Espumas inyectables de hidroxiapatita obtenidas por el método de espumado de la fase líquida de un cemento de fostato tricálcico alfa

Actualmente existe un gran número de lesiones y enfermedades que afectan el tejido óseo. El origen de estos problemas es diverso y su tratamiento también es diferente en cada caso. Uno de los principales problemas es la pérdida de masa ósea, que puede ser causada por la reabsorción de hueso, fracturas o extirpación de tumores; en estos casos el tratamiento requiere de un material de relleno óseo. Se espera que este material de relleno sea capaz de promover la regeneración de hueso; los materiales que cumplen con este requisito se les denomina injertos óseos. Otras aplicaciones de los injertos óseos son el aumento de hueso para corregir deformaciones o para fijar implantes dentales u ortopédicos. Se estima que en Europa los sustitutos óseos se utilizan en un millón de cirugías anualmente. El mercado mundial de estos materiales se estima en cinco billones de Euros con una tasa de crecimiento anual de 8-15 %.La hidroxiapatita, que corresponde a la fase mineral de hueso y dientes, es uno de los mejores materiales utilizados en el desarrollo de injertos óseos sintéticos. Las bioactividad y osteoconductividad de la hidroxiapatita son la razón de su éxito. Sin embargo, la hidroxiapatita es relativamente estable, por ello, los bloques sólidos no son reabsorbidos a largo plazo. Para incrementar la reabsorción de la hidroxiapatita, y permitir la formación de nuevo hueso hacia su interior, se incorporan macroporos abiertos en el material, mejorando también la osteointegración del implante. Un método para conseguir lo anterior es el espumado de los cementos de fosfatos de calcio; este enfoque tiene la ventaja de que la hidroxiapatita obtenida después del fraguado del cemento es más similar a la hidroxiapatita biológica que las hidroxiapatitas sinterizadas. Además, debido a que los cementos son inyectables cuando son una pasta, es posible obtener injertos óseos inyectables, macroporosos y autoconsolidables. El objetivo de la presente tesis es desarrollar espumas sólidas de hidroxiapatita a través del método de espumado de la fase líquida de un cemento de fosfato de calcio. Se espera que las espumas obtenidas puedan ser utilizadas como injertos óseos inyectables y autoconsolidables bajo condiciones fisiológicas. A lo largo de la tesis se estudian dos tipos de agentes espumantes. En primer lugar, el surfactante no iónico Tween 80 se estudia como agente espumante sintético, en segundo lugar, la gelatina, el alginato de sodio y el extracto de soja se estudian como agentes espumantes naturales. Estos polímeros se proponen como agentes espumantes con la intención de obtener espumas compuestas que imiten la composición y la estructura del nanocompuesto natural que es el tejido óseo.Los resultados mostraron que es posible obtener espumas inyectables de hidroxiapatita empleando contenidos de Tween 80 por debajo de la dosis máxima recomendada en administración parenteral. También se comprobó que la gelatina y el extracto de soja permiten obtener espumas inyectables y autofraguables in situ. Por el contrario, el alginato de sodio no mostró ser un buen agente espumante. Al comparar los dos tipos de agentes espumantes estudiados (sintético y natural) se puede concluir que el espumante sintético Tween 80 es más eficiente que los espumantes naturales; no obstante, la gelatina y el extracto de soja mejoran la Inyectabilidad y/o la cohesión de las espumas, ambas propiedades indispensables para utilizar estos materiales como injertos óseos inyectables y autoconsolidables.Los estudios in vitro e in vivo mostraron que las espumas obtenidas de hidroxiapatita no son toxicas, que pueden ser preparadas bajo condiciones estériles dentro del quirófano sin equipos especiales, y que las espumas seleccionadas tienen cohesión para endurecer in situ bajo condiciones fisiológicas, adaptando la forma y rellenando perfectamente el defecto cuando son implantadas por inyección.Nowadays, there are great number of injuries and illnesses that affect bone tissue. The origin of these diseases is diverse and their treatment is also different in each case. A great problem is bone loss, which can be caused by bone resorption, fractures or bone tumor extirpation; in these cases, a bone filler material will be required. It is expected that the filling material should be able to promote bone healing; materials that meet this properties are known as bone grafts. Other applications of bone grafting materials are the bone mass augmentation to correct deformations or to fix dental and orthopedic implants. It is estimated that in Europe bone substitutes are used in one million surgical procedures annually. The worldwide market of these materials is an estimated of five billion Euros with annual growth rate of 8-15%.One of the most successful materials used in the development of synthetic bone grafting materials is hydroxyapatite, which correspond to the mineral phase of bone and teeth. The bioactivity and the osteoconductivity properties of hydroxyapatite are the reasons of its success. Nevertheless, hydroxyapatite is relatively stable; therefore, solid blocks are not resorbed in the long term. In order to increase the hydroxyapatite resorption, and allow new bone ingrowth, open macropores are incorporated to the material, improving also implant osteointegration. One method to do this is by foaming calcium phosphate cements; this approach has the advantage that the resulting hydroxyapatite after cement setting is more similar to the biological apatite than sintered hydroxyapatite. In addition, since bone cements are injectable when they are a paste, it is possible to develop an injectable, macroporous, and self-setting bone graft.The objective of this thesis was to develop solid hydroxyapatite foams through the liquid phase foaming of a calcium phosphate bone cement. It is expected that the developed foams can be applied as injectable, self-setting, synthetic bone grafts under physiological conditions. Along with this thesis, two types of foaming agent were studied. In first place, non ionic surfactant Tween 80 was tested as syntactic foaming agent; and second, gelatin, sodium alginate and soy extract were tested as natural foaming agents. These biopolymers are proposed as foaming agents with the objective of obtaining composite foams that mimic the composition and structure of the natural nanocomposite that is bone tissue.The results showed that is possible to obtain injectable hydroxyapatite foams using Tween 80 amounts below the maximum dosage recommended for parenteral administration. Also, it was validated that gelatin and soy extract allow obtaining injectable, in situ self-setting foams. In contrast, sodium alginate did not show a good foaming capacity. Comparing the two types of studied foaming agents (synthetic and natural) it can be concluded that the synthetic foaming agent Tween 80 is more efficient than the natural foaming agents; however, gelatin and soy extract improved the injectability and/or the cohesion of the foams, both essential properties to use these materials as injectable and self-setting grafts.The in vitro and in vivo studies showed that the hydroxyapatite foams obtained were not toxic, they could be prepared under sterile conditions in the operating room without special staff, and the selected foams maintained their cohesion to set in situ under physiological conditions, adapting and filling perfectly the shape of the defect when implanted through injection

Focus ion beam/scanning electron microscopy characterization of osteoclastic resorption of calcium phosphate substrates

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low- and high-temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.Peer ReviewedPostprint (author's final draft

Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.Preprin

Brushite foams - the effect of Tween® 80 and Pluronic® F-127 on foam porosity and mechanical properties

Resorbable calcium phosphate based bone void fillers should work as temporary templates for new bone formation. The incorporation of macropores with sizes of 100 -300 µm has been shown to increase the resorption rate of the implant and speed up bone ingrowth. In this work, macroporous brushite cements were fabricated through foaming of the cement paste, using two different synthetic surfactants, Tween® 80 and Pluronic® F-127. The macropores formed in the Pluronic samples were both smaller and less homogeneously distributed compared with the pores formed in the Tween samples. The porosity and compressive strength (CS) were comparable to previously developed hydroxyapatite foams. The cement foam containing Tween, 0.5M citric acid in the liquid, 1 mass% of disodium dihydrogen pyrophosphate mixed in the powder and a liquid to powder ratio of 0.43 mL/g, showed the highest porosity values (76% total and 56% macroporosity), while the CS was >1 MPa, that is, the hardened cement could be handled without rupture of the foamed structure. The investigated brushite foams show potential for future clinical use, both as bone void fillers and as scaffolds for in vitro bone regenerationPeer ReviewedPostprint (author's final draft

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

Extent and mechanism of phase separation during the extrusion of calcium phosphate pastes.

The aim of this study was to increase understanding of the mechanism and dominant drivers influencing phase separation during ram extrusion of calcium phosphate (CaP) paste for orthopaedic applications. The liquid content of extrudate was determined, and the flow of liquid and powder phases within the syringe barrel during extrusion were observed, subject to various extrusion parameters. Increasing the initial liquid-to-powder mass ratio, LPR, (0.4-0.45), plunger rate (5-20 mm/min), and tapering the barrel exit (45°-90°) significantly reduced the extent of phase separation. Phase separation values ranged from (6.22 ± 0.69 to 18.94 ± 0.69 %). However altering needle geometry had no significant effect on phase separation. From powder tracing and liquid content determination, static zones of powder and a non-uniform liquid distribution was observed within the barrel. Measurements of extrudate and paste LPR within the barrel indicated that extrudate LPR remained constant during extrusion, while LPR of paste within the barrel decreased steadily. These observations indicate the mechanism of phase separation was located within the syringe barrel. Therefore phase separation can be attributed to either; (1) the liquid being forced downstream by an increase in pore pressure as a result of powder consolidation due to the pressure exerted by the plunger or (2) the liquid being drawn from paste within the barrel, due to suction, driven by dilation of the solids matrix at the barrel exit. Differentiating between these two mechanisms is difficult; however results obtained suggest that suction is the dominant phase separation mechanism occurring during extrusion of CaP paste

In vivo performance of novel soybean/gelatin-based bioactive and injectable hydroxyapatite foams

Major limitations of calcium phosphate cements (CPCs) are their relatively slow degradation rate and the lack of macropores allowing the ingrowth of bone tissue. The development of self-setting cement foams has been proposed as a suitable strategy to overcome these limitations. In previous work we developed a gelatine-based hydroxyapatite foam (G-foam), which exhibited good injectability and cohesion, interconnected porosity and good biocompatibility in vitro. In the present study we evaluated the in vivo performance of the G-foam. Furthermore, we investigated whether enrichment of the foam with soybean extract (SG-foam) increased its bioactivity. G-foam, SG-foam and non-foamed CPC were implanted in a critical-size bone defect in the distal femoral condyle of New Zealand white rabbits. Bone formation and degradation of the materials were investigated after 4, 12 and 20 weeks using histological and biomechanical methods. The foams maintained their macroporosity after injection and setting in vivo. Compared to non-foamed CPC, cellular degradation of the foams was considerably increased and accompanied by new bone formation. The additional functionalization with soybean extract in the SG-foam slightly reduced the degradation rate and positively influenced bone formation in the defect. Furthermore, both foams exhibited excellent biocompatibility, implying that these novel materials may be promising for clinical application in non-loaded bone defects. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd.Peer ReviewedPostprint (published version

Accelerated hardening of nanotextured 3D-plotted self-setting calcium phosphate inks

Direct ink writing (DIW) techniques open up new possibilities for the fabrication of patient-specific bone grafts. Self-setting calcium phosphate inks, which harden at low temperature, allow obtaining nanostructured scaffolds with biomimetic properties and enhanced bioactivity. However, the slow hardening kinetics hampers the translation to the clinics. Different hydrothermal treatments for the consolidation of DIW scaffolds fabricated with an a-tricalcium phosphate /pluronic F127 ink were explored, comparing them with a biomimetic treatment. Three different scaffold architectures were analysed. The hardening process, associated to the conversion of a-tricalcium phosphate to hydroxyapatite was drastically accelerated by the hydrothermal treatments, reducing the time for complete reaction from 7¿days to 30 minutes, while preserving the scaffold architectural integrity and retaining the nanostructured features. ß-tricalcium phosphate was formed as a secondary phase, and a change of morphology from plate-like to needle-like crystals in the hydroxyapatite phase was observed. The binder was largely released during the treatment. The hydrothermal treatment resulted in a 30% reduction of the compressive strength, associated to the residual presence of ß-tricalcium phosphate. Biomimetic and hydrothermally treated scaffolds supported the adhesion and proliferation of rat mesenchymal stem cells, indicating a good suitability for bone tissue engineering applications. Statement of Significance 3D plotting has opened up new perspectives in the bone regeneration field allowing the customisation of synthetic bone grafts able to fit patient-specific bone defects. Moreover, this technique allows the control of the scaffolds’ architecture and porosity. The present work introduces a new method to harden biomimetic hydroxyapatite 3D-plotted scaffolds which avoids high-temperature sintering. It has two main advantages: i) it is fast and simple, reducing the whole fabrication process from the several days required for the biomimetic processing to a few hours; and ii) it retains the nanostructured character of biomimetic hydroxyapatite and allows controlling the porosity from the nano- to the macroscale. Moreover, the good in vitro cytocompatibility results support its suitability for cell-based bone regeneration therapiesPeer ReviewedPostprint (author's final draft

In vivo efficiency of antimicrobial inorganic bone grafts in osteomyelitis treatments

Peer ReviewedPostprint (author's final draft