Sara M. Oliveira

Cell interactions with superhydrophilic and superhydrophobic surfaces

Interactions of cells with biomaterials dictate their biocompatibility and biofunctionality, and are strongly influenced by surface properties. Moreover, it is important to control cell adhesion to surfaces for biological studies and diagnosis. Surface properties influence protein adsorption in terms of conformation and quantity adsorbed that further affects cell adhesion and proliferation. Several works have demonstrated that wettability influences cell attachment and proliferation. However, most studies have reported the influence of the surface energy of smooth substrates within a limited range of wettabilities. By controlling the roughness and the hydrophilicity of the surface, one can obtain biomimetic substrates with a wettability ranging from superhydrophobic to superhydrophilic. This review intends to summarize recent works, where the interaction of cells with surfaces with extreme wettabilities was investigated. Such information may be relevant in different biomedical and biological applications including diagnosis, cell biology, or tissue engineering.

Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate.

Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P > 5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL.

Towards the design of 3D multiscale instructive tissue engineering constructs: Current approaches and trends

The design of 3D constructs with adequate properties to instruct and guide cells both in vitro and in vivo is one of the major focuses of tissue engineering. Successful tissue regeneration depends on the favorable crosstalk between the supporting structure, the cells and the host tissue so that a balanced matrix production and degradation are achieved. Herein, the major occurring events and players in normal and regenerative tissue are overviewed. These have been inspiring the selection or synthesis of instructive cues to include into the 3D constructs. We further highlight the importance of a multiscale perception of the range of features that can be included on the biomimetic structures. Lastly, we focus on the current and developing tissue-engineering approaches for the preparation of such 3D constructs: top-down, bottom-up and integrative. Bottom-up and integrative approaches present a higher potential for the design of tissue engineering devices with multiscale features and higher biochemical control than top-down strategies, and are the main focus of this review.

Development of an injectable system based on elastin-like recombinamer particles for tissue engineering applications

An elastin-like recombinamer (ELR) containing the RGD cell adhesion domain was used to fabricate microparticles by an innovative and affordable process based on the use of superhydrophobic surfaces. Two microparticles types with different crosslinking extents were prepared. The biological response was tested using an osteoblast-like cell line (SaOs-2) performing proliferation and alkaline phosphatase (ALP) quantification tests, as well as assessing cytotoxicity, morphology and cell distribution on the particles. The main goal of the work was the assessment of the in vitro formation of cell-induced microparticle aggregates that could provide indications for the possible formation of an in situ-forming scaffold upon implantation. ELR microparticles have been successfully obtained by deposition of a polymeric solution on bioinspired polystyrene superhydrophobic surfaces and two different crosslinking extents were achieved by controlling the time of exposure to the crosslinker. The crosslinking extent affected swelling behavior and the dynamic mechanical properties of the particles. SaOs-2 morphology, ALP expression, spatial distribution and ability to bind the microparticles together were dependent on the physicochemical properties of the microparticles: the more crosslinked condition was the most favorable for cell proliferation and to form a cell-induced aggregation scaffold, making these particles suitable to be applied in bone tissue engineering.

Hierarchical Fibrillar Scaffolds Obtained by Non-conventional Layer-By-Layer Electrostatic Self-Assembly

A new application of layer-by-layer assembly is presented, able to create nano/micro fibrils or nanocoatings inside 3D scaffolds using non-fibrillar polyelectrolytes for tissue-engineering applications. This approach shows promise for developing advanced scaffolds with controlled nano/micro environments, and nature and architectures similar to the natural extracellular matrix, leading to improved biological performance.

Nanocoatings containing sulfated polysaccharides prepared by layer-by-layer assembly as models to study cell–material interactions

The understanding of both cell-extracellular matrix (ECM) and cell-material interactions is crucial for the success of implantable biomaterials including tissue engineering devices. ECM is rich in sulfated and aminated glycosaminoglycans and proteoglycans. The development of synthetic models containing those chemical groups is thus of major interest. Thin coatings of polysaccharides with controlled sulfur and nitrogen contents were developed by layer-by-layer assembly. In particular, the multilayers were prepared by assembling chitosan with κ-, ι- and λ-carrageenan (increasing sulfur content). The nanostructured multilayers were characterized by quartz crystal microbalance with dissipation (QCM-D), atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle, X-ray photoelectron spectroscopy (XPS) and used as models to study the effect of the sulfate groups over the behavior of osteoblast-like cells. The biomimetic coatings increased the alkaline phosphatase (ALP) activity and proliferation compared with unmodified polycaprolactone surfaces. Biomineralization increase with the presence of the coatings is significantly higher on ι-carrageenan coatings, suggesting that the sulfate groups may interact positively with molecules involved in the osteoblastic activity as it occurs in the natural ECM. The developed nanocoatings can constitute an interesting model to understand the biological influence of the sulfate and amine groups existing on the surface of biomaterials.

Assembly of cell-laden hydrogel fiber into non-liquefied and liquefied 3D spiral constructs by perfusion-based layer-by-layer technique

In this work, three-dimensional (3D) self-sustaining, spiral-shaped constructs were produced through a combination of ionotropic gelation, to form cell-encapsulated alginate fibers, and a perfusion-based layer-by-layer (LbL) technique. Single fibers were assembled over cylindrical molds by reeling to form spiral shapes, both having different geometries and sizes. An uninterrupted nanometric multilayer coating produced by a perfusion-based LbL technique, using alginate and chitosan, generated stable 3D spiral-shaped macrostructures by gripping and affixing the threads together without using any crosslinking/binding agent. The chelation process altered the internal microenvironment of the 3D construct from the solid to the liquefied state while preserving the external geometry. L929 cell viability by MTS and dsDNA quantification favor liquefied 3D constructs more than non-liquefied ones. The proposed technique setup helps us to generate complex polyelectrolyte-based 3D constructs for tissue engineering applications and organ printing.

Extraction and characterization of collagen from Antarctic and Sub-Antarctic squid and its potential application in hybrid scaffolds for tissue engineering

Collagen is the most abundant protein found in mammals and it exhibits a low immunogenicity, high biocompatibility and biodegradability when compared with others natural polymers. For this reason, it has been explored for the development of biologically instructive biomaterials with applications for tissue substitution and regeneration. Marine origin collagen has been pursued as an alternative to the more common bovine and porcine origins. This study focused on squid (Teuthoidea: Cephalopoda), particularly the Antarctic squid Kondakovia longimana and the Sub-Antarctic squid Illex argentinus as potential collagen sources. In this study, collagen has been isolated from the skins of the squids using acid-based and pepsin-based protocols, with the higher yield being obtained from I. argentinus in the presence of pepsin. The produced collagen has been characterized in terms of physicochemical properties, evidencing an amino acid profile similar to the one of calf collagen, but exhibiting a less preserved structure, with hydrolyzed portions and a lower melting temperature. Pepsin-soluble collagen isolated from I. argentinus was selected for further evaluation of biomedical potential, exploring its incorporation on poly-ε-caprolactone (PCL) 3D printed scaffolds for the development of hybrid scaffolds for tissue engineering, exhibiting hierarchical features.

Platelet lysate-based pro-angiogenic nanocoatings

UNLABELLED Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenic-associated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. STATEMENT OF SIGNIFICANCE The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures.

Structural monitoring and modeling of the mechanical deformation of three-dimensional printed poly(ε-caprolactone) scaffolds

Three-dimensional (3D) printed poly(ε-caprolactone) (PCL) based scaffolds have being proposed for different tissue engineering applications. This study addresses the design and fabrication of 3D PCL constructs with different struts alignments at 90°, 45° and 90° with offset. The morphology and the mechanical behavior under uniaxial compressive load were assessed at different strain percentages. The combination of a new compressionCT device and micro computed tomography (micro-CT) allowed understanding the influence of pore geometry under controlled compressive strain in the mechanical and structural behavior of PCL constructs. Finite element analysis (FEA) was applied using the micro-CT data to modulate the mechanical response and compare with the conventional uniaxial compression tests. Scanning electron microscopic analysis showed a very high level of reproducibility and a low error comparing with the theoretical values, confirming that the alignment and the dimensional features of the printed struts are reliable. The mechanical tests showed that the 90° architecture presented the highest stiffness. With the compressionCT device was observed that the 90° and 90° with offset architectures presented similar values of porosity at same strain and similar pore size, contrary to the 45° architecture. Thus, pore geometric configurations affected significantly the deformability of the all PCL scaffolds under compression. The prediction of the FEA showed a good agreement to the conventional mechanical tests revealing the areas more affected under compression load. The methodology proposed in this study using 3D printed scaffolds with compressionCT device and FEA is a framework that offers great potential in understanding the mechanical and structural behavior of soft systems for different applications, including for the biomedical engineering field.