffolds†

Carbon nanotubes (CNTs) have lately attracted significant attention in the field of biomedicine. Although a wide repertoire of CNT-based composites has been explored as substrates for cell growth, the fabrication of 3D scaffolds has been more rarely accomplished. Additionally, concerns referred to CNT biocompatibility make their use in biomaterials still controversial. Herein we explore the interaction of three types of CNT-based 3D scaffolds - prepared with multi-walled CNTs and processed to show different architectural and morphological features at the microscale by using three different polymers (i.e., chitosan, chondroitin sulphate and gelatin) - with three types of mammalian cells displaying different sizes and adhesion patterns. Cell-material interaction has been assessed by studying cell viability, adhesion, morphology, and apoptosis. By means of time-lapse confocal laser scanning microscopy, we investigate, for the first time in CNT-based scaffolds, cell migration processes in real time. Scaffolds displaying both a pore size in range with that of cells and lower surface roughness reveal the highest viability values. In contrast, those with a smaller pore size and higher surface roughness account for the lowest cytocompatibility. Results from these studies benefit the fabrication of optimized biomaterials by varying scaffold-dependent parameters in accordance with those of target cells. Furthermore, they may serve to anticipate the response of other cell types sharing similar characteristics to those described herein when in contact with CNT-based scaffolds.

Immunomodulatory and angiogenic responses induced by graphene oxide scaffolds in chronic spinal hemisected rats.

Attractive physic-chemical features of graphene oxide (GO) and promising results inxa0vitro with neural cells encourage its exploration for biomedical applications including neural regeneration. Fueled by previous findings at the subacute state, we herein investigate for the first time chronic tissue responses (at 30 days) to 3D scaffolds composed of partially reduced GO (rGO) when implanted in the injured rat spinal cord. These studies aim to define fibrotic, inflammatory and angiogenic changes at the lesion site induced by the chronic implantation of these porous structures. Injured animals receiving no scaffolds show badly structured lesion zones and more cavities than those carrying rGO materials, thus pointing out a significant role of the scaffolds in injury stabilization and sealing. Notably, GFAP(+) cells and pro-regenerative macrophages are evident at their interface. Moreover, rGO scaffolds support angiogenesis around and, more importantly, inside their structure, with abundant and functional new blood vessels in whose proximities inside the scaffolds some regenerated neuronal axons are found. On the contrary, lesion areas without rGO scaffolds show a diminished quantity of blood vessels and no axons at all. These findings provide a foundation for the usefulness of graphene-based materials in the design of novel biomaterials for spinal cord repair and encourage further investigation for the understanding of neural tissue responses to this kind of materials inxa0vivo.