Saloplastic membranes as green devices for soft tissue regeneration

Implantable devices must exhibit mechanical properties similar to native tissues to promote appropriate cellular behavior and regeneration. Herein, we report a new membrane manufacture method based on the synthesis of polyelectrolyte complexes (PECs) that exhibit saloplasticity, i.e. variable physical-chemistry using salt as a plasticizer. This is a Green Chemistry approach, as PECs generate structures that are stabilized solely by reversible electrostatic interactions, avoiding the use of harmful crosslinkers completely. Furthermore, natural polyelectrolytes - chitosan and alginate - were used. Upon mixing them, membranes were obtained by drying the PECs at 37ºC, yielding compact PECs without resorting to organicsolvents. The plasticizing effect of salt after synthesis was shown by measuring tensile mechanical properties, which were lower when samples were immersed in high ionic strength solutions.Salt was also used during membrane synthesis in different quan- tities (0 M, 0.15 M and 0.5 M in NaCl) yielding structures with no significant differences in morphology and degradation (around 15% after 3 months in lysozyme). However, swelling was higher (about 10x) when synthesized in the presence of salt. In vitro cell studies using L929 fibroblasts showed that cells adhered and proliferated preferentially in membranes fabricated in the presence of salt (i.e. the membranes with lower tensile strength). Structures with physical-chemical properties controlled with precision open a path to tissue engineering strategies depending on fine tuning mechanical properties and cellular adhesion simply by changing ionic strength during membrane manufacture

Compact saloplastic membranes of natural polysaccharides for soft tissue engineering

The regeneration of soft biological tissues requires new substitutes that exhibit mechanical properties similar to the native tissue. Herein, thin saloplastic membranes with tunable physical properties are prepared by complexation of chitosan and alginate solutions containing different concentrations of sodium chloride. Polyelectrolyte complexes (PECs) are transferred to flat Petri dishes for compaction into membrane shapes by sedimentation and solvent evaporation. All membranes are resistant to degradation by lysozyme and are stable in solutions with pH values between 1 and 13. Immersing the different membranes in new doping solutions of increasing salt concentrations triggers the typical saloplastic behavior, with a high water absorption and decrease of the rigidity and ultimate tensile strength. The range of such variations is tuned by the sodium chloride amount used in the synthesis: high salt concentrations increase water uptake and tensile moduli, while decreasing the ultimate strength. Cellular assays demonstrate high proliferation rates and viability of L929 fibroblasts seeded onto the most rigid membranes. The results validate the use of saloplastic membranes as soft tissue substitutes for future biomedical applications.

Flow cytometry analyses of calcification propensity

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Extremely strong and tough hydrogels as prospective candidates for tissue repair – A review

Ideal candidates for the repair of robust biological tissues should exhibit diverse features such as biocompatibility, strength, toughness, self-healing ability and a well-defined structure. Among the available biomaterials, hydrogels, as highly hydrated 3D-crosslinked polymeric networks, are promising for Tissue Engineering purposes as result of their high resemblance with native extracellular matrix. However, these polymeric structures often exhibit a poor mechanical behavior, hampering their use in load-bearing applications. During the last years, several efforts have been made to create new strategies and concepts to fabricate strong and tough hydrogels. Although it is already possible to shape the mechanical properties of artificial hydrogels to mimic biotissues, critical issues regarding, for instance, their biocompatibility and hierarchical structure are often neglected. Therefore, this review covers the structural and mechanical characteristics of the developed methodologies to toughen hydrogels, highlighting some pioneering efforts employed to combine the aforementioned properties in natural-based hydrogels.

Modulation of the secretome of hBMSCs by tailoring the macromolecular gradient in hydrogels to generate tissue-to-tissue interfaces

Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and chemical gradients serving a wide range of physiological functions. These interfaces are responsible for mediation of load transfer between two adjacent tissues. They are also important structures in sustaining the cellular communications to retain tissueâ s functional integration and homeostasis. [1] All cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in three-dimensional (3D) environments they tend to perform their function better than in two-dimensional (2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional hydrogel system. Specifically, differential paracrine secretory profiles can be engineered using human Bone Marrow Stem Cells (hBMSCâ s). Hence, the specific objectives of this study are to: assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSCâ s encapsulation by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSCâ s over time. The gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding the two solutions consecutively on top of each other. The labelling of the macromolecules was performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA and Col degradation assay, degree of crosslinking and stability. hBMSCâ s at P3 were encapsulated into each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously described. The hBMSCâ s were observed under confocal microscopy to assess viability by Live/Dead® staining. Cellular behaviour concerning proliferation and matrix deposition was also performed. Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different macromolecules showed to be a suitable environment for hBMSCâ s. The hBMSCâ s gradient hydrogels supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated hBMSCâ s. References: 1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006 2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008

Advancing tendon regeneration through the development of bio-stimulating tissue engineering approaches

Tendon tissue engineering (TE) requires tailoring scaffolds designs and properties to the anatomical and functional requirements of tendons located in different regions of the body. Cell sourcing is also of utmost importance as tendon cells are scarce. Recently, we have found that it is possible to direct the tenogenic differentiation of Amniotic fluid and Adipose tissue derived stem cells (hAFSCs and hASCs), and also that there are hASCs subpopulations that might be more prone to tenogenic differentiation. Nevertheless, biochemical stimulation may not be enough to develop functional TE substitutes for a tissue that is known to be highly dependent on mechanical loading.

In vitro mechanical fatigue behavior of poly-ε-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and subjected to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behaviour of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow’s criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.

Polysaccharide-based freestanding multilayered membranes exhibiting reversible switchable properties

The design of self-standing multilayered structures based on biopolymers has been attracting increasing interest due to their potential in the biomedical field. However, their use has been limited due to their gel-like properties. Herein, we report the combination of covalent and ionic cross-linking, using natural and non-cytotoxic cross-linkers, such as genipin and calcium chloride (CaCl2). Combining both cross-linking types the mechanical properties of the multilayers increased and the water uptake ability decreased. The ionic cross-linking of multilayered chitosan (CHI)â alginate (ALG) films led to freestanding membranes with multiple interesting properties, such as: improved mechanical strength, calcium-induced adhesion and shape memory ability. The use of CaCl2 also offered the possibility of reversibly switching all of these properties by simple immersion in a chelate solution. We attribute the switch-ability of the mechanical properties, shape memory ability and the propensity for induced-adhesion to the ionic cross-linking of the multilayers. These findings suggested the potential of the developed polysaccharide freestanding membranes in a plethora of research fields, including in biomedical and biotechnological fields.

Highly functional and biodegradable nanofibrous scaffolds combined with stem cells for bone and cartilage tissue engineering

Among the various possible embodiements of Advanced Therapies and in particular of Tissue Engineering the use of temporary scaffolds to regenerate tissue defects is one of the key issues. The scaffolds should be specifically designed to create environments that promote tissue development and not merely to support the maintenance of communities of cells. To achieve that goal, highly functional scaffolds may combine specific morphologies and surface chemistry with the local release of bioactive agents. Many biomaterials have been proposed to produce scaffolds aiming the regeneration of a wealth of human tissues. We have a particular interest in developing systems based in nanofibrous biodegradable polymers1,2. Those demanding applications require a combination of mechanical properties, processability, cell-friendly surfaces and tunable biodegradability that need to be tailored for the specific application envisioned. Those biomaterials are usually processed by different routes into devices with wide range of morphologies such as biodegradable fibers and meshes, films or particles and adaptable to different biomedical applications. In our approach, we combine the temporary scaffolds populated with therapeutically relevant communities of cells to generate a hybrid implant. For that we have explored different sources of adult and also embryonic stem cells. We are exploring the use of adult MSCs3, namely obtained from the bone marrow for the development autologous-based therapies. We also develop strategies based in extra-embryonic tissues, such as amniotic fluid (AF) and the perivascular region of the umbilical cord4 (Whartonâ s Jelly, WJ). Those tissues offer many advantages over both embryonic and other adult stem cell sourcess. These tissues are frequently discarded at parturition and its extracorporeal nature facilitates tissue donation by the patients. The comparatively large volume of tissue and ease of physical manipulation facilitates the isolation of larger numbers of stem cells. The fetal stem cells appear to have more pronounced immunomodulatory properties than adult MSCs. This allogeneic escape mechanism may be of therapeutic value, because the transplantation of readily available allogeneic human MSCs would be preferable as opposed to the required expansion stage (involving both time and logistic effort) of autologous cells. Topics to be covered: This talk will review our latest developments of nanostructured-based biomaterials and scaffolds in combination with stem cells for bone and cartilage tissue engineering.

Chitosan-based scaffolds for tissue regeneration: preparation and microstructure characterization

Scaffolds are porous three-dimensional supports, designed to mimic the extracellular environment and remain temporarily integrated into the host tissue while stimulating, at the molecular level, specific cellular responses to each type of body tissues. The major goal of the research work entertained herein was to study the microstructure of scaffolds made from chitosan (Ch), blends of chitosan and sodium alginate (Ch/NaAlg), blends of chitosan, sodium alginate and calcium chloride (Ch/NaAlg/CaCl2) and blends of chitosan, sodium alginate and hydroxyapatite (Ch/NaAlg/HA). Scaffolds possessing ideal physicochemical properties facilitate cell proliferation and greatly increase the rate of recovery of a damaged organ tissue. Using CT three-dimensional images of the scaffolds, it was observed that all scaffolds had a porosity in the range 64%-92%, a radius of maximum pore occurrence in the range 95m-260m and a permeability in the range 1×10-10-18×10-10 m2. From the results obtained, the scaffolds based on Ch, Ch/NaAlg and Ch/NaAlg/CaCl2 would be most appropriate both for the growth of osteoid and for bone tissue regeneration, while the scaffold made with a blend of Ch/NaAlg/HA, by possessing larger pores size, might be used as a support for fibrovascular tissue.

Biomaterials for cartilage tissue engineering under mechanical stimulus

Tese de Doutoramento em Ciências (Especialidade de Física)

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Poly(ester-urethane) scaffolds: effect of structure on properties and osteogenic activity of stem cells

The present study aimed to investigate the effect of structure (design and porosity) on the matrix stiffness and osteogenic activity of stem cells cultured on poly(ester-urethane) (PEU) scaffolds. Different three-dimensional (3D) forms of scaffold were prepared from lysine-based PEU using traditional salt-leaching and advanced bioplotting techniques. The resulting scaffolds were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), mercury porosimetry and mechanical testing. The scaffolds had various pore sizes with different designs, and all were thermally stable up to 300â °C. In vitrotests, carried out using rat bone marrow stem cells (BMSCs) for bone tissue engineering, demonstrated better viability and higher cell proliferation on bioplotted scaffolds compared to salt-leached ones, most probably due to their larger and interconnected pores and stiffer nature, as shown by higher compressive moduli, which were measured by compression testing. Similarly, SEM, von Kossa staining and EDX analyses indicated higher amounts of calcium deposition on bioplotted scaffolds during cell culture. It was concluded that the design with larger interconnected porosity and stiffness has an effect on the osteogenic activity of the stem cells.

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of k-carrageenan hydrogels for the delivery of stem cells obt ained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation met hod and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v k-carrageenan solution at a cell density of 5  10 6 cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that k-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mec hanical analysis demonstrated an increase in stiffness and viscoelastic properties of k-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that k-carrageenan exhibits properties t hat enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects.

Marine Collagen/Apatite scaffolds envisaging tissue engineering applications

Despite the vast investigation and the large amount of products already available in the market to treat the different bone defects there is still a growing need to develop more advanced and complex therapeutic strategies. In this context, a mixture of Marine Hydroxyapatite-Fluorapatite:Collagen (HA-FP:ASC) seems to be a promising solution to overcome these bone defects, specifically, dental defects. HA-FP particles (20–63 μm) were obtained through pyrolysis (950°C, 12 h) of shark teeth (Isurus oxyrinchus, P. glauca), and Type I collagen was isolated from Prionace glauca skin as previously described (1). After the steps of purification, collagen was solubilized in 0.5 M acetic acid and HA-FP added producing three different formulations: were produced, 30:70, 50:50 and 70:30 of HA-FP:ASC, respectively. EDC/NHS and HMDI binding agents were used to stabilize the produced scaffolds. Mechanical properties were evaluated by compression tests. SEM analysis allowed observing the mineral deposition, after immersion in simulated body fluid and also permitted to evaluate how homogenous was the distribution of HA-FP in the different scaffold formulations, also confirmed by μ-CT assay. It was readily visible by Cytotoxicity and life/dead CLSM assays that cells were able to adhere and proliferate in the produced scaffolds. Scaffolds crosslinked with EDC/NHS showed lower cytotoxicity, being the ones chosen for further cellular evaluation.