Improvement of biomaterials used in tissue engineering by an ageing treatment.

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60% (scaffold without ageing treatment) to 80%. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.

Global transcriptome profiles provide insights into muscle cell development and differentiation on microstructured marine biopolymer scaffolds for cultured meat production.

Biomaterial scaffolds play a pivotal role in the advancement of cultured meat technology, facilitating essential processes like cell attachment, growth, specialization, and alignment. Currently, there exists limited knowledge concerning the creation of consumable scaffolds tailored for cultured meat applications. This investigation aimed to produce edible scaffolds featuring both smooth and patterned surfaces, utilizing biomaterials such as salmon gelatin, alginate, agarose and glycerol, pertinent to cultured meat and adhering to food safety protocols. The primary objective of this research was to uncover variations in transcriptomes profiles between flat and microstructured edible scaffolds fabricated from marine-derived biopolymers, leveraging high-throughput sequencing techniques. Expression analysis revealed noteworthy disparities in transcriptome profiles when comparing the flat and microstructured scaffold configurations against a control condition. Employing gene functional enrichment analysis for the microstructured versus flat scaffold conditions yielded substantial enrichment ratios, highlighting pertinent gene modules linked to the development of skeletal muscle. Notable functional aspects included filament sliding, muscle contraction, and the organization of sarcomeres. By shedding light on these intricate processes, this study offers insights into the fundamental mechanisms underpinning the generation of muscle-specific cultured meat.

Improvement of human skin cell growth by radiation-induced modifications of a Ge/Ch/Ha scaffold.

Gelatin-/chitosan-/hyaluronan-based biomaterials are used in tissue engineering as cell scaffolds. Three gamma radiation doses (1, 10 and 25 kGy) were applied to scaffolds for sterilization. Microstructural changes of the irradiated polymers were evaluated by using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A dose of 25 kGy produced a rough microstructure with a reduction of the porosity (from 99 to 96 %) and pore size (from 160 to 123 µm). Radiation also modified the glass transition temperature between 31.2 and 42.1 °C (1 and 25 kGy respectively). Human skin cells cultivated on scaffolds irradiated with 10 and 25 kGy proliferated at 48 h and secreted transforming growth factor ß3 (TGF-ß3). Doses of 0 kGy (non-irradiated) or 1 kGy did not stimulate TGF-ß3 secretion or cell proliferation. The specific growth rate and lactate production increased proportionally to radiation dose. The use of an appropriate radiation dose improves the cell scaffold properties of biomaterials.

Designing a gelatin/chitosan/hyaluronic acid biopolymer using a thermophysical approach for use in tissue engineering.

Cell culture on biopolymeric scaffolds has provided treatments for tissue engineering. Biopolymeric mixtures based on gelatin (Ge), chitosan (Ch) and hyaluronic acid (Ha) have been used to make scaffolds for wound healing. Thermal and physical properties of scaffolds prepared with Ge, Ch and Ha were characterized. Thermal characterization was made by using differential scanning calorimetry (DSC), and physical characterization by gas pycnometry and scanning electron microscopy. The effects of Ge content and cross-linking on thermophysical properties were evaluated by means of a factorial experiment design (central composite face centered). Gelatin content was the main factor that affects the thermophysical properties (microstructure and thermal transitions) of the scaffold. The effect of Ge content of the scaffolds for tissue engineering was studied by seeding skin cells on the biopolymers. The cell attachment was not significantly modified at different Ge contents; however, the cell growth rate increased linearly with the decrease of the Ge content. This relationship together with the thermophysical characterization may be used to design scaffolds for tissue engineering.

Design of a biodegradable UV-irradiated gelatin-chitosan/nanocomposed membrane with osteogenic ability for application in bone regeneration.

Guided bone regeneration membranes are used in oral surgery to protect the site of a lesion exposed to connective tissue invasion which, in turn, prevents new bone formation. Although non-degradable and degradable materials have been applied in clinical treatments, biodegradable membranes have the advantage that they do not require a secondary surgical procedure to be removed. However, they have a very low mechanical strength. As biodegradable membranes, biomaterials based on gelatin-chitosan have gained importance in clinical applications due to their unique properties. Gelatin contains RGD-like sequences, promoting cell adhesion/migration, and it can be blended with chitosan, which allows the immobilization of nanoparticles. In this work, we designed a new gelatin-chitosan polymeric membrane which contains hydroxyapatite and titania nanoparticles as two very well-documented osteoconductive materials. UV radiation was used as a non-toxic cross-linking agent to improve the thermophysical/mechanical characteristics and to control the biodegradability of the nanocomposed membrane. The microstructure, thermophysical and mechanical properties of the UV-irradiated material were studied by scanning electron microscopy, differential scanning calorimetry and Young's modulus, respectively. The in vitro biocompatibility of the new nanocomposite was evaluated by cell adhesion and proliferation assays. The osteoconductive ability was determined by an alkaline phosphatase production assay using mouse embryonic fibroblast (MEF) cells. The results show a homogeneous material with an appropriate distribution of nanoparticles. Cross-linking by UV radiation improved the mechanical and biological performance of the membrane. The presence of two osteoconductive nanoparticles, such as titania and hydroxyapatite, increased the osteogenic potential of the gelatin-based material in vitro, which confers a biological function, in addition to functioning as a physical barrier. The material obtained herein represents a good alternative to current guided bone regeneration membranes, with high potential for use in oral/orthopaedic applications in patients.

Cold-adaptation of a methacrylamide gelatin towards the expansion of the biomaterial toolbox for specialized functionalities in tissue engineering.

Tissue regeneration is witnessing a significant surge in advanced medicine. It requires the interaction of scaffolds with different cell types for efficient tissue formation post-implantation. The presence of tissue subtypes in more complex organs demands the co-existence of different biomaterials showing different hydrolysis rate for specialized cell-dependent remodeling. To expand the available toolbox of biomaterials with sufficient mechanical strength and variable rate of enzymatic degradation, a cold-adapted methacrylamide gelatin was developed from salmon skin. Compared with mammalian methacrylamide gelatin (GelMA), hydrogels derived from salmon GelMA displayed similar mechanical properties than the former. Nevertheless, salmon gelatin and salmon GelMA-derived hydrogels presented characteristics common of cold-adaptation, such as reduced activation energy for collagenase, increased enzymatic hydrolysis turnover of hydrogels, increased interconnected polypeptides molecular mobility and lower physical gelation capability. These properties resulted in increased cell-remodeling rate in vitro and in vivo, proving the potential and biological tolerance of this mechanically adequate cold-adapted biomaterial as alternative scaffold subtypes with improved cell invasion and tissue fusion capacity.

TGFß3 secretion by three-dimensional cultures of human dental apical papilla mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less-studied MSC source. These dental-derived MSCs are of great interest because of their potential as an accessible source for cell-based therapies and tissue-engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic-mediated repair and regenerative effects observed when they are implanted. TGFß3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP-derived MSCs (hSCAPs) can produce and secrete TGFß3 in response to micro-environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFß3 secretion. We found that dynamic changes of cell-matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two-dimensional, 2D) towards a three-dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFß3 secretion, while monolayer cultures showed almost 10-fold less secretion of TGFß3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright © 2015 John Wiley & Sons, Ltd.

Design of a hybrid biomaterial for tissue engineering: Biopolymer-scaffold integrated with an autologous hydrogel carrying mesenchymal stem-cells.

Biologically active biomaterials as biopolymers and hydrogels have been used in medical applications providing favorable results in tissue engineering. In this research, a wound dressing device was designed by integration of an autologous clot hydrogel carrying mesenchymal stem-cells onto a biopolymeric scaffold. This hybrid biomaterial was tested in-vitro and in-vivo, and used in a human clinical case. The biopolymeric scaffold was made with gelatin, chitosan and hyaluronic acid, using a freeze-drying method. The scaffold was a porous material which was designed evaluating both physical properties (glass transition, melting temperature and pore size) and biological properties (cell viability and fibronectin expression). Two types of chitosan (120 and 300kDa) were used to manufacture the scaffold, being the high molecular weight the most biologically active and stable after sterilization with gamma irradiation (25kGy). A clot hydrogel was formulated with autologous plasma and calcium chloride, using an approach based on design of experiments. The optimum hydrogel was used to incorporate cells onto the porous scaffold, forming a wound dressing biomaterial. The wound dressing device was firstly tested in-vitro using human cells, and then, its biosecurity was evaluated in-vivo using a rabbit model. The in-vitro results showed high cell viability after one week (99.5%), high mitotic index (19.8%) and high fibronectin expression. The in-vivo application to rabbits showed adequate biodegradability capacity (between 1 and 2weeks), and the histological evaluation confirmed absence of rejection signs and reepithelization on the wound zone. Finally, the wound dressing biomaterial was used in a single human case to implant autologous cells on a skin surgery. The medical examination indicated high biocompatibility, partial biodegradation at one week, early regeneration capacity at 4weeks and absence of rejection signs.

Senescent cultures of human dermal fibroblasts modified phenotype when immobilized in fibrin polymer.

One of the limitations in tissue engineering is the restricted ability to expand the number of cells, because somatic cells can duplicate a limited number of times before they lose the ability to divide, leading to a senescent state. Here we report that the interaction of senescent fibroblasts with fibrin polymer can modify the senescent phenotype and partially restore the ability of growth-arrested cells to continue replicating. Primary human dermal fibroblasts were grown to >90% SA/beta-Gal (senescence associated beta-galactosidase). The senescent cells were immobilized in fibrin-polymers by mixing fibrinogen and thrombin solutions. Immobilized senescent cell cultures grew, however, their growth arrested after 24 h of immobilization. The percentage of cells with a positive reaction at SA/beta-Gal did not decrease significantly after immobilization, but the intensity of the stain decreased. The glycolytic activity in immobilized senescent fibroblast was re-established at pre-senescent levels. In conclusion, fibrin induces changes in the phenotype of senescent human fibroblasts. This simple procedure could complement available tissue-engineering techniques to increase the amount of biomass seeded on a fibrin scaffold, which could be beyond senescence.

Characterization of a gelatin/chitosan/hyaluronan scaffold-polymer

Gelatin, chitosan and hyaluronic acid are natural components used to prepare polymeric scaffold in tissue engineering. The physical properties of these materials confer an appropriate microenvironment for cells, which can be used as a regeneration system for skin and cartilage. In this work, we prepared and characterized a Gelatin/Chitosan/Hyaluronan lyophilized-polymer. Physical properties of lyophilized-polymer changed slightly with moisture, but when polymer was totally hydrated the elasticity changed significantly. Thermophysical characterisation indicated that temperatures higher than 30ºC could modify irreversibly the polymeric matrix probably due to protein denaturation. Besides, we used the polymer as scaffold to prepare a biosynthetic-skin, reporting biological behaviour and its mechanical properties.