Clara R. Correia

Chitosan Scaffolds Containing Hyaluronic Acid for Cartilage Tissue Engineering

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.

Nanostructured Polymeric Coatings Based on Chitosan and Dopamine‐Modified Hyaluronic Acid for Biomedical Applications

In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick to a surface. These mussel proteins present an unusual amino acid 3,4-dihydroxyphenylalanine (known as DOPA). The outstanding adhesive properties of these materials in the sea harsh conditions have been attributed to the presence of the catechol groups present in DOPA. Inspired by the structure and composition of these adhesive proteins, dopamine-modified hyaluronic acid (HA-DN) prepared by carbodiimide chemistry is used to form thin and surface-adherent dopamine films. This conjugate was characterized by distinct techniques, such as nuclear magnetic resonance and ultraviolet spectrophotometry. Multilayer films are developed based on chitosan and HA-DN to form polymeric coatings using the layer-by-layer methodology. The nanostructured films formation is monitored by quartz crystal microbalance. The film surface is characterized by atomic force microscopy and scanning electron microscopy. Water contact angle measurements are also conducted. The adhesion properties are analyzed showing that the nanostructured films with dopamine promote an improved adhesion. In vitro tests show an enhanced cell adhesion, proliferation and viability for the biomimetic films with catechol groups, demonstrating their potential to be used in distinct biomedical applications.

Multilayered hierarchical capsules providing cell adhesion sites.

Liquified capsules featuring (i) an external shell by layer-by-layer assembly of poly(l-lysine), alginate, and chitosan, and encapsulating (ii) surface functionalized poly(l-lactic acid) (PLLA) microparticles were developed. We hypothesize that, while the liquified environment enhances the diffusion of essential molecules for cell survival, microparticles dispersed in the liquified core of capsules provide the physical support required for cellular functions of anchorage-dependent cells. The influence of the incorporation of PLL on the regime growth, thickness, and stability was analyzed. Results show a more resistant and thicker film with an exponential build-up growth regime. Moreover, capsules ability to support cell survival was assessed. Capsules containing microparticles revealed an enhanced biological outcome in cell metabolic activity and proliferation, suggesting their potential to boost the development of innovative biomaterial designs for bioencapsulation systems and tissue engineering products.

Liquified chitosan–alginate multilayer capsules incorporating poly(L-lactic acid) microparticles as cell carriers

We report the development of liquified multilayer hierarchical capsules capable of providing cell adhesion sites to the encapsulated cells. The proof of principle is demonstrated with the example of a chitosan–alginate shell via layer-by-layer assembly, encapsulating cells adhered to the functionalized surface of poly(L-lactic acid) microparticles.

Adhesive nanostructured multilayer films using a bacterial exopolysaccharide for biomedical applications

Medical adhesives and sealants often require that long-term adhesiveness is achieved. In this work, nanostructured coatings consisting of chitosan and the adhesive bacterial exopolysaccharide levan are fabricated using layer-by-layer (LbL) assembly. Taking advantage of the electrostatic self-assembly mechanism of LbL, the charges of both chitosan and a phosphonate-derivatized levan (Ph-levan) are measured and the feasibility of constructing hybrid films is monitored and confirmed using a quartz crystal microbalance with dissipation monitoring (QCM-D). The adhesive properties between two identical bonded films with a total of 100 layers are compared to control films in which Ph-levan is replaced by alginate, revealing that the detachment force of the former is about 3 times higher than the control. Scanning electron microscopy of the films surface shows that the surface of Ph-levan films is smooth and homogeneous. Cell adhesion tests were conducted using a L929 cell line. Early cell adhesion is significantly higher in chitosan/Ph-levan films when compared to chitosan/alginate controls. These findings establish levan derivatives as bioinspired ingredients for conceiving medical adhesive devices that allow achieving enhanced mechanical and biological performance.

Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation.

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Magnetically Labeled Cells with Surface-Modified Fe3O4 Spherical and Rod-Shaped Magnetic Nanoparticles for Tissue Engineering Applications

Magnetically targeted cells with internalized magnetic nanoparticles (MNPs) could allow the success of cell transplantation and cell-based therapies, overcoming low cell retention that occurs when delivering cells by intravenous or local injection. Upon magnetization, these cells could then accumulate and stimulate the regeneration of the tissue in situ. Magnetic targeting of cells requires a detailed knowledge between interactions of engineered nanomaterials and cells, in particular the influence of shape and surface functionalization of MNPs. For the first time, cellular internalization of amino surface-modified iron oxide nanoparticles of two different shapes (nanospheres or nanorods) is studied. MNPs show high cellular uptake and labeled cells could exhibit a strong reaction with external magnetic fields. Compared to nanorods, nanospheres show better internalization efficiency, and labeled cells exhibit strong transportation reaction with external magnetic fields. Contiguous viable cell-sheets are developed by magnetic-force-based tissue engineering. The results confirm that the developed magnetic-responsive nano-biomaterials have potential applicability in tissue engineering or cellular therapies.

Liquid Marbles for High‐Throughput Biological Screening of Anchorage‐Dependent Cells

Stable liquid marbles (LM) are produced by coating liquid droplets with a hydrophobic powder. The used hydrophobic powder is produced by fluorosi-lanization of diatomaceous earth, used before to produce superhydrophobic structures. Here, the use of LM is proposed for high-throughput drug screening on anchorage-dependent cells. To provide the required cell adhesion sites inside the liquid environment of LM, surface-modified poly(l-lactic acid) microparticles are used. A simple method that takes advantage from LM appealing features is presented, such as the ability to inject liquid on LM without disrupting (self-healing ability), and to monitor color changes inside of LM. After promoting cell adhesion, a cytotoxic screening test is performed as a proof of concept. Fe(3+) is used as a model cytotoxic agent and is injected on LM. After incubation, AlamarBlue reagent is injected and used to assess the presence of viable cells, by monitoring color change from blue to red. Color intensity is measured by image processing and the analysis of pictures takes using an ordinary digital camera. The proposed method is fully validated in counterpoint to an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo​xymethoxyphenyl)-2-(4-sulfophenyl)-2H-te​trazolium) colorimetric assay, a well-known method used for the cytotoxicity assessment.

Sequential ionic and thermogelation of chitosan spherical hydrogels prepared using superhydrophobic surfaces to immobilize cells and drugs

Chitosan is soluble in acidic media, which makes it incompatible for the encapsulation of cells and pH-sensitive molecules. In this work, a mild chitosan-based system with two sequential gelation steps is proposed, where the model drug dexamethasone and L929 cells are immobilized inside hydrogel beads. Superhydrophobic surfaces were used to produce the spherical hydrogel particles that provided favorable conditions to encapsulate cells or bioactive agents. First, the chitosan acidic solution was neutralized with β-glycerophosphate at room temperature to pH 6.2. Suspended cells (or dexamethasone) in the formulation were dispensed in controlled volumes onto biomimetic polystyrene superhydrophobic surfaces, to form spherical shapes. The addition of sodium tripolyphosphate on the top of each sphere induced an ionic gelation process of the chitosan through electrostatic interactions. At 37°C, the hydrophobicity of the chitosan-based formulations increased and a second gelation step occurred, which increased the elastic modulus. In addition, the pH-responsive behavior characteristic of chitosan was maintained. The softness and flexibility of the system can potentially be utilized to implant cells and therapeutic molecules using less invasive procedures.

Compartmentalized bioencapsulated liquefied 3D macro-construct by perfusion-based layer-by-layer technique

Self-supporting, millimeter length 3D constructs consisting of individualized liquefied compartments, were produced using cell encapsulated hydrogel beads as building blocks. A perfusion-based layer-by-layer approach was used that allowed bioencapsulated beads to assemble, pattern, hold and attach to produce non-liquefied 3D constructs with controlled shape, displaying the binding feature of a continuous nanometric multilayer coating. No binders or crosslinking strategies were used. The internal microenvironment of this 3D construct was modified from solid to liquefied state by chelation. MTS and live–dead assays showed enhanced L929 cell viability with liquefied 3D constructs, compared to non-liquefied ones. The proposed technique opens new prospects to create complex 3D polyelectrolyte based constructs for tissue engineering applications.