Intrinsic osteoinduction and osteogenesis of biomimetic calcium phosphate scaffolds with different nano-, micro- and macroporosities : ectopic and orthotopic implantation in a canine model

The development of synthetic bone substitutes with enhanced osteogenic properties is urged by the global ageing population. Sintered calcium-phosphate (CaP) ceramics are the most widely used synthetic biomaterials for bone regeneration. However, their clinical performance is inferior to those of autografts, which are still considered the gold standard, despite the serious drawbacks associated with the need of a harvesting surgery. This thesis aims at providing new insights in the development of CaP biomaterials with osteoinductive properties, this is, with the capacity to foster the differentiation of mesenchymal stem cells to bone forming cells, without the need of adding exogenous growth factors. Previous studies pointed to chemical composition, macropore architecture, microstructural topography and specific surface area (SSA) as critical factors in the intrinsic osteoinduction of biomaterials. However, only sintered ceramics with a limited range of porosities and low SSAs had been analyzed so far. In the present thesis, we were able to extend this range to the nanoscale by using biomimetic low-temperature processing routes. Foaming and 3D-printing methods allowed producing biomimetic CaP scaffolds with tailored macropore architectures together with controlled micro and nanoporosity and, hence, high SSAs. In order to evaluate the intrinsic osteoinduction of this new family of biomimetic bone substitutes, nanostructured calcium deficient hydroxyapatite (CDHA) scaffolds with needle-like crystal morphology were implanted intramuscularly in a canine model, and compared with two sintered ceramics, namely biphasic calcium phosphate and beta-tricalcium phosphate (Study I). The results showed that the high reactivity of nanostructured biomimetic CDHA, combined with a spherical concave macroporosity of foamed scaffolds, accelerated and enhanced the osteoinduction potential beyond the limits of conventional, microstructured, sintered ceramics. As a second step, the effect of macropore geometry of nanostructured CDHA on the bone healing capacity was analyzed. The same foamed and 3D-printed CDHA scaffolds were implanted intraosseoulsy in a canine model (Study II). Whereas nanostructured CDHA was shown to be highly osteoconductive irrespective of macropore geometry, a superior osteogenic capacity was observed in the foamed scaffolds, which correlated well with the higher intrinsic osteoinductive potential demonstrated previously. Moreover, foams showed a higher cell-mediated degradation than the 3D-printed constructs, with a simultaneous and progressive replacement of the scaffold by new bone, demonstrating that the control of macropore architecture allows tuning both material degradation and new bone formation. Finally, aiming to further mimic the natural bone apatite, the effect of nanocrystal morphology (plate vs. needle) and carbonate doping on the intrinsic bioactivity of biomimetic CDHA was investigated. To this end, CDHA foams with different nanostructures (Coarse/Fine-CDHA) and carbonated CDHA foams were compared, both in canine ectopic and orthotopic implantation models (Study III). Fine-CDHA foams showed a superior osteoinduction and bone healing potential, as well as a higher degradation than Coarse-CDHA foams, suggesting that there is a threshold value in terms of SSA necessary to activate the cell-mediated resorption and the associated osteoinduction, which determines in turn the osteogenic capacity of the materials in a bony enviroment. Moreover, carbonate dopping of CDHA accelerated both intrinsic osteoinduction and bone healing, simultaneously increasing the cell-mediated resorption. Thus, the increased biomimetism of CDHA allowed the material to enter the natural bone remodelling cycle, this resulting in a tight synchronization between material degradation and bone formation, and ultimately, obtaining bone substitutes with enhanced bone regeneration potential.L’envelliment global de la població exigeix el desenvolupament de nous substituts ossis sintètics amb capacitats osteogèniques optimitzades. Tot i que les ceràmiques de fosfats de calci (CaP) sinteritzades són els biomaterials sintètics més utilitzats en regeneració òssia, la seva eficiència és inferior a la dels empelts d’ós autòleg, els quals continuen sent el tractament de primera elecció malgrat presentar inconvenients importants associats a la necessitat d’una segona cirurgia. Aquesta tesi té com a objectiu optimitzar el desenvolupament de biomaterials de CaP amb propietats osteoinductives, fet que estimula la diferenciació de cèl·lules mare mesenquimals a cèl·lules osteogèniques, sense l’ús de factors de creixement exògens. Estudis recents han identificat diferents factors crítics en l’osteoinducció intrínseca dels biomaterials com ara la composició química, la macroporositat, la microestructura i la superfície específica (SSA). Fins al moment, només s’han analitzat ceràmiques sinteritzades amb un rang limitat de porositats i SSAs. Tanmateix, en la present tesi s’ha aconseguit augmentar aquest rang a la nanoescala per mitjà de rutes de processament biomimètiques a baixes temperatures. L’escumat i la impressió 3D de CaP biomimètics, ha permès l’obtenció d’implants amb arquitectures macroporoses específicament modulades conjuntament amb micro i nanoporositats controlades, i per tant, amb SSAs significativament superiors. Per tal d'avaluar l'osteoinducció intrínseca d'aquests nous materials biomimètics, es van implantar intramuscularment materials nanoestructurats (cristalls tipus agulla) de hidroxiapatita deficient en calci (CDHA) en un model caní, i es van comparar amb dues ceràmiques sinteritzades (Estudi I). Els resultats van mostrar que la gran reactivitat de la CDHA nanoestructurada, combinada amb una macroporositat esfèrica còncava de les escumes, van incrementar el potencial d'osteoinducció més enllà dels límits oferts per les ceràmiques sinteritzades microestructurades. El segon pas va consistir en l’anàlisi de l'efecte de la geometria de la macroporositat dels materials de CDHA sobre la seva capacitat de consolidació òssia, implantant els materials escumats i els impresos en 3D a nivell intraossi en un model caní (Estudi II). Tot i que la CDHA nanoestructurada va demostrar ser altament osteoconductiva independentment de la geometria macroporosa, les escumes van mostrar una capacitat osteogènica superior, correlacionant-se directament amb el major potencial osteoinductiu intrínsec demostrat anteriorment. A més, les escumes van mostrar una reabsorció cel·lular superior als implants obtinguts per impressió 3D, substituint progressivament el material per nou os i, demostrant així que el control de l'arquitectura de la macroporositat permet adequar tant la degradació del material com fomentar la regeneració òssia. Finalment, amb l'objectiu de mimetitzar encara més la fase mineral òssia, es va investigar l’efecte de la morfologia dels nanocristalls (placa vs. agulla) i del dopatge amb ions carbonat sobre la bioactivitat intrínseca de la CDHA biomimètica, implantant escumes de CDHA amb diferents nanoestructures (Coarse/Fine-CDHA) i escumes carbonatades a nivell ectòpic i ortotòpic en gos (Estudi III). Les escumes Fine-CDHA van mostrar un potencial osteoinductiu i osteogènic superiors, i una degradació incrementada respecte a les escumes Coarse-CDHA, suggerint que existeix una SSA mínima per activar la degradació cel·lular dels materials i la conseqüent resposta osteoinductiva, fet que determina la capacitat osteogènica dels materials en un defecte ossi. La carbonatació de la CDHA va accelerar tant el potencial osteoinductiu i osteogènic, com la degradació cel·lular dels materials, suggerint que l’increment del biomimetisme de la CDHA afavoreix la introducció del material dins del cicle de remodelació òssi

Regeneració residencial : habitatges de Cal Simon a Sant Bartomeu del Grau

Es proposa una intervenció sobre l’edifici d’habitatges de Cal Simon. El seu estat actual suposa un problema pels residents, per la propietat i pel poble de Sant Bartomeu degut a la seva desocupació i el mal envelliment arrel de la fallida de l’empresa de Filats i Texits Puigneró. Es projecta l’enderroc de l’edifici central i la generació d’una pell posterior buscant una millor integració amb les edificacions del poble i una transformació dels habitatges existents, minimitzant així la intervenció sobre el parc edificat. Mitjançant aquesta rehabilitació es pretén potenciar l’habitatge flexible i versàtil per donar resposta a les necessitats actuals del poble

El retorn del llop a Catalunya : anàlisi de les repercussions socials i legals /

Treball presentat a l'assignatura de Deontologia i Veterinària Legal (21223

Microbial community changes induced by Managed Aquifer Recharge activities: linking hydrogeological and biological processes

Managed Aquifer Recharge (MAR) is a technique used worldwide to increase the availability of water resources. We study how MAR modifies microbial ecosystems and its implications for enhancing biodegradation processes to eventually improve groundwater quality. We compare soil and groundwater samples taken from a MAR facility located in NE Spain during recharge (with the facility operating continuously for several months) and after 4 months of no recharge. The study demonstrates a strong correlation between soil and water microbial prints with respect to sampling location along the mapped infiltration path. In particular, managed recharge practices disrupt groundwater ecosystems by modifying diversity indices and the composition of microbial communities, indicating that infiltration favors the growth of certain populations. Analysis of the genetic profiles showed the presence of nine different bacterial phyla in the facility, revealing high biological diversity at the highest taxonomic range. In fact, the microbial population patterns under recharge conditions agree with the intermediate disturbance hypothesis (IDH). Moreover, DNA sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) band patterns revealed the existence of indicator species linked to MAR, most notably Dehalogenimonas sp., Nitrospira sp. and Vogesella sp.. Our real facility multidisciplinary study (hydrological, geochemical and microbial), involving soil and groundwater samples, indicates that MAR is a naturally based, passive and efficient technique with broad implications for the biodegradation of pollutants dissolved in water.Peer ReviewedPostprint (published version

Monitoring induced denitrification during managed aquifer recharge in an infiltration pond

Managed aquifer recharge (MAR) is a well-known technique for improving water quality and increasing groundwater resources. Denitrification (i.e. removal of nitrate) can be enhanced during MAR by coupling an artificial recharge pond with a permeable reactive layer (PRL). In this study, we examined the suitability of a multi-isotope approach for assessing the long-term effectiveness of enhancing denitrification in a PRL containing vegetal compost. Batch laboratory experiments confirmed that the PRL was still able to enhance denitrification two years after its installation in the infiltration pond. At the field scale, changes in redox indicators along a flow path and below the MAR-PRL system were monitored over 21 months during recharge and non-recharge periods. Results showed that the PRL was still releasing non-purgeable dissolved organic carbon five years after its installation. Nitrate concentration coupled with isotopic data collected from the piezometer network at the MAR system indicated that denitrification was occurring in the saturated zone immediately beneath the infiltration pond, where recharged water and native groundwater mix. Furthermore, longer operational periods of the MAR-PRL system increased denitrification extent. Multi-isotope analyses are therefore proved to be useful tools in identifying and quantifying denitrification in MAR-PRL systems

Monitoring induced denitrification during managed aquifer recharge in an infiltration pond

Managed aquifer recharge (MAR) is a well-known technique for improving water quality and increasing groundwater resources. Denitrification (i.e. removal of nitrate) can be enhanced during MAR by coupling an artificial recharge pond with a permeable reactive layer (PRL). In this study, we examined the suitability of a multi-isotope approach for assessing the long-term effectiveness of enhancing denitrification in a PRL containing vegetal compost. Batch laboratory experiments confirmed that the PRL was still able to enhance denitrification two years after its installation in the infiltration pond. At the field scale, changes in redox indicators along a flow path and below the MAR-PRL system were monitored over 21¿months during recharge and non-recharge periods. Results showed that the PRL was still releasing non-purgeable dissolved organic carbon five years after its installation. Nitrate concentration coupled with isotopic data collected from the piezometer network at the MAR system indicated that denitrification was occurring in the saturated zone immediately beneath the infiltration pond, where recharged water and native groundwater mix. Furthermore, longer operational periods of the MAR-PRL system increased denitrification extent. Multi-isotope analyses are therefore proved to be useful tools in identifying and quantifying denitrification in MAR-PRL systems.Peer ReviewedPostprint (author's final draft

Are dominant microbial sub-surface communities affected by water quality and soil characteristics?

Subsurface microorganisms must deal with quite extreme environmental conditions. The lack of light, oxygen, and potentially nutrients are the main environmental stresses faced by subsurface microbial communities. Likewise, environmental disruptions providing an unbalanced positive input of nutrients force microorganisms to adapt to varying conditions, visible in the changes in microbial community diversity. In order to test microbial community adaptation to environmental changes, we performed a study in a surface Managed Aquifer Recharge facility, consisting of a settlement basin (two-day residence time) and an infiltration pond. Data on groundwater hydrochemistry, soil texture, and microbial characterization was compiled from surface water, groundwater, and soil samples at two distinct recharge operation conditions. Multivariate statistics by means of Principal Component Analysis (PCA) was the technique used to map the relevant dimensionality reduced combinations of input variables that properly describe the system behavior. The methodology selected allows including variables of different nature and displaying very different range values. Strong differences in the microbial assemblage under recharge conditions were found, coupled to hydrochemistry and grain-size distribution variables. Also, some microbial groups displayed correlations with either carbon or nitrogen cycles, especially showing abundant populations of denitrifying bacteria in groundwater. A significant correlation was found between Methylotenera mobilis and the concentrations of NO3 and SO4, and also between Vogesella indigofera and the presence of DOC in the infiltrating water. Also, microbial communities present at the bottom of the pond correlated with representative descriptors of soil grain size distribution.Peer ReviewedPostprint (author's final draft

Microbial community changes induced by Managed Aquifer Recharge activities: linking hydrogeological and biological processes

Managed Aquifer Recharge (MAR) is a technique used worldwide to increase the availability of water resources. We study how MAR modifies microbial ecosystems and its implications for enhancing biodegradation processes to eventually improve groundwater quality. We compare soil and groundwater samples taken from a MAR facility located in NE Spain during recharge (with the facility operating continuously for several months) and after 4 months of no recharge. The study demonstrates a strong correlation between soil and water microbial prints with respect to sampling location along the mapped infiltration path. In particular, managed recharge practices disrupt groundwater ecosystems by modifying diversity indices and the composition of microbial communities, indicating that infiltration favors the growth of certain populations. Analysis of the genetic profiles showed the presence of nine different bacterial phyla in the facility, revealing high biological diversity at the highest taxonomic range. In fact, the microbial population patterns under recharge conditions agree with the intermediate disturbance hypothesis (IDH). Moreover, DNA sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) band patterns revealed the existence of indicator species linked to MAR, most notably Dehalogenimonas sp., Nitrospira sp. and Vogesella sp.. Our real facility multidisciplinary study (hydrological, geochemical and microbial), involving soil and groundwater samples, indicates that MAR is a naturally based, passive and efficient technique with broad implications for the biodegradation of pollutants dissolved in water

Generalized tuberculosis due to Mycobacterium caprae in a red fox phylogenetically related to livestock breakdowns

Ajuts: Generalitat de Catalunya. Departament d'Acció Climàtica, Alimentació i Agenda Rural EFA357/INNOTUBTuberculosis (TB) due to Mycobacterium caprae is endemic in goat herds and free-ranging wild boars in Spain, causing infections in other livestock or wild animals to a lesser extent. TB infection in foxes is infrequently reported and they are usually considered spillover hosts of TB

Searching for animal models and potential target species for emerging pathogens : Experience gained from Middle East respiratory syndrome (MERS) coronavirus

Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013-2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in 2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV), which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV), associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species in the transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed