Novel phosphate-based cements for clinical applications

This Thesis aims at the development of two novel families of inorganic phosphate cements with suitable characteristics for clinical applications in hard tissue regeneration or replacement. It is organized in two distinct parts. The first part focuses at the development of silicon-doped a-tricalcium phosphate and the subsequent preparation of a silicon-doped calcium phosphate cement for bone regeneration applications. For this purpose, silicon-doped a-tricalcium phosphate was synthesized by sintering a calcium-deficient hydroxyapatite at 1250ºC with different amounts of silicon oxide. The high temperature polymorph a-tricalcium phosphate was stabilized by the presence of silicon, which inhibited reversion of the b-a transformation, whereas in the Si-free a-tricalcium phosphate completely reverted to the b-polymorph. It was observed that the presence of Si did not alter the b-a transformation temperature. Both the Si-doped a-tricalcium phosphate and its Si-free counterpart were used as reactants in the formulation of calcium phosphate cements. While Si-doped a-tricalcium phosphate showed faster hydrolysis to calcium deficient hydroxyapatite, the composition, morphology and mechanical properties of both cements were similar upon completion of the reaction. When the samples were immersed in simulated body fluid, the Si-doped cement exhibited a faster deposition of an apatite layer on its surface than its Si-free counterpart, suggesting an enhanced bioactivity of the doped-cement. An in vitro cell culture study, in which osteoblast-like cells were exposed to a medium modified by the materials, showed a delay in cell proliferation and a stimulation of cell differentiation, the differentiation being more marked for the Si-containing cement. These results were attributed to the Ca depletion from the medium by both cements and to the continuous Si release detected for the Si-containing cement. The second part of this Thesis is focused on the development of a new family of inorganic phosphate-based cements for biomedical applications, namely magnesium phosphate cements. The magnesium phosphate cements have been extensively used in civil engineering due to their fast setting, early strength acquisition and adhesive properties, properties that can be also of use for biomedical applications. However, there are some aspects that should be improved before they can be used in the human body, namely their high exothermic setting reaction and the release of potentially harmful ammonium ions. Therefore, a new family of magnesium phosphate cements was explored as candidate biomaterials for hard tissue applications. These cements were prepared by mixing magnesium oxide with either sodium dihydrogen phosphate, ammonium dihydrogen phosphate or an equimolar mixture of both. The exothermia and the setting kinetics of the new cement formulations were tailored. The ammonium-containing magnesium phosphate cements resulted in struvite as the major reaction product, whereas the magnesium phosphate cement prepared with sodium dihydrogen phosphate resulted in an amorphous product. The magnesium phosphate cements studied showed an early compressive strength substantially higher than that of conventional apatitic calcium phosphate cements. Moreover, they showed antimicrobial properties against bacteria present in dental infections, which were attributed to the synergistic effect of a high osmolarity and high pH of the cement extracts. These properties make magnesium phosphate cements good candidates for endodontic applications. It is with this latter point in mind that some of the most relevant physico-chemical properties were further optimized and characterized. Particularly, their radiopacity was enhanced by the addition of bismuth oxide. The sealing efficiency of the magnesium phosphate cements and their adhesion to dentin were shown to be comparable or even higher than those presented by other inorganic cements used for endodontic treatments.Aquesta Tesi té com a objectiu el desenvolupament de dues noves famílies de ciments inorgànics de base fosfat amb propietats adequades per a aplicacions clíniques en regeneració o substitució de teixits durs. La Tesi està organitzada en dues parts. La primera part està centrada en el desenvolupament de fosfat tricàlcic a dopat amb silici i la subseqüent preparació de ciments de fosfat de calci dopats amb silici. Per a aquest objectiu, es va obtenir fosfat tricàlcic a dopat amb silici mitjançant la sinterització d’una hidroxiapatita deficient en calci amb diferents quantitats d’òxid de silici a 1250°C. La presència de silici va estabilitzar el polimorf d’alta temperatura (fosfat tricàlcic a), inhibint-se la reversió de la transformació b-a, mentre que el fosfat tricàlcic a sense silici va revertir completament a polimorf b. La presència de silici no va alterar la temperatura de la transformació b-a. Tant el fosfat tricàlcic a dopat amb silici com el seu homòleg sense silici van ser utilitzats com a reactius en la formulació de ciments de fosfat de calci. Si bé el fosfat tricàlcic a dopat amb silici va mostrar en les fases inicials una hidròlisi més ràpida a hidroxiapatita deficient en calci, un cop completada la reacció, la composició, morfologia i propietats mecàniques d’ambdós ciments van ser similars. L’estudi de bioactivitat mitjançant la immersió de les mostres en fluid corporal simulat va donar com a resultat la formació d’una capa d’apatita a la superfície del ciment dopat amb silici, més ràpida que al seu homòleg sense silici, fet que va suggerir una bioactivitat millorada del ciment dopat. L’estudi in vitro, en el qual cèl·lules osteoblàstiques es van exposar a un medi de cultiu que havia estat prèviament en contacte amb els ciments estudiats, va mostrar un retràs en la proliferació cel·lular i un estímul de la diferenciació cel·lular, aquest últim més marcat pel ciment que contenia silici. Aquests resultats es van atribuir a la reducció de calci en els medis en els quals estaven introduïts els ciments i a l’alliberament continu d’ions silici per part del ciment que en contenia.Postprint (published version

Determinació de radiocarboni d'origen antropogènic

Tots els materials, tant vivents corn inerts, poden contenir traces de radioactivitat natural a causa de l'existència de radionúclids presents a la terra des del moment de la seva formació (series de l'U-235, U-238, Th-232 i els radionúclids independents K-40, Sr-87 i Sm-187) o bé de la formació continua per interacció dels raigs còsmics amb els elements terrestres (H-3, Be-7, C-14). El radiocarboni es produeix segons la reacció nuclear següent: 14 7N+0 1n ---------- 14 6C+ 1 1H i ha mantingut constant la seva proporció en relació amb el carboni total al llarg del temps perquè s'ha establert un equilibri entre la seva formació i la seva desaparició. Darrerament l'home ha alterat aquest equilibri principalment a causa de l'ús de 1'energia nuclear per a diverses finalitats, i cal pensar que en el futur hi haurà un increment de l'ús d'aquest tipus d'energia. Els vegetals incorporen el radiocarboni atmosfèric a llurs teixits a través de la fotosíntesi, d'aquests passa als teixits animals i finalment s'incorpora a l'home, que clou la cadena tròfica. Un augment del contingut de radiocarboni en els dipòsits naturals produeix un increment del contingut de radiocarboni en cadascuna de les baules de la cadena tròfica, i finalment pot causar efectes cancerígens en l'home. Són necessaris, doncs, cada vegada més, el mesurament i el control dels nivells de concentració d'aquest radionúclid. En aquest treball presentem els materials que cal analitzar amb aquesta finalitat, i llur tractament per a l'obtenció de CO2 i posterior transformació d'aquest en benzè per a poder procedir a la determinació de 1'activitat per centelleig líquid. Tamb´r avaluem les condicions òptimes d'aquesta determinació tal corn són dutes a terme en el laboratori instal•lat al Departament de Química Anàlitica de la Universitat de Barcelona

Functionalized silk promotes cell migration into calcium phosphate cements by providing macropores and cell adhesion motifs

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano- and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.Peer ReviewedPostprint (published version

Influence of Si substitution on the reactivity of a-tricalcium phosphate

Silicon substituted calcium phosphates have been widely studied over the last ten years due to their enhanced osteogenic properties. Notwithstanding, the role of silicon on a-TCP reactivity is not clear yet. Therefore, the aim of this work was to evaluate the reactivity and the properties of Si-a-TCP in comparison to a-TCP. Precursor powders have similar properties regarding purity, particle size distribution and specific surface area, which allowed a better comparison of the Si effects on their reactivity and cements properties. Both Si-a-TCP and a-TCP hydrolyzed to a calcium-deficient hydroxyapatite when mixed with water but their conversion rates were different. Si-a-TCP exhibited a slower setting rate than a-TCP, i.e. kSSA for Si-TCP (0.021 g·m- 2·h- 1) was almost four times lower than for a-TCP (0.072 g·m- 2·h- 1). On the other hand, the compressive strength of the CPC resulting from fully reacted Si-a-TCP was significantly higher (12.80 ± 0.38 MPa) than that of a-TCP (11.44 ± 0.54 MPa), due to the smaller size of the entangled precipitated apatite crystals.Preprin

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen-fixing alfalfa

The contribution of carbon and nitrogen reserves to regrowth following shoot removal and the processes involved in the reduction of nodule functioning were studied in alfalfa plants (Medicago sativa L.). To do so, isotopic labelling was conducted at root and canopy level with both 15N2 and 13C-depleted CO2 on exclusively nitrogen fixing alfalfa plants. Our results indicate that the roots were the main sink organs before shoot removal as expected. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules in order to sustain respiratory activity. However, this carbon and nitrogen derived from the root did not overcome carbon and nitrogen limitation in nodules and leaves. Together with the limited carbohydrate availability, the up-regulation of nodule peroxidases indicates the involvement of oxidative stress in a worse nodule performance. Fourteen days after shoot removal, leaf and nodule performance were completely reestablished

Effect of moisture on the reactivity of alpha-tricalcium phosphate

The ability of the high-temperature polymorph of tricalcium phosphate, a-TCP, to hydrolyse to calcium-deficient hydroxyapatite underlies many developments in the field of synthetic bone grafts, including calcium phosphate cements, foams, and self-setting inks. The objective of the present work was to investigate the effect of humidity on a-TCP powder reactivity. The results showed that a 3-week incubation at high relative humidity (80%) had no impact on reactivity, but, as the incubation was prolonged, the powder started to hydrolyse. This reactivity was associated to the presence of defects and to an amorphous phase induced during powder milling. Moisture studies performed under static/dynamic conditions gave comparable water adsorption percentages. The dynamic studies further proved irreversible water sorption, indicating that some water molecules reacted with the powder after short incubation times. Taken together, these results demonstrate that, although a-TCP powder adsorbs water from moisture immediately, the impact on reactivity appears only after several weeks of storage.Peer ReviewedPostprint (published version

In vivo efficiency of antimicrobial inorganic bone grafts in osteomyelitis treatments

Peer ReviewedPostprint (author's final draft