Facultad de Ciencias - Tesis Doctorales y Tesinas

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    Análisis de los efectos causados en la ventana redonda y áreas anatómicas adyacentes, por la inserción de una guía de electrodos de implante coclear en Macaca fascicularis
    (Universidad de Navarra, 2024-04-15) Picciafuoco Destefanis, S.E. (Sebastian Eduardo); Manrique-Rodríguez, M.J. (Manuel Jesús); Manrique-Huarte, R. (Raquel); Raquel
    Se define a la Hipoacusia como la dificultad de la capacidad de oír. Según el Instituto Nacional de Estadística estima que, en España, de una población total de 43.198.000, hay alrededor de 1.771.118 personas sufren de algún grado de hipoacusia, y 259.188 la padecen en un grado severo a profundo. En Navarra, donde la población, según la misma fuente es de alrededor de 593.472 personas, se estima que 24.332 personas sufren de algún grado de hipoacusia, y 3.560 sufrirían de hipoacusia por encima de los 71 dB. Además, la esperanza de vida de los ancianos está aumentando. Sobre los 60 años, se estima que alrededor de 1,8% de las personas tendrán una audición no funcional, es decir, pérdidas auditivas severas o profundas. Por otro lado, considerando a la población infantil, se estima que 5 de cada 1000 nacidos padecen una sordera de gravedad variable y 1 de cada 1000 recién nacidos presenta sordera severa o profunda, lo que en España supone que, cada año, hay en torno a 2.500 nuevas familias con un hijo/a con sordera (Comisión para la Detección Precoz de la Sordera Infantil-CODEPEH 2022).
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    Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma
    (Universidad de Navarra, 2024-02-28) Etxebeste-Mitxeltorena, A. (Amaia); Martinez-Climent, J.A. (José Ángel); Larrayoz, M. (Marta)
    La falta de modelos experimentales que reflejen la heterogeneidad genética del mieloma múltiple (MM) ha obstaculizado históricamente el avance de los descubrimientos terapéuticos preclínicos. Para superar esta limitación, examinamos ratones diseñados para expresar lesiones genéticas comunes del MM encontradas en pacientes, incluyendo NF-kB, BCL2, MYC, TP53, KRAS, ciclina D1, MMSET/NSD2 y c-MAF. Estas lesiones fueron activadas en células B maduras del centro germinal mediante el alelo cγ1-cre, lugar donde se cree que se origina la enfermedad. Después de nuestro estudio, encontramos dos modelos llamados BIcγ1 y MIcγ1, los cuales desarrollaron tumores de médula ósea (MO) que cumplían con los elementos clave de la patogénesis del MM. Además, los ratones mostraron respuesta in vivo a lenalidomida y combinaciones de lenalidomida, las cuales actualmente son parte de la terapia estándar en pacientes con MM. La caracterización de ambos modelos a través de ensayos celulares, moleculares e inmunológicos reveló que la adquisición de la expresión de MYC condicionó el tiempo de progresión y, a su vez, dictó mecanismos de evasión inmunológica que remodelaron de manera diferente el microambiente de la MO. Los ratones MIcγ1 con progresión rápida impulsada por la sobreexpresión inicial de MYC, exhibieron un alto número de células T CD8+ activadas y una disminución de las células T reguladoras (Treg) inmunosupresoras en la MO, mientras que los ratones BIcγ1, con adquisición tardía de la expresión de MYC, mostraron una menor infiltración de células T CD8+ en la MO y una infiltración superior de células Treg. De manera inesperada, los ratones MIcγ1 con progresión rápida respondieron a la terapia de bloqueo de puntos de control inmunitario (ICB), mientras que el modelo de progresión lenta BIcγ1 fue completamente refractario a esta misma terapia. El estudio de las diferencias en la respuesta entre ambos modelos murinos nos llevó a la conclusión de que una alta proporción de células T CD8+ frente a células Treg en la MO predijo la respuesta a la terapia ICB. A partir de estos nuevos modelos murinos, intentamos establecer líneas celulares de MM derivadas de muestras de tumor primario, con el objetivo de utilizar un panel de líneas celulares genéticamente diversas para probar las inmunoterapias. Entre ellas, las líneas celulares MM5080 y MM8273 pudieron ser injertadas en ratones singénicos inmunocompetentes, lo cual permitió probar la inmunoterapia in vivo. En estas líneas celulares, evaluamos la hipótesis de si modular la proporción de células CD8+ /Treg podría revertir la resistencia a los ICB. Nuestros hallazgos experimentales indicaron que al aumentar la citotoxicidad de las células T CD8+ mediante la combinación de anticuerpos monoclonales (moAbs) anti-PD-1 y anti-TIGIT, o al eliminar las células T reguladoras (Treg) con un moAb anti-CD25, se revirtió la resistencia a la terapia PD-1/PD-L1, lo cual resultó en un control prolongado del MM. En conclusión, hemos generado modelos de MM similares a los encontrados en pacientes, lo cual nos permite correlacionar los rasgos genéticos e inmunológicos del MM con las respuestas a la terapia preclínica. Esto resulta fundamental para los ensayos clínicos de nuevas inmunoterapias.
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    Evaluation of Modified Vaccinia virus Ankara based locoregional immunotherapy in peritoneal carcinomatosis models
    (Universidad de Navarra, 2024-02-28) Bella-Carreño, Á. (Ángela); Berraondo, P. (Pedro); Aranda, F. (Fernando)
    The peritoneum is a serous membrane of mesodermal origin that coats the abdominal wall and forms a lining on most abdominal organs. It consists of a thin layer of mesothelial cells over a basal lamina and is divided into the parietal peritoneum, which covers the abdominal and pelvic walls and the visceral peritoneum which surrounds the visceral organs (stomach, spleen, liver and some parts of the intestine) (1–3). The space found between the parietal and visceral peritoneum is called the peritoneal cavity and, in physiological conditions, contains between 50-100 mL of peritoneal fluid that serves as lubricant reducing friction among intraperitoneal organs during peristalsis and is provided with nutrients, growth factors, cytokines and chemokines as well as immune cells (1, 2). Thus, the peritoneum plays a crucial role in the maintenance of homeostasis in the peritoneal cavity mediating antigen presentation, inflammatory responses, fibrosis and tissue repair (1).
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    Advances in precision and safety of CRISPR-based gene targeting for Primary Hyperoxaluria Type 1
    (Universidad de Navarra, 2024-02-22) Torella, L. (Laura); González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea)
    Primary hyperoxaluria type 1 (PH1) is a rare metabolic disorder caused by pathogenic mutations in the AGXT gene. It is characterized by excessive oxalate production in the liver, resulting in its toxic accumulation in the kidneys. CRISPR-Cas9 targeting the Hao1 gene, encoding the glycolate oxidase (GO) protein, has proven effective as a substrate reduction therapy (SRT) in a preclinical PH1 model. This thesis aims to refine this gene targeting strategy to enhance the predictability, precision, and overall safety of this therapeutic approach. First, we compared the editing outcomes of single Cas9 nuclease and paired Cas9 nucleases guided by two gRNAs, 64 nucleotides apart and targeting opposite strands, aiming for a more precise disruption of Hao1. Utilizing adeno-associated viral vectors serotype 8 (AAV8) to deliver editing tools into mouse liver, and confining Cas9 expression to hepatocytes via a liver-specific promoter, we demonstrated that paired Cas9 nucleases yielded more predictable modifications, primarily resulting in a precise deletion between the two cuts. Subsequently, we explored the use of paired Cas9 nickases to minimize off-target effects and achieve a more accurate gene disruption. Our findings revealed that paired Cas9 nickases, delivered by two independent AAV vectors, exhibited efficacy comparable to individual and paired Cas9 nucleases in reducing the target enzyme level in vivo. Notably, we observed that paired nick-induced double-strand breaks were likely repaired through the microhomology-mediated end-joining pathway, resulting in heterogeneous modifications of variable sizes. Conversely and as expected, single nicks failed to disrupt the target gene. Importantly, the use of paired Cas9 nickases significantly reduced AAV integration at the target site compared to Cas9 nucleases, potentially due to differences in repair mechanisms. To facilitate clinical translation, we developed an all-in-one AAV vector for nickase-mediated targeted cleavage, maintaining therapeutic efficacy with a reduced AAV dose. Our comprehensive off-target analysis confirmed the specificity of selected gRNAs with no off-target activity or chromosomal translocations. Finally, paired gRNAs targeting the HAO1 human orthologous gene were tested in human cells demonstrating efficiency and laying the groundwork for future studies. In parallel, we explored the use of CRISPR-CasRx, a programmable RNA editor, for a safer SRT avoiding permanent DNA modifications. The successful reduction of GO expression in vitro paved the way for in vivo testing. However, AAV8 vectors delivering CasRx with single or multiple gRNAs did not result in significant GO protein reduction in the livers of mice, despite indications of collateral effects. This emphasizes the need for further investigation to improve the efficiency of this technology. In conclusion, this thesis addresses the refinement of CRISPR-Cas9-mediated gene targeting strategies for treating PH1. It demonstrates the efficacy of paired Cas9 nucleases in achieving more predictable modifications. Additionally, it underscores the promise of paired Cas9 nickases for in vivo gene disruption in a therapeutic context, with minimized on-target AAV integration and off-target effects. Finally, it highlights the need for further optimization of CRISPR-CasRx application for therapeutic purposes.
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    Modelling, design, fabrication and characterization of engineered human myocardium made with melt electrowriting and cardiac cells derived from hiPSCs
    (Universidad de Navarra, 2024-02-22) Flandes-Iparraguirre, M. (María); Mazo, M. (Manuel); Prosper-Cardoso, F. (Felipe); Prosper, F. (Felipe)
    The adult human heart has evolved to become a highly specialized organ, whose continuous pumping of blood is critical for survival. However, its ability to regenerate or self-repair following injury is very limited, so consequently any event or disease resulting in damage to the heart poses a serious threat to the patient. Moreover, cardiovascular diseases represent one of the most pressing healthcare concerns nowadays, as they are the leading cause of death worldwide, and the number of cases is only expected to increase in the following years. Despite great progress made over the years to treat cardiovascular diseases, to date there is no therapy able to fully cure a heart that has been damaged. In consequence, there is a dire need to generate new strategies to repair the heart damage and restore the lost cardiac function, as well as to develop accurate modelling platforms to advance in the understanding of disease progression and assess the effectiveness of new drugs. Since its advent, cardiac tissue engineering and regenerative medicine has been regarded as a promising candidate to realise this enormous challenge. Given its interdisciplinary nature, scientific breakthroughs in different areas such as cellular reprogramming, polymer chemistry, and additive manufacturing technologies have resulted in the advancement of cardiac tissue engineering and regenerative medicine over the years. One of such cornerstone discoveries was the generation of induced pluripotent stem cells and subsequent differentiation to different cardiac phenotypes, and the present Thesis revolves around their application to generate patient-specific cardiac disease models and humanised engineered functional cardiac minitissues. Firstly, we reprogrammed peripheral blood mononuclear cells from a transthyretin amyloid cardiomyopathy patient, resulting in the generation of a new cell line carrying a c.128G>A (p.Ser43Asn) mutation in the transthyretin gene. Experiments demonstrated the efficacy and safety of the approach, confirming the pluripotency of the cells, the presence of the disease-causing mutation, and the removal of reprogramming vectors. This cell line, which is now available in a repository, can be used to investigate disease biology, molecular mechanisms and progression; as well as an advanced cellular model to test novel therapeutic strategies. Secondly, we aimed to generate functional human minitissues by combining human cardiomyocytes derived from induced pluripotent stem cells and tridimensional fibrillar scaffolds generated with the technology of melt electrowriting. Compared to conventional two-dimensional cell culture, the cardiac minitissues demonstrated enhanced maturation, with a significant increase in conduction velocity, presence of connexin 43 and expression of cardiac-associated genes such as MYL2, GJA5 and SCN5A, and isoform ratios MYH7/MYH6 and MYL2/MYL7 after 28 days in culture. When investigating the effect of the scaffold fibres on the cells, we found that cardiomyocytes placed close to the fibre were arranged parallel to it, but that alignment was progressively lost towards the centre of the scaffold pore. We then used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition and intercellular connectivity allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture. We next aimed at increasing the biological representativity of the cardiac minitissues, by implementing a few changes in cellular (addition of induced pluripotent stem cell-derived cardiac fibroblasts) and hydrogel (substitution of Matrigel for fibrin) composition. We also sought to control cardiomyocyte behaviour based on melt electrowritten scaffold geometry. For this, we hypothesized that diamond-based scaffolds would induce cardiomyocyte contraction in the direction of least mechanical resistance, i.e., the small diagonal of the diamonds. The characterization of the new cardiac minitissues demonstrated functional maturation consistent with the previous work in terms of gene expression and conduction velocity, although the observed low initial cell retention within the scaffold highlighted the need of new strategies to improve cell seeding efficiency. When comparing contractile dynamics between melt electrowritten scaffolds made with square, rectangular, and diamond-shaped pores, we found that the latter resulted in significantly faster, stronger and aligned contraction in the direction that we had anticipated. The potential use of the cardiac minitissues as therapy agents was tested by implanting the constructs in a murine model of chronic myocardial infarction. Compared to controls, implanted animals showed significant improvement, including higher left ventricular ejection fraction and greater wall thickness. Finally, in another attempt to enhance the biological representativity of the constructs, a proof of concept was made to generate melt electrowritten ellipsoidal scaffolds with controlled pore architecture. In summary, the present Thesis revolves around human induced pluripotent stem cells and melt electrowriting as cornerstone tools for cardiac tissue engineering and regenerative medicine efforts. By combining both and iteratively optimising the design and experimental conditions, we were able to generate human functional cardiac minitissues of increased biological relevance.
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    Towards inmunotherapeutic exploitation strategies of rotumor and antitumor cytokines
    (Universidad de Navarra, 2024-02-13) Olivera, I. (Irene); Melero, I. (Ignacio)
    In the last two decades, cancer immunotherapy has become one of the most promising strategies for cancer treatment. Understanding the link between the immune responses and cancer was essential to the development of these novel therapies. In the late 1950’s, Burnet and Thomas suggested the role of the immune system in the repression of carcinomas (1,2). In 2013, Chen and Mellman (3) published “the cancerImmunity cycle” that conceptually described the indispensable steps for an effective antitumor response (figure 1). The cycle starts with the release of neoantigens by the cancer cells which are captured by the dendritic cells (DCs) for processing. DCs migrate to lymph nodes where they present processed antigens to T cells, activating effector T cell responses. Finally, the activated T cells traffic to the tumor bed where they specifically kill tumor cells which releases additional tumor antigens (3). If this cycle works properly, the immune cells can eliminate or control the tumor. However, cancer cells have developed different mechanisms to resist the immune system attack.
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    TIM-3 blockade as a therapeutic approach for diffuse intrinsic pontine glioma
    (Universidad de Navarra, 2024-02-13) Ausejo-Mauleón, I. (Iker); Alonso-Roldán, M.M. (Marta María); Pastor, F. (Fernando)
    Diffuse intrinsic pontine gliomas (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in understanding this disease, prognosis is dismal, with over 90% of patients dying within two years of diagnosis and a median overall survival time of 9-12 months. These grim statistics underscore that DIPG is an unmet clinical need. In this doctoral thesis, I have evaluated the local administration of an anti-TIM-3 antibody in anti-TIM-3 antibody in syngeneic orthotopic models of DIPG as a therapeutic approach for this disease. Our work uncovered, through in silico studies in patient datasets (whole RNAseq and scRNAseq) and samples (by multiplex IF) that TIM-3 was robustly expressed in tumor cells and tumor microenvironment, mainly in microglia and macrophages, suggesting this molecule as a potential therapeutic target in DIPGs. Mechanistic studies showed that TIM-3 provided intrinsic survival cues to the tumor cell while modulating the tumor microenvironment when expressed in the myeloid compartment. In vivo studies showed that TIM-3 blockade significantly increased the overall survival of DIPG immunocompetent orthotopic models, led to long-term survivors, and showed immune memory. TIM-3 inhibition resulted in an increase in the number and proliferative state of microglia, NK cells, and CD8+ T cells and higher levels of IFN, GrzB, and TNF&945; corresponding to NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population, which caused an increase in the pro-inflammatory CD8/Treg ratio. Chemokine studies demonstrated an augmentation of CCL5, CCL2 chemotactic chemokines, and CXCL10, IL-1;, and IFN- pro-inflammatory cytokines in the tumor microenvironment of treated mice. Additionally, DCs, CD4+, and CD8+ T cells were increased in treated draining lymph nodes and of functional significance, expressed higher amounts of pro-inflammatory cytokines than in control mice. Interestingly, the depletion of NK cells, CD4+, and CD8+ T cells immune populations did not completely abrogate the treatment efficacy. However, microglia and macrophages depletion with an anti-CSF1R resulted in a total loss due to a loss of microglia and CD8 T cells pro-inflammatory populations, chemokines, and cytokines indicating a critical role of these populations in the therapeutic effect of TIM-3 blockade. This study provides a new and previously unstudied view of DIPG treatment. TIM-3 blockade emerges as an exciting alternative to classical immune checkpoints, such as PD-1, that did not obtain the desired results in DIPG clinical trials. Moreover, the lack of other effective therapies for these devastating pediatric brain tumors makes these pre-clinical results especially promising and offers strong support for initiating a clinical trial with an anti-TIM-3 antibody for the treatment of DIPG.
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    Functional study of chromatin factors uncovers strong lineage determining roles and divergent behaviours between normal and malignant haematopoiesis
    (Universidad de Navarra, 2024-01-18) Goñi-Salaverri, A. (Ainhoa); Pineda-Lucena, A. (Antonio); Lara-Astiaso, D. (David)
    Haematopoiesis relies on the coordinated activities of transcription and chromatin factors (TFs and CFs), which interact to form Genome Regulatory Complexes (GRCs). These dynamic complexes precisely control lineage-specific transcriptional patterns and steer cellular phenotypes. Furthermore, the significance of TFs and CFs is strongly supported by a wealth of evidence from recent studies, which consistently reveal the high recurrence and almost ubiquitous presence of mutations affecting TFs and CFs in haematological malignancies, including acute myeloid leukaemia (AML). During the last decade, researchers in the field of haematopoiesis have greatly benefited from a comprehensive understanding of normal differentiation roadmaps and the transformation events occurring in malignancies. However, our understanding of CFs contribution to haematopoiesis remains limited. We have yet to determine the main factors (CFs and TFs) within GRCs that govern haematopoietic differentiation, unravel their intricate functional interactions and whether CFs have specific roles or redundantly contribute to lineage determination. Furthermore, we still need to discern which epigenetic mechanisms are disrupted in leukaemia and determine their impact on disease initiation and/or maintenance. We hypothesised that CFs within GRCs exert distinct roles during haematopoietic lineage determination and that specific CF disruption might contribute to the initiation and/or maintenance of AML. Firstly, we systematically assessed the lineage-specifying potential of chromatin regulators (CFs and TFs) in murine haematopoietic differentiation trajectories, both ex vivo and in vivo. Ex vivo bulk results unveiled the nuanced and stage-specific roles of GRCs involved in fundamental epigenetic processes during haematopoietic differentiation, including COMPASS methyl-transferases and BAF remodellers. However, a higher level of functional consistency was observed among epigenetic repressor complexes and corepressors. Consistent with the ex vivo effects, in vivo findings at single-cell resolution revealed pronounced lineage-specific trends and functional variability for COMPASS and BAF subcomplexes and suggested that multiple epigenetic repressors, like NuRD, ISWI and N-CoR, help maintain progenitor diversity and balanced lineage distribution by mitigating excessive myelopoiesis. Our second goal was to unveil the potential interactions amongst TFs and CFs to unravel the composition of key GRCs, shifting the current regulatory paradigm from a single factor to a protein complex-centric view. We examined the dynamic patterns of chromatin accessibility upon TF/CF disruptions and selected representative TF motifs of haematopoietic cell fates to analyse global TF footprints upon every knockout. Disruption of COMPASS and BAF showed downregulation of myeloid TF motif accessibility. In contrast, NuRD and other repressors induced increased accessibility of myeloid TFs associated with inflammatory responses upon knockout. Lastly, we aimed to dissect the corruption of CFs function in a Npm1c/Flt3-ITD leukaemia model and provide leukaemic-specific vulnerabilities that suggest potential epigenetic therapeutic avenues. scRNA-seq and CITE-seq analyses unveiled leukaemia transcriptomic heterogeneity, with distinct subpopulations exhibiting varying degrees of growth potential. Afterwards, Perturb-seq analysis of the chromatin regulators loss of function across leukaemia clusters confirmed tumour vulnerabilities upon specific perturbations, facilitating the transition of AML-infected cells towards leukaemic differentiated populations with limited cell proliferation and fitness capacities. These observations revealed that leukaemias subvert the function of CFs involved in homeostatic differentiation by aberrantly blocking these processes to sustain their malignant state.
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    Nuevos mecanismos oncogénicos de la calreticulina mutante: identificación de nuevas dianas terapéuticas en las neoplasias mieloproliferativas crónicas
    (Universidad de Navarra, 2024-01-18) Guijarro-Hernández, A. (Ana); Vizmanos-Pérez, J.L. (José Luis)
    Hasta la fecha, la investigación en los efectos oncogénicos de la calreticulina en las NMPs Ph-negativas se ha enfocado principalmente en el eje MPL/JAK2, ya que su activación constituye el principal mecanismo oncogénico subyacente a estas enfermedades. Sin embargo, la calreticulina mutante también desencadena mecanismos independientes de la activación de este eje que colaboran en el desarrollo y progresión de la enfermedad. La identificación de estos mecanismos no ha sido sencilla debido a que la propia activación de las vías relacionadas con JAK2 es muy potente o por la falta de modelos experimentales que permitan este análisis. En este trabajo, se ha utilizado un modelo en C. elegans con mutaciones en la calreticulina homólogas a las que aparecen en las NMPs, que carece de manera natural de ortólogos de MPL y JAK2. En este modelo, hemos observado una alteración transcripcional de genes y procesos que tienen un papel en el cáncer y en las NMPs en particular, mostrando que estos genes y procesos pueden estar perturbados directamente en el gusano por la calreticulina mutante sin la intervención de JAK2 o MPL. Aunque la alteración de estos procesos había sido detectada con anterioridad en pacientes con NMPs, nunca antes se había planteado como una consecuencia directa de las mutaciones en calreticulina sin la intervención del eje MPL/JAK2 en humanos. Según nuestros resultados, la mayor parte de los efectos de la calreticulina mutante en C. elegans son consecuencia de una pérdida de función de la calreticulina, aunque otros, como la subexpresión de algunos genes cuyos productos participan en la vía Hh (como ptc-3/PTCH1) o la sobrexpresión de los genes flh-3/FLYWCH1 o nhr-2/RXRA, codificantes de factores de transcripción, parecen ser consecuencia de una función neomórfica de la calreticulina mutante. La validación de estas alteraciones en líneas celulares humanas y pacientes con TE y mutaciones en CALR, junto con la comparación del transcriptoma de células CD34+ de MO y neutrófilos de SP de pacientes con TE ha permitido revelar a RXRA como una potencial diana terapéutica para pacientes con TE. Hasta donde sabemos, esta es la primera vez que una cepa de C. elegans con mutaciones similares a las de los pacientes es propuesta como modelo para la investigación en leucemias.
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    Optimization of mimetic periosteum autografts for the treatment of nonunions
    (Universidad de Navarra, 2024-01-18) Romero-Torrecilla, J.A. (Juan Antonio); Granero-Moltó, F. (Froilán)
    Bone presents truly regenerative capacity being able to regenerate into a native state in response to injuries. Despite this self-renewal potential, bone healing is not absent of complications and different conditions can interfere with the regenerative process, leading to delayed fracture and in some cases fracture nonunion. Fracture nonunion is a major cause of chronic pain and disability and, despite the low incidence of nonunion and delayed union fractures (5-10%), the numerous fractures that take place globally (~180 million every year) emphasizes the huge economic burden that fracture nonunion represents. Once detected, fracture nonunion requires a surgical approach, and the use of bone autografts that provide and osteoinductive, osteogenic and osteoconductive environment for a successful repair. However, the availability of bone grafts is limited. The scarcity of bone tissue that can be used for autografts have consolidated the need for novel tissue engineering approaches as potential candidates for the treatment of nonunion and for long bone defects, prone to evolve to nonunions. Tissue engineering strategies allow for the combination of novel tunable materials along with different biological adjuvants, including growth factors and cells. During the bone regenerative response, the periosteum, a fibrous layer surrounding the bone, plays a key role delivering osteochondroprogenitor cells and crucial growth factors into the injured tissue. Thus, we developed a tissue engineering strategy where biocompatible, 3D melt-electro-written polycaprolactone membrane would act as a mimetic periosteum. The engineered mimetic periosteum allows vascularization of the construct either when implanted ectopically or orthotopically. Additionally, we demonstrated its capacity to be functionalized with rhBMP-2, the most important morphogen for bone regeneration, both exposed on the membrane surface attached through PEA-hFN or encapsulated in microparticles covalently bound to the PCL membrane. When functionalized with low doses of rhBMP-2 the mimetic periosteum demonstrated great osteogenic potential in vitro, inducing human MSCs differentiation into osteoblasts. More importantly, in vivo results indicate that the functionalization of the mimetic periosteum with rhBMP-2 allows regenerative properties able to heal critical size femoral defects in SD rats with high efficiency and reproducibility using unpreceded low doses of rhBMP-2. Ultimately, the mimetic periosteum demonstrated its ability to deliver key mesenchymal progenitor cells into the injured site. All these results indicate that our engineered mimetic periosteum represents an efficient system for rhBMP-2 and progenitor cells delivery with important translational potential.