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.

Hind limb ischemia in type 1 diabetic mice as useful tool to evaluate the neovascularization of tissue engineering constructs

Hind-limb ischemia has been used in type 1 diabetic mice to evaluate treatments for peripheral arterial disease or mechanisms of vascular impairment in diabetes [1]. Vascular deficiency is not only a pathophysiological condition, but also an obvious circumstance in tissue regeneration and in tissue engineering and regenerative medicine (TERM) strategies. We performed a pilot experiment of hind-limb ischemia in streptozotocin(STZ)-induced type 1 diabetic mice to hypothesise whether diabetes influences neovascularization induced by biomaterials. The dependent variables included blood flow and markers of arteriogenesis and angiogenesis. Type 1 diabetes was induced in 8-week-old C57BL/6 mice by an i.p. injection of STZ (50 mg/kg daily for 5 days). Hind-limb ischemia was created under deep anaesthesia and the left femoral artery and vein were isolated, ligated, and excised. The contralateral hind limb served as an internal control within each mouse. Non-diabetic ischaemic mice were used as experiment controls. At the hind-limb ischemia surgical procedure, different types of biomaterials were placed in the blood vessels gap. Blood flow was estimated by Laser Doppler perfusion imager, right after surgery and then weekly. After 28 days of implantation, surrounding muscle was excised and evaluated by histological analysis for arteriogenesis and angiogenesis. The results showed that implanted biomaterials were promote faster restoration of blood flow in the ischemic limbs and improved neovascularization in the diabetic mice. Therefore, we herein demonstrate that the combined model of hind-limb ischemia in type 1 diabetes mice is suitable to evaluate the neovascularization potential of biomaterials and eventually tissue engineering constructs.  

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

Metabolic engineering with multi-objective optimization of kinetic models

Kinetic models have a great potential for metabolic engineering applications. They can be used for testing which genetic and regulatory modifications can increase the production of metabolites of interest, while simultaneously monitoring other key functions of the host organism. This work presents a methodology for increasing productivity in biotechnological processes exploiting dynamic models. It uses multi-objective dynamic optimization to identify the combination of targets (enzymatic modifications) and the degree of up- or down-regulation that must be performed in order to optimize a set of pre-defined performance metrics subject to process constraints. The capabilities of the approach are demonstrated on a realistic and computationally challenging application: a large-scale metabolic model of Chinese Hamster Ovary cells (CHO), which are used for antibody production in a fed-batch process. The proposed methodology manages to provide a sustained and robust growth in CHO cells, increasing productivity while simultaneously increasing biomass production, product titer, and keeping the concentrations of lactate and ammonia at low values. The approach presented here can be used for optimizing metabolic models by finding the best combination of targets and their optimal level of up/down-regulation. Furthermore, it can accommodate additional trade-offs and constraints with great flexibility.

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.

Bioactive ceramics for tissue engineering and regenerative medicine derived from marine sponges

Publicado em "Journal of tissue engineering and regenerative medicine". Vol. 8, suppl. s1 (2014)

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.

Introducing biomimetic approaches to materials development and product design for engineering students

In this paper, we present a new course entitled “Biomimicry: from life to nanotechnological innovations” at the Mines Nancy Engineering School, Nancy, France, and explain how we developed a specific curriculum covering biomimicry. We discuss strategies that can be followed by teachers to explain selected contents in the multi-disciplinary field of biomimicry and/or bioinspiration to undergraduate students and how practical classroom activities can be conducted as individual or team work. We hope that sharing our experience will help teachers and senior researchers disseminate useful concepts and real examples of biomimetic principles and tools for the development of new materials, new/improved design and fabrication strategies, and innovation methodologies.

Current approaches and future perspectives on strategies for the development of personalized tissue engineering therapies

Personalized tissue engineering and regenerative medicine (TERM) therapies propose patient-oriented effective solutions, considering individual needs. Cell-based therapies, for example, may benefit from cell sources that enable easier autologous set-ups or from recent developments on IPS cells technologies towards effective personalized therapeutics. Furthermore, the customization of scaffold materials to perfectly fit a patientâ s tissue defect through rapid prototyping technologies, also known as 3D printing, is now a reality. Nevertheless, the timing to expand cells or to obtain functional in vitrotissue substitutes prior to implantation prevents advancements towards routine use upon patient´s needs. Thus, personalized therapies also anticipate the importance of creating off-the-shelf solutions to enable immediately available tissue engineered products. This paper reviews the main recent developments and future challenges to enable personalized TERM approaches and to bring these technologies closer to clinical applications.

Engineering a commercial cold-adapted xylanase for improved low pH stability

Dissertação de mestrado em Genética Molecular

Development of composite auxetic structures for civil engineering applications

Tese de Doutoramento em Engenharia Têxtil

Ingeniería de glicanos: diseño de inmunógenos y ligandos de lectinas. Glycan engineering: design of immunogens and ligand of lectins.

La ingeniería de glicanos es un área de investigación emergente, la que posee múltiples aplicaciones en medicina. Mediante esta herramienta se intentará reducir la flexibilidad de las uniones glicosídicas de antígenos tumorales, como la del antígeno T (Galbeta3GalNAcalfa-Ser/Thr). Aquí se realizarán las menores alteraciones posibles en la topología de glicanos que generen la mejor respuesta inmune hacia el antígeno de interés. Por otra parte, se buscará ligandos de alta afinidad que interaccionen con lectinas involucradas en diseminación de metástasis. Mediante ensayos teóricos de Docking se tratará de hallar modificaciones topológicas de glicanos que potencialmente tengan propiedades anti-adhesivas para células tumorales. Este proyecto constará de tres etapas: una teórica, utilizando programas de cálculos para ensayos de Docking y mínimos energéticos de glicanos. Otra de síntesis, generando los glicoconjugados sugeridos en la etapa anterior. En la última, se verificará si estos glicanos rediseñados adquirieron las propiedades biológicas deseadas. Así se determinará si generan una respuesta inmune que reconozca antígenos y células tumorales. También, se analizarán las propiedades anti-adhesivas de los glicanos utilizando diferentes modelos experimentales. Finalmente, se determinará si los inmunógenos producidos y/o glicoconjugados rediseñados poseen efecto en el desarrollo tumoral y sobrevida animal.

Los entornos virtuales como herramienta sustituta del laboratorio "real" y como complemento de la enseñanza de Química a alumnos de Ingeniería. Virtual environments as substitute tools for the "conventional" laboratories and as a complement for Chemistry teaching to Engineering students.

El presente proyecto se plantea el siguiente problema de investigación:¿Cuál es la eficacia de los entornos virtuales de enseñanza para optimizar los aprendizajes de Química? Se sostiene la hipótesis de que los entornos virtuales de enseñanza, empleados como mediación instrumental, son eficaces para optimizar los aprendizajes de química, particularmente facilitando la vinculación y reversibilidad entre "mundo micro y macroscópico"; capacidad que usualmente sólo se atribuye al trabajo experimental de laboratorio. Los objetivos propuestos son: Determinar la eficacia de entornos virtuales de enseñanza, como mediaciones instrumentales, para optimizar los aprendizajes de química en estudiantes de ingeniería. Implementar un entrono virtual de enseñanza de química, diseñado como mediación instrumental y destinado a estudiantes de dos carreras de ingeniería del IUA. Evaluar el desarrollo y los resultados de la innovación introducida. Comparar los resultados de la innovación con los resultados de la enseñanza usual. Derivar conclusiones acerca de la eficacia de la innovación propuesta. Socializar el conocimiento producido en ámbitos científico-tecnológicos reconocidos. Se generará un aula virtual en plataforma Educativa y utilidzando el laboratorio de computación de la institución se buscará desarrollar laboratorios virtuales donde se propondrán actividades de simulación de trabajo experimental. Los resultados esperados son: - Un Aula Virtual que cumpla funciones análogas a las de un laboratorio experimental. - Información válida y confiable acerca de la eficacia de la misma como medio para optimizar los aprendizajes de química. - Publicaciones en ámbitos científico-tecnológicos reconocidos que sometan a juicio público la innovación y la investigación efectuadas. La importancia del proyecto radica principalmente en poner a prueba la eficacia de los entornos virtuales para optimizar los aprendizajes de química, analogando tareas usualmente limitadas al trabajo experimental de laboratorio. Su pertinencia apunta a un replanteo del curriculo de los cursos de Química para estudiantes de Ingeniería.

Sistemas de Tipos para Verificación de Programas Concurrentes. Type Systems for Concurrent Programs Verification.

La programación concurrente es una tarea difícil aún para los más experimentados programadores. Las investigaciones en concurrencia han dado como resultado una gran cantidad de mecanismos y herramientas para resolver problemas de condiciones de carrera de datos y deadlocks, problemas que surgen por el mal uso de los mecanismos de sincronización. La verificación de propiedades interesantes de programas concurrentes presenta dificultades extras a los programas secuenciales debido al no-determinismo de su ejecución, lo cual resulta en una explosión en el número de posibles estados de programa, haciendo casi imposible un tratamiento manual o aún con la ayuda de computadoras. Algunos enfoques se basan en la creación de lenguajes de programación con construcciones con un alto nivel de abstración para expresar concurrencia y sincronización. Otros enfoques tratan de desarrollar técnicas y métodos de razonamiento para demostrar propiedades, algunos usan demostradores de teoremas generales, model-checking o algortimos específicos sobre un determinado sistema de tipos. Los enfoques basados en análisis estático liviano utilizan técnicas como interpretación abstracta para detectar ciertos tipos de errores, de una manera conservativa. Estas técnicas generalmente escalan lo suficiente para aplicarse en grandes proyectos de software pero los tipos de errores que pueden detectar es limitada. Algunas propiedades interesantes están relacionadas a condiciones de carrera y deadlocks, mientras que otros están interesados en problemas relacionados con la seguridad de los sistemas, como confidencialidad e integridad de datos. Los principales objetivos de esta propuesta es identificar algunas propiedades de interés a verificar en sistemas concurrentes y desarrollar técnicas y herramientas para realizar la verificación en forma automática. Para lograr estos objetivos, se pondrá énfasis en el estudio y desarrollo de sistemas de tipos como tipos dependientes, sistema de tipos y efectos, y tipos de efectos sensibles al flujo de datos y control. Estos sistemas de tipos se aplicarán a algunos modelos de programación concurrente como por ejemplo, en Simple Concurrent Object-Oriented Programming (SCOOP) y Java. Además se abordarán propiedades de seguridad usando sistemas de tipos específicos. Concurrent programming has remained a dificult task even for very experienced programmers. Concurrency research has provided a rich set of tools and mechanisms for dealing with data races and deadlocks that arise of incorrect use of synchronization. Verification of most interesting properties of concurrent programs is a very dificult task due to intrinsic non-deterministic nature of concurrency, resulting in a state explosion which make it almost imposible to be manually treat and it is a serious challenge to do that even with help of computers. Some approaches attempts create programming languages with higher levels of abstraction for expressing concurrency and synchronization. Other approaches try to develop reasoning methods to prove properties, either using general theorem provers, model-checking or specific algorithms on some type systems. The light-weight static analysis approach apply techniques like abstract interpretation to find certain kind of bugs in a conservative way. This techniques scale well to be applied in large software projects but the kind of bugs they may find are limited. Some interesting properties are related to data races and deadlocks, while others are interested in some security problems like confidentiality and integrity of data. The main goals of this proposal is to identify some interesting properties to verify in concurrent systems and develop techniques and tools to do full automatic verification. The main approach will be the application of type systems, as dependent types, type and effect systems, and flow-efect types. Those type systems will be applied to some models for concurrent programming as Simple Concurrent Object-Oriented Programming (SCOOP) and Java. Other goals include the analysis of security properties also using specific type systems.