Mechanical stretch and chronotherapeutic techniques for progenitor cell transplantation and biomaterials

In the body, mesenchymal progenitor cells are subjected to a substantial amount external force from different mechanical stresses, each potentially influences their behaviour and maintenance differentially. Tensile stress, or compression loading are just two of these forces, and here we examine the role of cyclical or dynamic mechanical loading on progenitor cell proliferation and differentiation, as well as on other cellular processes including cell morphology, apoptosis and matrix mineralisation. Moreover, we also examine how mechanical stretch can be used to optimise and ready biomaterials before their implantation, and examine the role of the circadian rhythm, the body’s innate time keeping system, on biomaterial delivery and acceptance. Finally, we also investigate the effect of mechanical stretch on the circadian rhythm of progenitor cells, as research suggests that mechanical stimulation may be sufficient in itself to synchronise the circadian rhythm of human adult progenitor cells alone, and has also been linked to progenitor cell function. If proven correct, this could offer a novel, non- intrusive method by which human adult progenitor cells may be activated or preconditioned, being readied for differentiation, so that they may be more successfully integrated within a host body, thereby improving tissue engineering techniques and the efficacy of cellular therapies

Design and characterization of modified powder metallurgy titanium surfaces obtained by ß-Stabilizing elements diffusion treatments for biomedical applications

Esta tesis contiene artículos de investigación en anexoTitanium (Ti) is a metal highly recognized by its employment in the biomedical sector since 1950. Nevertheless, in these last two decades from 1990 till date, a second generation of Ti biomaterials has received high attention. They are known as β-Ti alloys and its strong interest comes from their excellent combination of properties for biomaterials. Currently, due to the great demand to develop new biomaterials, the surface modification is one of the main alternatives for the design of new Ti biomaterials with improved properties for the biomedical sector. In this PhD Thesis, the design of new Ti surfaces modified by diffusion of niobium (Nb) and molybdenum (Mo) as β-stabilizing elements is presented as alternative to Cp-Ti or fully β-Ti alloys; preserving their lightness of Ti in the core. These diffusion elements share their capacity of decreasing the elastic modulus of titanium as well as their biocompatibility character. Thus, the global idea of this work is the design and study of these modified Ti surfaces produced by powder metallurgy with their evaluation on mechanical performance, tribological properties, corrosion and biocompatibility character to be considered as possible candidates for biomedical applications. Regarding this matter, different modified Ti surfaces were designed with several conditions: i) titanium substrate (green or sintered), ii) diffusion element (Nb or Mo), and iii) diffusion treatments (co-sintering plus diffusion, diffusion or thermo-reactive diffusion). These systems were compared with the behaviour of the commonly employed Ti biomaterial, the commercially pure titanium (Cp-Ti) obtained through powder metallurgy (PM). The β-stabilizing elements were deposited by means of aqueous suspensions through spraying. The gradients in microstructure and composition were analyzed by spectroscopy and diffraction techniques, and their differences were related to the designing parameters. The final surface conditions were investigated to obtain the most suitable ones in function of the final properties measured: mechanical properties according to hardness and elastic modulus, wear, corrosion and tribocorrosion behaviour and biocompatibility features. Therefore, this doctoral work covers the whole process from the design of the materials employing preparation and deposition of suspensions, diffusion treatments, surface conditions selection and microstructure investigations to their final characterization of hardness, modulus of elasticity, wear, corrosion and their synergistic effect (tribocorrosion), and bioactivity and cell-material response. Although some questions were found during different stages that should be further investigated, these new Ti surfaces have demonstrated some suitable characteristics for biomaterials, providing improvements with respect to titanium with a promising character for bone replacements.El titanio (Ti) es un metal altamente reconocido y empleado en el sector biomédico desde 1950. Sin embargo, una nueva generación de biomateriales de titanio ha recibido gran atención en estas dos últimas décadas. Esta se conoce con el nombre de segunda generación de biomateriales de titanio y su alto interés deriva de la excelente combinación de propiedades que presentan para biomateriales. Actualmente, debido a la gran demanda del desarrollo de nuevos biomateriales, la modificación superficial es una de las principales alternativas para el diseño de nuevos biomateriales de Ti con mejores propiedades para aplicaciones del sector biomédico. En esta tesis doctoral, se presenta una alternativa al titanio comercialmente puro (Cp-Ti) y a las aleaciones completamente beta (β-Ti) mediante el diseño de nuevas superficies de titanio modificadas con elementos betágenos, niobio (Nb) y molibdeno (Mo), mediante procesos de difusión. Estos elementos de difusión comparten la capacidad de disminuir el módulo elástico del titanio, mientras que son elementos biocompatibles. Por tanto, la idea global de este trabajo se centra en el diseño y estudio de superficies modificadas de titanio pulvimetalúrgico, junto con la evaluación de las propiedades mecánicas y comportamiento a desgaste, corrosión y tribocorrosión. Además, de la evaluación de la biocompatibilidad de estos para considerarlos como posibles candidatos para aplicaciones biomédicas. Para ello se diseñaron diferentes superficies modificadas de Ti basadas en las siguientes condiciones: i) substrato de titanio (prensado o sinterizado), ii) elemento de difusión (Nb o Mo), y iii) tratamiento de difusión (co-sinterización + difusión, difusión o difusión termo-reactiva). Todos los materiales se compararon con el material de titanio obtenido mediante pulvimetalurgia (PM). Los elementos betágenos se depositaron mediante pulverización de suspensiones acuosas. Los gradientes de microestructura y composición se analizaron con técnicas de espectroscopia y difracción. Se estudiaron las condiciones superficiales más idóneas para la caracterización mecánica (dureza y módulo elástico), comportamiento a desgaste, corrosión, tribocorrosión y biocompatibilidad. Por tanto, esta tesis doctoral abarca todo el proceso desde el diseño de los materiales, preparación y pulverización de las suspensiones, tratamientos de difusión, caracterización microestructural y superficial, hasta la evaluación de dureza, módulo de elasticidad, desgaste, corrosión, tribocorrosión y biocompatibilidad de las superficies modificadas de Ti. Aunque se necesita la evaluación más profunda de algunos aspectos, estas superficies modificadas de Ti han demostrado que presentan características adecuadas para biomateriales con mejoras con respecto al titanio, por tanto con propiedades prometedoras para reemplazo óseo.Programa Oficial de Doctorado en Ciencia e Ingeniería de MaterialesPresidente: José Luis González Carrasco.- Secretario: Sandra Carolina Cifuentes Cuéllar.- Vocal: Yaiza González Garcí

Effects of Antidepressants on Human Mesenchymal Stem Cell Differentiation on Clinically Relevant Titanium Surfaces

Selective Serotonin Reuptake Inhibitors (SSRIs) are the most frequently prescribed class of drugs worldwide and are implemented in the treatment of depression and other psychiatric disorders. SSRIs relieve depressive symptoms by modulating levels of the neurotransmitter serotonin in the brain. SSRIs block the function of the serotonin transporter, thereby increasing concentrations of extracellular serotonin. However, serotonin levels in the neurons of the brain only account for 5% while the remaining 95% is present outside the brain. Serotonin receptors and transporter are located on bone resident cells (mesenchymal stem cells (MSCs)), osteoblasts and osteoclasts, and serotonergic activity is believed to affect bone homeostasis. Consequently, alterations in serotonin levels by SSRI treatment have the potential to alter bone formation and remodeling. Clinical reports correlate increase risk of bone fractures and delayed bone healing with SSRI use. Metallic implants are commonly used as orthopedic and dental implants to fix bony defects. Surface modifications have been used to increase the level of bone to implant contact by controlling the differentiation of MSCs into an osteoblastic linage and facilitate bone production. However, it is not known if SSRIs can affect MSCs osteoblastic differentiation and bone remodeling signaling in response to microstructured biomaterials. The aims of this study were: 1) Investigate the effects of SSRIs on MSCs differentiation on microstructured titanium (Ti), 2) Determine the effects of SSRIs on bone remodeling signaling and osteoclast activation, and 3) Elucidate the effects of SSRIs on serotonin receptors and their effect on bone remodeling. To investigate this, human MSCs were grown on tissue culture polystyrene (TCPS), smooth Ti (PT) or microstructured Ti (SLA) surfaces under exposure to therapeutic concentrations of commonly prescribed antidepressants (SSRIs (fluoxetine, sertraline, paroxetine), Selective Norepinephrine Reuptake Inhibitor (SNRI) (duloxetine) and other regularly prescribed antidepressants (bupropion)) during differentiation toward osteoblasts. Osteoblastic differentiation was assessed in MSCs after treatment with the drugs (0.1μM, 1μM, 10μM) by alkaline phosphatase activity and osteocalcin levels. Antidepressant treatment decreased levels of MSC differentiation markers on microstructured Ti surfaces. Furthermore, treatment dose-dependently decreased protein levels secreted by MSCs which are important for bone formation (BMP2, VEGF, Osteoprotegerin), and increased those involved in bone resorption (RANKL). To determine the effect of SSRIs on bone remodeling signaling and osteoclast activation, human osteoclasts were either directly exposed to antidepressants or conditioned media obtained from MSCs treated with antidepressants on Ti surfaces, after which, enzymatic tartrate-resistant acid phosphatase (TRAP) activity was assessed. Antidepressants increased TRAP activity both directly and through treated MSCs, with the highest levels evident after treatment with conditioned media from MSCs on microstructured Ti surfaces. To elucidate the effects of serotonin receptors and their effect on bone remodeling, receptors were pharmacologically inhibited. Surface roughness decreased gene expression of HTR2A, HTR1B, and HTR2B, and antidepressant treatment increased their expression. Inhibition of HTR2A decreased RANKL protein levels, while inhibition of other serotonin receptors had no effect on RANKL or OPG levels. These studies suggest that antidepressants inhibit MSCs differentiation on microstructured Ti surfaces and increase levels of proteins associated with bone resorption. Additionally, our results showed that RANKL is regulated by serotonin receptor HTR2A. Taken together, our results suggest that antidepressants have a negative effect on osteoblastic differentiation, compromising bone formation and enhancing bone resorption, which can be detrimental to patients under orthopedic and dental treatment

The effect of adding Sn on the mechanical properties and microstructure of the titanium

Ti-XSn (X = 0, 5, 10, 15) (wt. %) alloys were prepared by blending TiH2 and Sn powder compact extrusion. The effect of adding tin (Sn) on the microstructure and mechanical properties of titanium has been investigated by optical microscopy and tensile tests. The lamellar thickness (10 to 6.6 µm) of the Ti reduced after adding Sn and according to the Hall-Petch equation the yield strength (548.2 to 801.3 MPa) and microhardness (233.2 to 310 HV) of the Ti is increased with increasing the tin (Sn) content.</p

Evaluation of the mechanical properties of powder metallurgy Ti-6Al-7Nb alloy

Titanium and its alloys are common biomedical materials owing to their combination ofmechanical properties, corrosion resistance and biocompatibility. Powder metallurgy (PM) techniques can be used to fabricate biomaterials with tailored properties because changing theprocessing parameters, such as the sintering temperature, products with different level ofporosity and mechanical performances can be obtained. This study addresses the productionof the biomedical Ti-6Al-7Nb alloy by means of the master alloy addition variant of the PMblending elemental approach. The sintering parameters investigated guarantee that thecomplete diffusion of the alloying elements and the homogenization of the microstructure isachieved. The sintering of the Ti-6Al-7Nb alloy induces a total shrinkage between 7.4% and10.7% and the level of porosity decreases from 6.2% to 4.7% with the increment of thesintering temperature. Vickers hardness (280-300 HV30) and tensile properties (differentcombination of strength and elongation around 900 MPa and 3%) are achieved.The authors want to acknowledge the financial support from New Zealand Ministry of Business, Innovation and Employment (MBIE) through the UOWX1402 research contract (TiTeNZ - Titanium Technologies New Zealand)

Spiked Nanostructures Disrupt Fungal Biofilm and Impart Increased Sensitivity to Antifungal Treatment

There is a globally increasing demand for medically implanted devices, partly spurred by an aging population. In parallel, there is a proportionate increase in implant associated infection. Much focus has been directed toward the development of techniques to fabricate nanostructured antimicrobial biomaterials to mitigate infection. The present study investigates the interaction of the fungal pathogen Candida albicans with an antimicrobial surface bearing nanoscale protrusions. C. albicans cells were observed to be affected by cell wall stress, which impeded its ability to switch to a hyphal phenotype. There are significant differences in the expression of C. albicans virulence-associated genes between the untreated and nanostructured surfaces. To determine whether the observed inhibition of C. albicans would also sensitize it to antifungal drugs, a culture is established for 3 days on the nanostructured surface before being treated with the antifungal drug amphotericin B. The drug was able to kill all cells on the nanostructured surface at sub-clinical concentrations, while remaining ineffective against cultures grown on a smooth control surface. These findings may eventually prove to be impactful in the clinic, as clinicians may be able to reduce antifungal drug dosages and minimize the effects of drug associated toxicity

Recent developments of metallic implants for biomedical applications

Medical implants have undoubtedly made an indelible mark on our world during the last century. More than 100 million humans carry at least one major internal medical device. The prosthesis industry has topped 50 billion US$ in annual sales, with approximately 150 universities throughout the world proposing an undergraduate program in bioengineering or biomedical engineering. Despite that, however, most medical devices have been constructed using a significantly restricted number of conventional metallic, ceramic, polymeric, and composite biomaterials. In this study, recent developments of metallic implants are summarized for biomedical applications. To do this, first desired properties for biomaterials are defined. Then, types of metallic biomaterials are classified as stainless steel, Mg, Co, Ti, nobble and biodegradable ones. After that, surface modifications are defined for corrugation, topographies and chemical modification. Finally, future perspective is outlined for the sake of development new materials as well as production point of view

Atypical Mesenchymal Stromal Cell Responses to Topographic Modifications of Titanium Biomaterials Indicate Cytoskeletal- and Genetic Plasticity-Based Heterogeneity of Cells

Titanium (Ti) is widely used as a biomaterial for endosseous implants due to its relatively inert surface oxide layer that enables implanted devices the ability of assembling tissue reparative components that culminate in osseointegration. Topographic modifications in the form of micro- and nanoscaled structures significantly promote osseointegration and enhance the osteogenic differentiation of adult mesenchymal stromal cells (MSCs). While the biological mechanisms central to the differential responses of tissues and cells to Ti surface modifications remain unknown, adhesion and morphological adaptation are amongst the earliest events at the cell-biomaterial interface that are highly influenced by surface topography and profoundly impact the regulation of stem cell fate determination. This study correlated the effects of Ti topographic modifications on adhesion and morphological adaptation of human MSCs with phenotypic change. The results showed that modified Ti topographies precluded the adhesion of a subset of MSCs while incurring distinct morphological constraints on adherent cells. These effects anomalously corresponded with a differential expression of stem cell pluripotency and Wnt signalling-associated markers on both modified surfaces while additionally differing between hydrophobic and hydrophilic surface modifications—though extent of osteogenic differentiation induced by both modified topographies yielded similarly significant higher levels of cellular mineralisation in contrast to polished Ti. These results suggest that in the absence of deposited proteins and soluble factors, both modified topographies incur the selective adhesion of a subpopulation of progenitors with relatively higher cytoskeletal plasticity. While the presence of deposited proteins and soluble factors does not significantly affect adherence of cells, nanotopographic modifications enhance expression of pluripotency markers in proliferative conditions, which are conversely overridden by both modified topographies in osteogenic inductive conditions. Further deciphering the mechanisms underlying cellular selectivity and Ti topographic responsiveness will improve our understanding of stem cell heterogeneity and advance the potential of MSCs in regenerative medicine.</jats:p