Rúben Pereira

Development of novel alginate based hydrogel films for wound healing applications.

Alginate and Aloe vera are natural materials widely investigated and used in the biomedical field. In this research work, thin hydrogel films composed by alginate and Aloe vera gel in different proportions (95:5, 85:15 and 75:25, v/v) were prepared and characterized. The films were evaluated regarding the light transmission behavior, contact angle measurements, and chemical, thermal and mechanical properties. These thin hydrogel films, prepared by crosslinking reaction using 5% calcium chloride solution, were also investigated relatively to their water solubility and swelling behavior. Results showed that Aloe vera improved the transparency of the films, as well their thermal stability. The developed films present adequate mechanical properties for skin applications, while the solubility studies demonstrated the insolubility of the films after 24h of immersion in distilled water. The water absorption and swelling behavior of these films were greatly improved by the increase in Aloe vera proportion.

Advanced biofabrication strategies for skin regeneration and repair

Skin is the largest organ of human body, acting as a barrier with protective, immunologic and sensorial functions. Its permanent exposure to the external environment can result in different kinds of damage with loss of variable volumes of extracellular matrix. For the treatment of skin lesions, several strategies are currently available, such as the application of autografts, allografts, wound dressings and tissue-engineered substitutes. Although proven clinically effective, these strategies are still characterized by key limitations such as patient morbidity, inadequate vascularization, low adherence to the wound bed, the inability to reproduce skin appendages and high manufacturing costs. Advanced strategies based on both bottom-up and top-down approaches offer an effective, permanent and viable alternative to solve the abovementioned drawbacks by combining biomaterials, cells, growth factors and advanced biomanufacturing techniques. This review details recent advances in skin regeneration and repair strategies, and describes their major advantages and limitations. Future prospects for skin regeneration are also outlined.

Preparation and Characterization of Films Based on Alginate and Aloe Vera

Sodium alginate films with aloe vera extract were prepared by the casting/solvent evaporation technique. The resulting films were characterized by differential scanning calorimetry, Fourier transform-infrared spectroscopy, scanning electron microscopy, and mechanical and water absorption tests. The in vitro degradation of the films was also investigated over 14 days. Results show that aloe vera contributes to both enhancing the thermal and mechanical properties of the films and decreasing the weight loss during in vitro degradation.

Influence of Aloe vera on water absorption and enzymatic in vitro degradation of alginate hydrogel films.

This study investigates the influence of Aloe vera on water absorption and the in vitro degradation rate of Aloe vera-Ca-alginate hydrogel films, for wound healing and drug delivery applications. The influence of A. vera content (5%, 15% and 25%, v/v) on water absorption was evaluated by the incubation of the films into a 0.1 M HCl solution (pH 1.0), acetate buffer (pH 5.5) and simulated body fluid solution (pH 7.4) during 24h. Results show that the water absorption is significantly higher for films containing high A. vera contents (15% and 25%), while no significant differences are observed between the alginate neat film and the film with 5% of A. vera. The in vitro enzymatic degradation tests indicate that an increase in the A. vera content significantly enhances the degradation rate of the films. Control films, incubated in a simulated body fluid solution without enzymes, are resistant to the hydrolytic degradation, exhibiting reduced weight loss and maintaining its structural integrity. Results also show that the water absorption and the in vitro degradation rate of the films can be tailored by changing the A. vera content.

3D Photo-Fabrication for Tissue Engineering and Drug Delivery

ABSTRACT The most promising strategies in tissue engineering involve the integration of a triad of biomaterials, living cells, and biologically active molecules to engineer synthetic environments that closely mimic the healing milieu present in human tissues, and that stimulate tissue repair and regeneration. To be clinically effective, these environments must replicate, as closely as possible, the main characteristics of the native extracellular matrix (ECM) on a cellular and subcellular scale. Photo-fabrication techniques have already been used to generate 3D environments with precise architectures and heterogeneous composition, through a multi-layer procedure involving the selective photocrosslinking reaction of a light-sensitive prepolymer. Cells and therapeutic molecules can be included in the initial hydrogel precursor solution, and processed into 3D constructs. Recently, photo-fabrication has also been explored to dynamically modulate hydrogel features in real time, providing enhanced control of cell fate and delivery of bioactive compounds. This paper focuses on the use of 3D photo-fabrication techniques to produce advanced constructs for tissue regeneration and drug delivery applications. State-of-the-art photo-fabrication techniques are described, with emphasis on the operating principles and biofabrication strategies to create spatially controlled patterns of cells and bioactive factors. Considering its fast processing, spatiotemporal control, high resolution, and accuracy, photo-fabrication is assuming a critical role in the design of sophisticated 3D constructs. This technology is capable of providing appropriate environments for tissue regeneration, and regulating the spatiotemporal delivery of therapeutics.

Cell-instructive pectin hydrogels crosslinked via thiol-norbornene photo-click chemistry for skin tissue engineering

Cell-instructive hydrogels are attractive for skin repair and regeneration, serving as interactive matrices to promote cell adhesion, cell-driven remodeling and de novo deposition of extracellular matrix components. This paper describes the synthesis and photocrosslinking of cell-instructive pectin hydrogels using cell-degradable peptide crosslinkers and integrin-specific adhesive ligands. Protease-degradable hydrogels obtained by photoinitiated thiol-norbornene click chemistry are rapidly formed in the presence of dermal fibroblasts, exhibit tunable properties and are capable of modulating the behavior of embedded cells, including the cell spreading, hydrogel contraction and secretion of matrix metalloproteases. Keratinocytes seeded on top of fibroblast-loaded hydrogels are able to adhere and form a compact and dense layer of epidermis, mimicking the architecture of the native skin. Thiol-ene photocrosslinkable pectin hydrogels support the in vitro formation of full-thickness skin and are thus a highly promising platform for skin tissue engineering applications, including wound healing and in vitro testing models. STATEMENT OF SIGNIFICANCE Photopolymerizable hydrogels are attractive for skin applications due to their unique spatiotemporal control over the hydrogel formation. This study reports the design of a promising photo-clickable pectin hydrogel which biophysical and biochemical properties can be independently tailored to control cell behavior. A fast method for the norbornene-functionalization of pectin was developed and hydrogels fabricated through UV photoinitiated thiol-norbornene chemistry. This one-pot click reaction was performed in the presence of cells using cell-adhesive and matrix metalloproteinase-sensitive peptides, yielding hydrogels that support extensive cell spreading. Keratinocytes seeded on top of the fibroblast-loaded hydrogel formed a compact epidermis with morphological resemblance to human skin. This work presents a new protease-degradable hydrogel that supports in vitro skin formation with potential for skin tissue engineering.

Photocrosslinkable Materials for the Fabrication of Tissue-Engineered Constructs by Stereolithography

Stereolithography is an additive technique that produces three-dimensional (3D) solid objects using a multi-layer procedure through the selective photo-initiated curing reaction of a liquid photosensitive material. Stereolithographic processes have been widely employed in Tissue Engineering for the fabrication of temporary constructs, using natural and synthetic polymers, and polymer-ceramic composites. These processes allow the fabrication of complex structures with a high accuracy and precision at physiological temperatures, incorporating cells and growth factors without significant damage or denaturation. Despite recent advances on the development of novel biomaterials and biocompatible crosslinking agents, the main limitation of these techniques are the lack number of available photocrosslinkable materials, exhibiting appropriate biocompatibility and biodegradability. This chapter gives an overview of the current state-of-art of materials and stereolithographic techniques to produce constructs for tissue regeneration, outlining challenges for future research.

Polyethylene Glycol and Polyethylene Glycol/Hydroxyapatite Constructs Produced through Stereo-Thermal Lithography

Stereo-thermal lithography (STLG) is an innovative system that uses ultraviolet (UV) radiation and near-infrared (IR) radiation simultaneously to initiate the curing reaction in a liquid solution, containing both UV and IR photoinitiator. In this research work, poly (ethylene glycol) dimethacrylate (PEGDMA) and PEGDMA/hydroxyapatite (HA) constructs were produced using the STLG system, operating in the UV mode, and characterised regarding the morphology and water absorption properties. Constructs were produced with different geometries and shapes by introducing several variations in the processing parameters, such as the irradiation time and the layer thickness. Results show the ability of the system to produce constructs at the micro-scale with very good definition and resolution. The irradiation time is the critical processing parameter, strongly affecting the water absorption and the structural integrity of the constructs.

Biofabrication of Hydrogel Constructs

Hydrogels are three dimensional (3D) hydrophilic networks with the ability to absorb and retain large amounts of water without dissolution, as a result of the establishment of physical or chemical bonds between the polymeric chains. Hydrogels obtained from either natural or synthetic polymers are attractive materials for tissue engineering applications due to their excellent biocompatibility, biodegradability, elasticity and compositional similarities to the extracellular matrix. Several techniques have been explored to produce hydrogel meshes, films or 3D constructs for cell attachment, differentiation and proliferation or to release drugs and growth factors according to specific release profiles. This chapter describes the current state-of-the-art of biomanufacturing additive processes to produce hydrogel constructs for tissue engineering. Biomanufacturing processes are described in detail and the major advantages and limitations outlined.

Computer modelling and simulation of a bioreactor for tissue engineering

A conventional approach to tissue engineering involves the implantation of porous, biodegradable and biocompatible scaffolds seeded with cells into the defect site. In some strategies, tissue engineering requires the in vitro culture of tissue-engineering constructs for implantation later. In this case, bioreactors are used to grow 3D tissues under controlled and monitored conditions. However, the quality of the resulting 3D tissue is highly dependent on the design and dimensions of the bioreactor, as well on the operating conditions. In this work, a computational fluid dynamic software package was used to investigate the influence of cylindrical bioreactor dimensions (length and diameter) on the fluid flow and scaffold shear stress. Computer simulations were performed using three different rotational movements (horizontal, vertical and biaxial rotation) and appropriate boundary conditions. Results show that the effect of the bioreactor length on the scaffold shear stress is more important than the diameter, while high length is associated to low scaffold shear stress. On the other hand, the fluid flows within the bioreactor and scaffold shear stresses are dependent on the rotational movement, being more uniform in the biaxial rotation due to the combination of rotational movements.