60 % of school children have some form of untreated tooth decay or have suffered trauma to the front teeth which results in pulp damage. If left untreated, these teeth are susceptible to premature fracture/loss under daily stresses. In cases of adolescent tooth loss, teenagers cannot get dental implants until after the growth spurts; their only option is using removable dentures which lowers their quality of life. Conventional endodontic treatment (root canal treatment) is used in cases of pulp necrosis, but cannot be performed in immature permanent teeth due to major differences in tooth anatomy. Currently the American Dental Academy has approved a procedure called Regenerative Endodontic Treatment (RET) for such cases, but the outcomes are still unpredictable and the method is largely unreliable. One issue that we are trying to address in this work is the regeneration of the pulp-dentin complex (PDC), specifically the interface. Endeavors in regenerating either pulp or dentin have been successful individually, but the interface region is the anatomical and physiologic hallmark of the PDC and has not been addressed. We have proposed a biomimetic scaffold to facilitate early stage stratification of these different tissues and allow recapitulation of their interface. Tissue engineering principles and biomaterial processing techniques were used simultaneously to encourage dental pulp stem cells into mineralize selectively only on one side. This effectively allows the scaffold to serve as the interface region between the hard dentin and the soft vascular pulp. / Bioengineering

Polymeric bone scaffolds are a promising tissue engineering approach for the repair of critical-size bone defects. Porous three-dimensional (3D) scaffolds play an essential role as templates to guide new tissue formation. However, there are critical challenges arising from the poor mechanical properties and low bioactivity of bioresorbable polymers, such as poly(epsilon-caprolactone) (PCL) in bone tissue engineering applications. This research investigates the potential use of cellulose nanocrystals (CNCs) as multi-functional additives that enhance the mechanical properties and increase the biomineralization rate of PCL. To this end, an in vitro biomineralization study of both sulfuric acid hydrolyzed-CNCs (SH-CNCs) and surface oxidized-CNCs (SO-CNCs) has been performed in simulated body fluid in order to evaluate the bioactivity of the surface functional groups, sulfate and carboxyl groups, respectively. PCL nanocomposites were prepared with different SO-CNC contents and the chemical/physical properties of the nanocomposites were analyzed. 3D porous scaffolds with fully interconnected pores and well-controlled pore sizes were fabricated from the PCL nanocomposites with a 3D printer. The mechanical stability of the scaffolds were studied using creep test under dry and submersion conditions. Lastly, the biocompatibility of CNCs and 3D printed porous scaffolds were assessed in vitro. The carboxyl groups on the surface of SO-CNCs provided a significantly improved calcium ion binding ability which could play an important role in the biomineralization (bioactivity) by induction of mineral formation for bone tissue engineering applications. In addition, the mechanical properties of porous PCL nanocomposite scaffolds were pronouncedly reinforced by incorporation of SO-CNCs. Both the compressive modulus and creep resistance of the PCL scaffolds were enhanced either in dry or in submersion conditions at 37 degrees Celsius. Lastly, the biocompatibility study demonstrated that both the CNCs and material fabrication processes (e.g., PCL nanocomposites and 3D printing) were not toxic to the preosteoblasts (MC3T3 cells). Also, the SO-CNCs showed a positive effect on biomineralization of PCL scaffolds (i.e., accelerated calcium or mineral deposits on the surface of the scaffolds) during in vitro study. Overall, the SO-CNCs could play a critical role in the development of scaffold materials as a potential candidate for reinforcing nanofillers in bone tissue engineering applications. / Ph. D.

Partial or complete tears of the anterior cruciate ligament (ACL) can greatly afflict quality of life and often require surgical reconstruction with autograft or allograft tissue to restore native knee biomechanical function. However, limitations exist with these treatments that include donor site pain and weakness found with autografts, and longer "ligamentization" and integration times due to the devitalization of allograft tissue. Alternatively, a tissue engineering approach has been proposed for the fabrication of patient-specific grafts that can more rapidly and completely heal after ACL reconstruction. Electrospun micro-fiber networks have been widely utilized as biomaterial scaffolds to support the growth and differentiation of mesenchymal stem cells toward many tissue lineages including ligament. However, these micro-fiber networks do not possess suitable sizes and shapes for a ligament application and cannot support cell infiltration. The objective of this work was to develop techniques to 1) rapidly cellularize micro-fiber networks, 2) assemble micro-fiber networks into cylindrical composites, 3) provide cues to mesenchymal stem cells (MSCs) to guide their differentiation toward a ligament phenotype. The cellularization of micro-fiber networks was performed utilizing a co-electrospinning/electrospraying technique. Cells deposited within a cell culture medium solution remained where they were deposited and did not proliferate. The inclusion of space-filling hydrogel network such as collagen was necessary to reduce the density of the micro-fiber network to facilitate spreading. However, it became apparent that the incorporation of significant collagen phase was necessary for long-term MSC survival within the micro-fiber network. Next, two approaches were developed to fabricate large cylindrical, composites. The first approach utilized a co-electrospinning/electrospraying technique to generate micro-fiber/collagen composites that were subsequently rolled into cylinders. These cylindrical composites exhibited greater diameters and water weight percentages as collagen content increased. However, the high micro-fiber content of these composites was inhibitory to cell survival. In the second approach, thin layers (~5-10 fibers) of aligned electrospun PEUR fibers were encapsulated within a collagen gel and subsequently rolled the composites into cylinders. These sparse-fiber composites were nearly 98% by weight water and confocal imaging revealed the presence of sparse fiber layers (~5 fibers thick) separated by approximately 200 μm thick collagen layers. We hypothesize that the proliferation and migration of MSCs within these micro-fiber/collagen composites may not be restricted by the presence of a dense, non-manipulatable electrospun fiber network present in traditionally rolled fiber composites. Simple model platforms were then developed to study the influence of sparse micro-fibers on MSCs differentiation within a collagen hydrogel. MSCs in the presence of the softest (5.6 MPa) micro-fibers elongated and oriented to the underlying network and exhibited greater expression of scleraxis, and α-smooth muscle actin compared to the stiffest (31 MPa) fibers. Additionally, preliminary results revealed that the incorporation of fibroblast growth factor-2 and growth and differentiation factor-5 onto micro-fibers through chemical conjugation enhanced expression of the ligamentous markers collagen I, scleraxis, and tenomodulin. In conclusion, micro-fiber/collagen composite materials must possess sufficient space to support the infiltration and differentiation of MSCs. The strategies described in this document could be combined to fabricate large, micro-fiber/collagen composites that can support cell infiltration and provide relevant cues to guide the formation of an engineered ligament tissue. / Ph. D.

Grafts currently used for the repair of anterior cruciate ligament (ACL) ruptures integrate poorly with bone due to a significant mismatch in properties between graft and bone. Specifically, conventional grafts (e.g., hamstring tendon) are unable to recapitulate intricate gradients in mechano-chemical properties and extracellular matrix (ECM) architecture found at natural bone-ligament (B-L) transitions, and thus result in stress-concentrations at the graft-bone interface leading to graft failure. In contrast, tissue-engineered scaffolds possessing gradients in properties can potentially guide the establishment of phenotypic gradients in bone marrow stromal cells (BMSCs), and thus aid the regeneration of B-L transitions in the long-term. Towards the eventual goal of regenerating complex tissue transitions, this project employs three complementary strategies to fabricate graded scaffolds. The three strategies involve the presentation of gradients in 1) mineral content, 2) scaffold architecture and 3) growth factor (GF) concentration within scaffolds to control BMSC morphology and phenotype. The first strategy involved co-electrospinning two polymers (one doped with hydroxyapatite) from offset spinnerets onto a rotating drum to produce scaffolds possessing a gradient in mineral content. Post-electrospinning, these graded scaffolds were treated with a simulated body fluid to further enhance the gradient. Analysis of mRNA expression of osteoblastic makers by BMSCs and the deposition of bone-specific ECM proteins indicated that the scaffolds could guide the formation of an osteoblastic phenotypic gradient. The second strategy involved electrospinning two polymer solutions onto a custom-designed dual-drum collector to fabricate scaffolds possessing region-wise differences in fiber alignment, diameter and chemistry. Specifically, electrospinning onto the dual-drum collector resulted in the deposition of aligned fibers from one polymer solution in the gap region between the drums, randomly oriented fibers from the other polymer solution on one of the drums and a mixture of fibers from both polymer solutions in the overlap region in between. The topographical cues within these scaffolds were shown to result in region-dependent BMSC morphology and orientation. Although the long-term goal of the third strategy was to create a co-electrospun scaffold possessing a gradient in GF concentration, a new technique to protect GF activity within electrospun scaffolds via the use of gelatin microspheres was first validated. Preliminary results from these studies indicate that microspheres can protect and deliver a model protein (lysozyme) in active conformation from electrospun scaffolds. These results further suggest that gradients of GF concentration can be achieved in the long-term by protecting GFs within microspheres and co-electrospinning as described in the first strategy. In conclusion, the results from this project suggest that graded scaffolds can help guide the formation of gradients in cell morphology, orientation and phenotype, and thus potentially promote the regeneration of B-L transitions in the long-term. The three strategies described in this project can be employed in concert to create scaffolds intended for the regeneration of complex tissue transitions. / Ph. D.

Damage to dura mater may occur during intracranial or spinal surgeries, which can result in cerebrospinal fluid leakage as well as other potentially fatal physiological changes. As a result, biological scaffolds derived from xenogeneic materials are typically used to repair and regenerate dura mater post intracranial or spinal surgeries. In this study we explore the mechanics, structure, and immunological capacity of xenogeneic dura mater to be considered as a replacement for human dura. A comparative analysis is done between native porcine dura and a commercially available bovine collagen-based dura graft. Native porcine dura mater was decellularized and subjected to mechanical and histological analysis. Our decellularized porcine dura was able to maintain the overall morphological/structural integrity and held an increased extensibility without sacrificing strength, which provides a solid foundation as a functional grafting material. The histological observations showed that the orientation of fibers was maintained after decellularization. We investigated the biocompatibility of native and decellularized porcine dura reseeded with fibroblast cells for in vitro study. Cell proliferation, cell viability, and mechanical properties of dural grafts were evaluated post reseeding on days 3, 7, and 14. Live-dead staining and resazurin salts quantified cell viability and cell proliferation, respectively. This in vitro study showed that the acellular porcine dural graft provided a favorable environment for rat fibroblast cell infiltration. The results of micro indentation testing show that the cell-seeded porcine dural graft provides a favorable environment for rat fibroblast cell infiltration. The mechanics and biocompatibility results provide promising insight for the potential use of porcine dura in future cranial dura mater graft applications. Lastly, a subcutaneous in vivo study of dura graft compared with the market available Lyoplant®. Grafts were evaluated for inflammation by evaluating macrophage and leukocyte invasion on 3, 7, and 14 days post implantation. Histological analysis of both implants revealed macrophage (and leukocyte infiltration, supporting reabsorption, and thus encouraging the regeneration at 14 days. Cell markers also revealed that inflammation and leukocytes decreased as the number of days increased. Future work will involve a long-term subcutaneous implantation up to 30 days and 60 days to determine the long-term immune response.

[EN] Articular cartilage is a tissue that consists of chondrocytes surrounded by a dense extracellular matrix (ECM). The ECM is mainly composed of type II collagen and proteoglycans. The main function of articular cartilage is to provide a lubricated surface for articulation. Articular cartilage damage is common and may lead to osteoarthritis. Articular cartilage does not have blood vessels, nerves or lymphatic vessels and therefore has limited capacity for intrinsic healing and repair. Tissue engineering (TE) is a powerful approach for healing degenerated cartilage. TE uses three-dimensional (3D) scaffolds as cellular culture supports. The scaffold provides a structure that facilitates chondrocyte adhesion and expansion while maintaining a chondrocytic phenotype and limiting dedifferentiation, which is a problem in two-dimensional (2D) systems. Cell attachment to the scaffolds depends on the physical and chemical characteristics of their surface (morphology, rigidity, equilibrium water content, surface tension, hydrophilicity, presence of electric charges). The primary aim of this thesis was to study the influence of different kinds of biomaterials on the response of chondrocytes to in vitro culture. 3D scaffold constructs must have an interconnected porous structure in order to allow cell development through the network, to maintain their differentiated function, as well as to allow the entry and exit of nutrients and metabolic waste removal. Therefore, the effect of the hydrophilicity and pore architecture of the scaffolds was studied. A series of polymer and copolymer networks with varying hydrophilicity was synthesised and biologically tested in monolayer culture. Cell viability, proliferation and aggrecan expression were quantified. When human chondrocytes were cultured on polymer substrates in which the hydrophilic groups were homogeneously distributed, adhesion, proliferation and viability decreased with the content of hydrophilic groups. Nevertheless, copolymers in which hydrophilic and hydrophobic domains alternate showed better results than the corresponding homopolymers. Biostable and biodegradable scaffolds with different hydrophilicity and porosity were synthesised using a template of sintered microspheres of controlled size. This technique allows the interconnectivity between pores and their size to be controlled. Periodic and regular pore architectures and reproducible structures were obtained. The mechanical behaviour of the porous samples was significantly different from that of the bulk material of the same composition. Cells fully colonised the scaffolds when the pores' size and their interconnection were sufficiently large. Another objective was to assess the chondrogenic redifferentiation in a biodegradable 3D scaffold of polycaprolactone (PCL) of human autologous chondrocytes previously expanded in monolayer. This study demonstrated that chondrocytes cultured in PCL scaffolds without fetal bovine serum (FBS) efficiently redifferentiated, expressing a chondrocytic phenotype characterised by their ability to synthesise cartilage-specific ECM proteins. The influence that pore connectivity and hydrophilicity of caprolactone-based scaffolds has on the chondrocyte adhesion to the pore walls, proliferation and composition of the ECM produced was studied. The number of cells inside polycaprolactone scaffolds increased as porosity was increased. A minimum of around 70% porosity was necessary for this scaffold architecture to allow seeding and viability of the cells within. The results suggested that some of the cells inside the scaffold adhered to the pore walls and kept the dedifferentiated phenotype, while others redifferentiated. In conclusion, the findings of this thesis provide valuable insight into the field of cartilage regeneration using TE techniques. The studies carried out shed light on the right composition, porosity and hydrophilicity of the scaffolds to be used for optimal cartilage production. / [ES] El cartílago articular es un tejido compuesto por condrocitos rodeados por una densa matriz extracelular (MEC). La MEC se compone principalmente de colágeno tipo II y de proteoglicanos. La función principal del cartílago articular es proporcionar una superficie lubricada para las articulaciones. Las lesiones en el cartílago articular son comunes y pueden derivar a osteoartritis. El cartílago articular no tiene vasos sanguíneos, nervios o vasos linfáticos y, por tanto, tiene una capacidad limitada de auto-reparación. La ingeniería tisular (IT) es un área prometedora en la regeneración de cartílago. En la IT se utilizan "andamiajes" (scaffolds) tridimensionales (3D) como soportes para el cultivo celular y tisular. Los scaffolds proporcionan una estructura que facilita la adhesión y la expansión de los condrocitos, manteniendo un fenotipo condrocítico limitando su desdiferenciación; que es el mayor problema en los sistemas bidimensionales (2D). La adhesión celular a los scaffolds depende de las características físicas y químicas de su superficie (morfología, rigidez, contenido de agua en equilibrio, tensión superficial, hidrofilicidad, presencia de cargas eléctricas). El objetivo general de esta tesis fue estudiar la influencia de diferentes tipos de biomateriales en la respuesta de los condrocitos en cultivo in vitro. Los scaffolds deben tener una estructura porosa interconectada para permitir el desarrollo celular a través de toda la estructura 3D, potenciando que los condrocitos mantengan su fenotipo, así como permitiendo entrada de nutrientes y eliminación de desechos metabólicos. Se estudió el efecto de la hidrofilicidad y de la arquitectura de poro. Se cuantificó la viabilidad celular, la proliferación y la expresión de agrecano. Cuando los condrocitos humanos se cultivaron en sustratos poliméricos donde los grupos hidrófilos se distribuyeron de manera homogénea, la adhesión, la proliferación y la viabilidad disminuyó con el contenido de grupos hidrófilo. Sin embargo, los copolímeros en los que los dominios hidrófilos e hidrófobos se alternaban mostraron mejores resultados que los homopolímeros correspondientes. Se sintetizaron series de scaffolds bioestables y series biodegradables con diferente hidrofilicidad y porosidad utilizando plantillas de microesferas sinterizadas. Se obtuvieron arquitecturas de poros regulares y reproducibles. Las células colonizaron el scaffold en su totalidad cuando los poros y la interconexión entre ellos era lo suficientemente grande. Se evaluó la rediferenciación condrogénica de condrocitos autólogos humanos, previamente expandidos en monocapa, sembrados en un scaffold biodegradable de policaprolactona (PCL). Se demostró que los condrocitos cultivados en scaffolds de PCL con medio sin suero bovino fetal (FBS), se rediferenciaban de manera eficiente; expresando un fenotipo condrocítico, caracterizado por su capacidad de sintetizar proteínas de la MEC específicas de cartílago hialino. Se estudió la influencia de la hidrofilicidad y la conectividad de los poros de los scaffolds de caprolactona sobre la adhesión de los condrocitos a las paredes de los poros, su capacidad proliferativa y la composición de MEC sintetizada. Se observó que un mínimo de 70% de porosidad era necesario para permitir la siembra de los condrocitos en el scaffold y su posterior viabilidad. El número de células aumentaba a medida que aumentaba la porosidad del scaffold. Los resultados sugieren que parte de las células que se adherían a las paredes internas de los poros mantenían el fenotipo desdiferenciado de condrocitos cultivados en monocapa, mientras que otros se rediferenciaban. En conclusión, los resultados de esta tesis aportan un avance en el campo de la regeneración de cartílago articular utilizando técnicas de IT. Los estudios realizados proporcionan directrices sobre la composición, la porosidad y la hidrofilicidad más adecuada para l / [CA] El cartílag articular és un teixit format per condròcits envoltats per una densa matriu extracel·lular (MEC). La MEC es compon principalment de col·lagen tipus II i de proteoglicans. La funció principal del cartílag articular és proporcionar una superfície lubricada a les articulacions. Les lesions en el cartílag articular són comuns i poden derivar en osteoartritis. El cartílag articular no té vasos sanguinis, nervis ni vasos limfàtics i, per tant, té una capacitat limitada d'auto-reparació. L'enginyeria tissular (IT) és una àrea prometedora en la regeneració del cartílag. A la IT s'utilitzen "bastiments" (scaffolds) tridimensionals (3D) com a suports per al cultiu cel·lular i tissular. Els scaffolds proporcionen una estructura que facilita l'adhesió i l'expansió dels condròcits, mantenint un fenotip condrocític limitant la seua desdiferenciació; que és el major problema en els sistemes bidimensionals (2D). L'adhesió cel·lular als scaffolds depèn de les característiques físiques i químiques de la superfície (morfologia, rigidesa, contingut d'aigua en equilibri, tensió superficial, hidrofilicitat i presència de càrregues elèctriques). L'objectiu general d'aquesta tesi va ser estudiar la influència de diferents tipus de biomaterials en la resposta dels condròcits en cultiu in vitro. Els scaffolds han de tindre una estructura porosa interconnectada per a permetre el desenvolupament cel·lular a través de tota l'estructura 3D, potenciant que els condròcits mantinguen el seu fenotip així com permetent l'entrada de nutrients i l'eliminació de productes metabòlics. S'ha estudiat l'efecte de la hidrofilicitat i de l'arquitectura de porus dels scaffolds. Es va quantificar la viabilitat cel·lular, la proliferació i l'expressió de agrecà. Quan els condròcits humans es van cultivar en substrats polimèrics en els quals els grups hidròfils es van distribuir de manera homogènia, l'adhesió, la proliferació i la viabilitat van disminuir amb el contingut de grups hidròfils. No obstant això, els copolímers en els quals els dominis hidròfils i hidròfobs s'alternaven van mostrar millors resultats que els homopolímers corresponents. Es van sintetitzar sèries de scaffolds bioestables i sèries biodegradables amb diferent hidrofilicitat i porositat utilitzant plantilles de microesferes sinteritzades. Es van obtindre arquitectures de porus regulars i reproduïbles. Les cèl·lules van colonitzar el scaffold en la seua totalitat quan els porus i la interconnexió entre ells era suficientment gran. Es van avaluar la rediferenciació condrogènica de condròcits autòlegs humans, prèviament expandits en monocapa, en un scaffold biodegradable de policaprolactona (PCL). Es va demostrar que els condròcits cultivats en scaffolds de PCL sense sèrum boví fetal (FBS) es rediferenciaven de manera eficient, expressant un fenotip condrocític caracteritzat per la seua capacitat de sintetitzar proteïnes de la MEC específiques de cartílag hialí. També es va estudiar la influència de la hidrofilicitat i la connectivitat dels porus dels scaffolds de caprolactona sobre l'adhesió dels condròcits a les parets dels porus, la seua capacitat proliferativa i la composició de MEC sintetitzada. Es va observar que un mínim del 70% de porositat sembla ser necessari per permetre la sembra dels condròcits i la seua posterior viabilitat en el scaffold. El nombre de cèl·lules augmentava a mesura que augmentava la porositat del scaffold. Els resultats suggereixen que part de les cèl·lules que s'adherien a les parets internes dels porus mantenien el fenotip desdiferenciat de condròcits cultivats en monocapa, mentre que altres es rediferenciaven. En conclusió, els resultats d'aquesta tesi proporcionen informació valuosa en el camp de la regeneració de cartílag utilitzant tècniques d'IT. Els estudis realitzats proporcionen directrius sobre la composició, la porositat i la hidrofilicitat m / Pérez Olmedilla, M. (2015). Tissue engineering techniques to regenerate articular cartilage using polymeric scaffolds [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58987

Yes / Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype. / BBSRC

Yes / The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide. / University of Turin (Ricerca Locale grant 2014 and 2015) and Prostate Cancer UK grant S12-027

Yes / The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds is described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide. / This research was financially supported by the University of Turin (Ricerca Locale grants BOSD_RILO_20_01, LOLM_RILO_21_01, PIPA_RILO_20_01 and PIPA_RILO_21_01), Fondazione Cassa di Risparmio di Torino (Grant BOSD_CRT_17_2) and TUBITAK (The Scientific and Technological Research Council of Turkey-2219 program).

No / The isotopic effect of exchanging deuterium with hydrogen on the mechanical and surface properties of agar gel is examined. The elastic modulus of the D2O gels obtained by AFM nanoindentation is significantly higher (factor of 1.5¿2) than the modulus found in H2O agar gels. Furthermore, the modulus is independent of loading rate. Surface imaging reveals that the surface roughness gets progressively smaller with increasing agar concentration. All these data suggest that the isotopic replacement of deuterium enhances the mechanical properties of the agar gel, with significant advantages in its use as a biphasic scaffold. / MRC