Development, characterization and sterilisation of Nanocellulose-alginate-(hyaluronic acid)- bioinks and 3D bioprinted scaffolds for tissue engineering.

3D-bioprinting is an emerging technology of high potential in tissue engineering (TE), since it shows effective control over scaffold fabrication and cell distribution. Biopolymers such as alginate (Alg), nanofibrillated cellulose (NC) and hyaluronic acid (HA) offer excellent characteristics for use as bioinks due to their excellent biocompatibility and rheological properties. Cell incorporation into the bioink requires sterilisation assurance, and autoclave, ß-radiation and γ-radiation are widely used sterilisation techniques in biomedicine; however, their use in 3D-bioprinting for bioinks sterilisation is still in their early stages. In this study, different sterilisation procedures were applied on NC-Alg and NC-Alg-HA bioinks and their effect on several parameters was evaluated. Results demonstrated that NC-Alg and NC-Alg-HA bioinks suffered relevant rheological and physicochemical modifications after sterilisation; yet, it can be concluded that the short cycle autoclave is the best option to sterilise both NC-Alg based cell-free bioinks, and that the incorporation of HA to the NC-Alg bioink improves its characteristics. Additionally, 3D scaffolds were bioprinted and specifically characterized as well as the D1 mesenchymal stromal cells (D1-MSCs) embedded for cell viability analysis. Notably, the addition of HA demonstrates better scaffold properties, together with higher biocompatibility and cell viability in comparison with the NC-Alg scaffolds. Thus, the use of MSCs containing NC-Alg based scaffolds may become a feasible tissue engineering approach for regenerative medicine.

Pore geometry influences growth and cell adhesion of infrapatellar mesenchymal stem cells in biofabricated 3D thermoplastic scaffolds useful for cartilage tissue engineering.

The most pressing need in cartilage tissue engineering (CTE) is the creation of a biomaterial capable to tailor the complex extracellular matrix of the tissue. Despite the standardized used of polycaprolactone (PCL) for osteochondral scaffolds, the pronounced stiffness mismatch between PCL scaffold and the tissue it replaces remarks the biomechanical incompatibility as main limitation. To overcome it, the present work was focused in the design and analysis of several geometries and pore sizes and how they affect cell adhesion and proliferation of infrapatellar fat pad-derived mesenchymal stem cells (IPFP-MSCs) loaded in biofabricated 3D thermoplastic scaffolds. A novel biomaterial for CTE, the 1,4-butanediol thermoplastic polyurethane (b-TPUe) together PCL were studied to compare their mechanical properties. Three different geometrical patterns were included: hexagonal (H), square (S), and, triangular (T); each one was printed with three different pore sizes (PS): 1, 1.5 and 2 mm. Results showed differences in cell adhesion, cell proliferation and mechanical properties depending on the geometry, porosity and type of biomaterial used. Finally, the microstructure of the two optimal geometries (T1.5 and T2) was deeply analyzed using multiaxial mechanical tests, with and without perimeters, µCT for microstructure analysis, DNA quantification and degradation assays. In conclusion, our results evidenced that IPFP-MSCs-loaded b-TPUe scaffolds had higher similarity with cartilage mechanics and T1.5 was the best adapted morphology for CTE.

Advances of hyaluronic acid in stem cell therapy and tissue engineering, including current clinical trials.

Hyaluronic acid (HA), as one of the main components of the extracellular matrix (ECM), plays a significant role in a multitude of biological processes involving cell migration, proliferation, differentiation, wound healing and inflammation. Thanks to its excellent biocompatibility, biodegradability and hygroscopic properties, HA has been used in its natural form for joint lubrication and ocular treatment. The chemical structure of HA can be easily modified by direct reaction with its carboxyl and hydroxyl groups. Recently, HA derivatives have been synthesised with the aim of developing HA-based materials with increased mechanical strength, improved cell interactions and reduced biodegradation and studied for regenerative medicine purposes, including cell therapy and tissue engineering. In this context, the present manuscript reviews HA applications from a basic point of view - including chemical modifications and cellular biology aspects related to clinical translation - and future perspectives of using biofabrication technologies for regenerative medicine. A detailed description of current clinical trials, testing advanced therapies based on combination of stem cells and HA formulations, is included. The final goal was to offer an integral portrait and a deeper comprehension of the current applications of HA from bench to bedside.

Cartilage biomechanics: A key factor for osteoarthritis regenerative medicine.

Osteoarthritis (OA) is a joint disorder that is highly extended in the global population. Several researches and therapeutic strategies have been probed on OA but without satisfactory long-term results in joint replacement. Recent evidences show how the cartilage biomechanics plays a crucial role in tissue development. This review describes how physics alters cartilage and its extracellular matrix (ECM); and its role in OA development. The ECM of the articular cartilage (AC) is widely involved in cartilage turnover processes being crucial in regeneration and joint diseases. We also review the importance of physicochemical pathways following the external forces in AC. Moreover, new techniques probed in cartilage tissue engineering for biomechanical stimulation are reviewed. The final objective of these novel approaches is to create a cellular implant that maintains all the biochemical and biomechanical properties of the original tissue for long-term replacements in patients with OA.

Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration.

IMPACT STATEMENT: 3D bioprinting represents a novel advance in the area of regenerative biomedicine and tissue engineering for the treatment of different pathologies, among which are those related to cartilage. Currently, the use of different thermoplastic polymers, such as PLA or PCL, for bioprinting processes presents an important limitation: the high temperatures that are required for extrusion affect the cell viability and the final characteristics of the construct. In this work, we present a novel bioprinting process called volume-by-volume (VbV) that allows us to preserve cell viability after bioprinting. This procedure allows cell injection at a safe thermoplastic temperature, and also allows the cells to be deposited in the desired areas of the construct, without the limitations caused by high temperatures. The VbV process could make it easier to bring 3D bioprinting into the clinic, allowing the generation of tissue constructs with polymers that are currently approved for clinical use.

Therapeutic strategies for skin regeneration based on biomedical substitutes.

Regenerative medicine and tissue engineering (TE) have experienced significant advances in the development of in vitro engineered skin substitutes, either for replacement of lost tissue in skin injuries or for the generation of in vitro human skin models to research. However, currently available skin substitutes present different limitations such as expensive costs, abnormal skin microstructure and engraftment failure. Given these limitations, new technologies, based on advanced therapies and regenerative medicine, have been applied to develop skin substitutes with several pharmaceutical applications that include injectable cell suspensions, cell-spray devices, sheets or 3Dscaffolds for skin tissue regeneration and others. Clinical practice for skin injuries has evolved to incorporate these innovative applications to facilitate wound healing, improve the barrier function of the skin, prevent infections, manage pain and even to ameliorate long-term aesthetic results. In this article, we review current commercially available skin substitutes for clinical use, as well as the latest advances in biomedical and pharmaceutical applications used to design advanced therapies and medical products for wound healing and skin regeneration. We highlight the current progress in clinical trials for wound healing as well as the new technologies that are being developed and hold the potential to generate skin substitutes such as 3D bioprinting-based strategies.

Aplicación clínica de las terapias con células, genes y tejidos en España / Clinical application of cell, gene and tissue therapies in Spain

Los avances científicos y técnicos en el área biomédica y la medicina regenerativa han permitido el desarrollo de nuevos tratamientos, denominados «terapias avanzadas», que engloban la terapia celular, la génica y la ingeniería tisular. Los productos biológicos que pueden fabricarse a partir de estos elementos se clasifican desde el punto de vista de la Agencia Española del Medicamento y Productos Sanitarios en medicamentos de terapias avanzadas, productos derivados de la sangre y trasplantes. Esta revisión pretende aportar información científica y administrativa, de utilidad para el clínico, sobre el uso de estos recursos biológicos

Scientific and technical advances in the areas of biomedicine and regenerative medicine have enabled the development of new treatments known as "advanced therapies", which encompass cell therapy, genetics and tissue engineering. The biologic products that can be manufactured from these elements are classified from the standpoint of the Spanish Agency of Medication and Health Products in advanced drug therapies, blood products and transplants. This review seeks to provide scientific and administrative information for clinicians on the use of these biologic resources

Clinical application of cell, gene and tissue therapies in Spain. / Aplicación clínica de las terapias con células, genes y tejidos en España.

Scientific and technical advances in the areas of biomedicine and regenerative medicine have enabled the development of new treatments known as "advanced therapies", which encompass cell therapy, genetics and tissue engineering. The biologic products that can be manufactured from these elements are classified from the standpoint of the Spanish Agency of Medication and Health Products in advanced drug therapies, blood products and transplants. This review seeks to provide scientific and administrative information for clinicians on the use of these biologic resources.

Desarrollo de una técnica para la microencapsulación de probióticos / Development of a technique for microencapsulation of probiotics

INTRODUCCIÓN: Los probióticos son “microorganismos vivos que administrados en cantidades adecuadas confieren beneficios en la salud del hospedador”. Sin embargo, el principal problema que se presenta a la hora de incorporar bacterias probióticas en un producto cualquiera, es la escasa resistencia de estos microorganismos a los procesos tecnológicos y ambientales, lo que supone un gran inconveniente para su inclusión en productos alimentarios o farmacéuticos. En este sentido, las técnicas de microencapsulación son de un gran interés porque permiten aislar a las bacterias mediante una cubierta, evitando así, su exposición a las condiciones adversas. Por tanto, el objetivo de este trabajo ha sido desarrollar una técnica de microencapsulación compatible con una cepa probiótica destinada a formas farmacéuticas para el tratamiento de la vaginitis. MATERIALES Y MÉTODOS: La técnica propuesta es la emulsificación-gelificación iónica interna 2. Una vez obtenidas las micropartículas, se procede al análisis de la supervivencia de los probióticos microencapsulados, mediante el método desarrollado por Sheu y cols 3. La evaluación del tamaño de partícula se llevo a cabo mediante microscopia óptica. RESULTADOS: La viabilidad del probiótico a lo largo del proceso de síntesis de las partículas se reduce en 1 log UFC. El tamaño de partícula está comprendido entre 40-240 micrómetros, o el predomínando, el intervalo de 120 a 160 micrómetros. DISCUSIÓN: La técnica desarrollada es compatible con el probiótico propuesto, obteniéndose una viabilidad, que permitiría alcanzar la dosis necesaria para el restablecimiento de la microbiota vaginal con tan solo un gramo de producto. Así mismo, el tamaño de las partículas, se considera aceptable para la terapéutica tópica(AU)

INTRODUCTION: Probiotics are "live microorganisms administered in adequate amounts conferhealth benefits on the host". But the main problem, when probiotics are incorporated in some products is their low resistance to technological and environmental processes. In this sense, microencapsulation techniques are good methods to protect the bacteria from adverse conditions since, it isolates bacteria from the environment and protect them from detrimental conditions. The aim of this study was to perform a microencapsulation technique compatible with the probiotic strain to develop a form for vaginal administration. MATERIALS AND METHODS: The ionic emulsification-internal gelation was used. After obtaining the microparticles, it was analyzed the survival of microencapsulated probiotics, using the method developed by Sheu et al. The evaluation of particle size was carried out by optical microscopy. RESULTS: During the process of microencapsulation, the viability of probiotic was reduced by 1 log CFU. The range of particle size is between 40-240 micrometers, although most of the particles were between 120 and 160 micrometers. DISCUSSION: The developed technique is compatible with the probiotic, since the viability of microencapsulated product, allows us to obtain a pharmaceutical form optimal for vaginal, administration with 1g of particles. Likewise, particle size is also considered acceptable for topical therapy(AU)

Desarrollo farmacéutico de un medicamento convencional versus medicamento celular / Pharmaceutical development of a conventional medicine versus cellular medicine

INTRODUCCIÓN: El advenimiento de la biología molecular ha agregado una nueva dimensión al proceso de descubrimiento de nuevos fármacos dirigidos hacia el concepto de las terapias avanzadas donde medicamentos clásicos coexistirán con células madre, genes y tejidos transformados ex vivo. Existen muchos tipos de células madre y todos ellos comparten las características de ser capaces de renovarse y dar lugar a progenie diferenciada. El presente trabajo, compara y reflexiona sobre el desarrollo farmacéutico de un medicamento convencional con un medicamento celular. Ambos presentan la misma forma de dosificación, suspensión de un principio activo: de síntesis para el medicamento convencional y células para el medicamento celular; en un medio acuoso, contenido en una jeringa como acondicionamiento primario. METODOLOGÍA: La fabricación de inyectables requiere de más cuidados que cualquier otra forma farmacéutica clásica debido a su vía de administración. Un inyectable debe cumplir, entre otras, con las siguientes características: isotonía, ausencia de pirógenos, y de esterilidad. Sin embargo en la preparación de una suspensión celular, el producto final no puede ser esterilizado, ya que las células deben ser viables para poder mantener sus características terapéuticas. La esterilidad del medicamento celular, deberá ser aportada en el proceso de producción, clase A sobre un entorno clase B. CONCLUSIÓN / DISCUSIÓN: Los métodos de preparación y control de inyectables convencionales son procedimientos que no pueden utilizarse en el medicamento celular ya que dichas técnicas son incompatibles con la viabilidad de células mesenquimales, por lo que el método de elaboración delactivo celular para la misma forma de dosificación debe realizarse en condiciones controladas. Así pues el desarrollo galénico de ambos medicamentos difiere y deberá ser considerado en cuanto a la tecnología farmacéutica se refiere(AU)

INTRODUCTION: The advent of molecular biology has added a new dimension to the process of discovering new drugs directed towards the concept of advanced therapies where traditional medicineco-exists with stem cells, genes and tissues processed ex vivo. There are many types of stem cells and all of them share the characteristics of being able to renew them selves as well as give rise to differentiated progeny 3.This paper discusses and compares the pharmaceutical development of a conventional medicine (injection) with a cellular medicine (suspension of mesenchymal cells). Both of them have the samedosage form, suspension of an active (synthesis / cell) in an aqueous solution (excipient) and are both contained in a syringe. METHODOLOGY: The manufacture of an injectable medicine requires more care than any other classical pharmaceutical form due to its route of administration. An injection should meet, the following features 4: sterility, pyrogen and isotonicity. However, in the preparation of a cell suspension, the final product can not be sterilized, since the cells must be viable to maintain their therapeutic properties. The sterility of cellular medicine must be provided in the production process, class A on class B environment. CONCLUSION / DISCUSSION: The methods of preparation and control of conventional injection sare procedures that can not be used in cellular medicine since these techniques are incompatible with the viability of mesenchymal cells. So the pharmaceutical development of the two products differs and should be considered in terms of pharmaceutical technology(AU)