Development of nanomaterial based scaffolds for bone tissue regenaration

Abstract

Las fracturas y defectos óseos a menudo son producidos por lesiones traumáticas, cáncer u otras enfermedades. Cuando un defecto aparece, el proceso de curado natural se inicia. Sin embargo, en ocasiones, los defectos óseos no pueden curarse espontáneamente. Esta situación ocurre especialmente en defectos óseos grandes o de infección, cuando alcanzan el tamaño crítico. En estos casos, se requieren intervenciones quirúrgicas para promover el curado del hueso. Los autoinjertos representan la solución más empleada para el tratamiento de defectos óseos de tamaño crítico. Sin embargo, puede producir muchos problemas, desde morbilidad del sitio dador hasta la limitada cantidad de hueso disponible. Para evitar los problemas que aparecen al usar métodos convencionales, se han realizado muchos esfuerzos para crear matrices 3D porosas, conocidas como andamios (scaffolds). Estos andamios se basan en la regeneración ósea guiada, cuyo objetivo es la regeneración y crecimiento óseos por la superficie del andamio. Para fabricarlos, se han usado técnicas como la colada con disolvente, liofilización o lixiviación de sales. Sin embargo, la geometría, porosidad y tamaño de poro no son controlables con estas técnicas. Este control se puede conseguir con tecnologías de fabricación aditiva, que permite imitar el tejido natural y los órganos para controlar el comportamiento celular. Los nanomateriales base grafeno son candidatos prometedores para ser usados como refuerzo en aplicaciones de reparación y regeneración ósea por su excelente combinación de propiedades mecánicas, térmicas y eléctricas, junto con su probada actividad antimicrobiana y su capacidad de regeneración celular. Además, los materiales nanocompuestos con nanomateriales carbonosos han demostrado favorecer la adhesión y la proliferación celulares, promoviendo el crecimiento óseo. Sin embargo, para producir andamios reforzados con nanomateriales base grafeno por fabricación aditiva, es importante estudiar la modificación de los materiales disponibles para ajustar las propiedades a las necesidades de esta aplicación. Además, en el caso de los polímeros fotocurables empleados en algunas tecnologías, los nanomateriales usados para modificar sus propiedades pueden afectar también a otros parámetros, como la imprimibilidad, polimerización, propiedades mecánicas y/o biológicas. El objetivo principal de esta tesis doctoral es desarrollar andamios óseos tridimensionales reforzados con nanomateriales base grafeno (NBG) utilizando tecnologías de fabricación aditiva de fotopolimerización, como estereolitografía (SLA), que permita obtener andamios con propiedades mecánicas mejoradas y alta capacidad de proliferación celular. Para ello, se propusieron algunos objetivos parciales: 1. Definir un procedimiento para preparar resinas fotocurables reforzadas con NBG que permitan obtener estructuras por tecnologías de fabricación aditiva basadas en fotopolimerización. 2. Estudiar el efecto de los NBG en la polimerización térmica y UV de la resina. 3. Optimizar el proceso de impresión y post-impresión de las resinas reforzadas con NBG para obtener unas propiedades mecánicas adecuadas. 4. Estudiar la eficiencia de la resina reforzada en términos de citotoxicidad y proliferación celular. Esta tesis ha establecido unos cimientos sólidos para fabricar andamios óseos con NBG por tecnologías de fabricación aditiva basadas en fotopolimerización, i.e. SLA, DLP, LCD, aprovechando la buena precisión y la fácil esterilización que ofrecen dichas tecnologías.

Nowadays, orthopaedic surgery is one of the areas of greatest interest in medicine, and within it, there are more and more surgical interventions carried out to solve problems related to the repair of large bone defects or damaged cellular tissue. They appear either as a result of disease, some type of trauma or the natural aging process. It is expected that in the near future this type of intervention will be more and more frequent, necessary and serious, due not only to the aging of the population, but also to the appearance of these ailments at younger ages. The decrease of the age when these problems appear is a consequence of a change in lifestyle and an increase in obesity in the population. Although bone tissue has the ability to self-regenerate, in the case of severe fractures with critical sizes (≥ 7 mm) or in the case of irregular fractures, this natural regeneration process is inhibited, which is a great challenge for orthopedic surgeons. In these cases, when the natural process of healing and bone regeneration is inhibited, the use of support materials, also called scaffolds, has proven to be very effective. These scaffolds act as an extracellular matrix, which provides structural and mechanical support to bone cells for their attachment, viability and growth, thus favouring their regeneration. The main advantage of tissue engineering is the potential elimination of donor scarcity, pathogen transfer, and graft rejection. To highlight the social impact, it is worth mentioning that in the case of osteoporosis alone, it is known that this disease causes more than 8.9 million fractures per year. One out of three women and one out of five men over the age of 50 will suffer an osteoporotic fracture at some moment in their lives. By 2050, the global incidence of hip fractures is expected to increase by 310% in men and 240% in women, compared to 1990. Consequently, in recent years, an important effort in the development of new repair techniques and in the use of new materials that favour cell regeneration is being done. Scaffolds have been conventionally manufactured by techniques like solvent casting, freeze-drying and salt leaching. However, these technologies allow to obtain porous structures, but their geometry, porosity and pore size are not controlled. This control can be achieved by Additive Manufacturing (AM) technologies. Graphene and graphene-based nanomaterials (GBN) are nanomaterials that have generated enormous interest in the scientific community in recent years due to their unique properties (high electrical and thermal conductivity, high mechanical strength, high hardness, flexibility, large surface area, etc). In relation to the field that concerns us, it has been proven that GBN are biocompatible materials with low toxicity, having been discovered that they have very interesting properties from the biological point of view and especially from the point of view of cell regeneration. It has antimicrobial properties and has been shown to promote cell adhesion and osteoblast growth. Several studies show that the incorporation of GBN in relatively low concentrations to different materials considerably favours the proliferation of bone cells on them. Furthermore, it has been shown that the addition of this material as a reinforcing agent is capable of greatly improving the mechanical properties of many materials. Therefore, GBN are promising candidates to be used as reinforcement for bone repair and regeneration applications due to the exceptional combination of excellent mechanical, thermal and electrical properties along with their proven antimicrobial and cell regeneration capabilities. However, to produce scaffolds reinforced with GBN by AM technologies, it is urgent to study the modification of available raw materials to adjust their properties to the needed for this purpose. Besides, in the case of photocurable polymers used in some AM technologies, nanofillers may modify their properties and could also affect other parameters, like printability, polymerization, mechanical and/or biological performance. Therefore, the main objective of this doctoral thesis is to develop 3D bone scaffolds reinforced with GBN using Vat Photopolymerization techniques, i.e. stereolithography (SLA), that allow to obtain scaffolds with improved mechanical properties and high cell proliferation activity. The addition of GBN affected the resin performance from different points of view. Therefore, to reach the final objective, it is important to understand the effect of GBN on polymerization, mechanical performance, and cell response. To achieve the final objective, some partial objectives are proposed: 1. To define a procedure to prepare photocurable resins reinforced with GBN that allows to obtain structures by vat polymerization technologies. 2. To study GBN effect on the thermal and UV polymerization process of acrylic photopolymer. 3. To optimize the printing and post-printing processes of the GBN-reinforced resins to reach good mechanical properties. 4. To study the efficacy of reinforced photocurable resins in terms of cytotoxicity and cell proliferation. In this doctoral thesis, a commercial acrylic resin (R) and three different GBN were used, which differ in size, degree of exfoliation and surface functionalization. Firstly, the GBN dispersion was optimized by adding monomer i.e., methyl methacrylate (MMA), to the resin. MMA is a liquid that reduces viscosity, favouring homogeneous dispersion. It was found that MMA does not produce significant changes in the mechanical properties of the resin and catalyzes the polymerization. Therefore, the use of MMA is a very effective technique for obtaining a good dispersion of GBN in acrylic-based resins. It was found that the effect of GBN on resin properties highly depended on the functionalization and the size of the nanofiller: - Graphene (G) inhibits UV polymerization due to its high absorbance, which results in a poor printing process, which was improved by increasing the exposure time. Furthermore, this inhibition of polymerization leads to a lack of improvement in mechanical properties when polymerization occurs by UV. However, when the samples were polymerized by heat, this improvement was found. Despite its high absorbency, interlayer adhesion appears to be adequate, but internal stresses occur that can be relieved by annealing after printing. - Graphene oxide (GO) catalyzes polymerization thanks to the presence of oxygenated groups on its surface. However, it was observed that it gives rise to a less cross-linked polymeric structure than the resin. Despite this, it was observed that GO improves the properties when the samples were post-cured. Therefore, with the adequate post-treatment, the GO shows a reinforcing effect from the mechanical point of view. - Graphite nanoplatelets (GoxNP) inhibit thermal polymerization but were not found to affect polymerization during printing. However, the mechanical properties of the printed specimens did not achieve the expected improvements. Analyzing the layers by microscopy, it was found that the presence of GoxNP affects the adhesion between layers due to its large size and low degree of exfoliation. From the biological point of view, the cellular response of MC3T3 preosteoblasts was studied. It was observed that all the nanocomposites made with R and GBN show low cell viability, which can be improved by washing the samples with cell culture medium. Washing improved cell viability because residual photoinitiator and monomer were removed, which were released into the medium, producing a slight decrease in cell viability. However, washing procedure should be optimized in future works to assure the biocompatibility of the nanocomposites. Based on the results found in this study, future work would involve optimizing the percentage of GBN added to achieve a more pronounced effect on both the mechanical and biological properties. Besides, the study could be extended to the electrical properties of the scaffolds, which are very interesting in biomedical applications. On the other hand, the functionalization of the GBN could also improve the interaction between the resin and the nanofillers, resulting in better final performance of the composite material. Besides, ideally, the scaffolds should be biodegradable to be reabsorbed once the bone regeneration process is complete. This biodegradation and control over the rate of disappearance of the scaffold can be achieved using a resin synthesized in the laboratory, in which the molecular weight and polymeric structure could be controlled in order to have this control over the rate of degradation. The study carried out in this doctoral thesis opens the door to being able to modify resins synthesized in the laboratory with GBN, understanding the effect that these nanomaterials can have on the process of polymerization, printing and the appearance of internal stresses. Taking into account all the aspects exposed above, it can be concluded that this thesis has established solid foundations for manufacturing bone scaffolds with GBN by Vat photopolymerization additive manufacturing technologies, i.e. stereolithography (SLA), Digital Light Processing (DLP), Liquid Crystal Display (LCD), taking advantage of the good precision and easy sterilization offered by these technologies.