Regulating the antibiotic drug release from ß-tricalcium phosphate ceramics by atmospheric plasma surface engineering

Calcium phosphate (CaP) ceramics are of interest in bone substitution due to their good biocompatibility and bioresorbability. Currently certain CaPs in the market are loaded with antibiotics in order to prevent infections but further control is needed over antibiotic release patterns. Cold plasmas have emerged as a useful means of modifying the interactions with drugs through surface modification of polymer materials. In this work we explore the possibility of using atmospheric pressure plasmas as a tool for the surface modification of these CaP materials with newly populated bonds and charges, with views on enabling higher loading and controlled drug release. Herein the surface modification of ß-tricalcium phosphate ceramics is investigated using an atmospheric pressure helium plasma jet as a tool for tuning the controlled release of the antibiotic doxycycline hyclate, employed as a drug model. The surface chemistry is tailored mainly by plasma jet surface interaction with an increasing O/C ratio without changes in the topography as well as by build-up of surface charges. With this surface tailoring it is demonstrated that the atmospheric plasma jet is a new promising tool that leads to the design of a control for drug release from bioceramic matrices.Peer ReviewedPostprint (author's final draft

Desarrollo y caracterización de un cemento óseo basado en fosfato tricácico para aplicaciones quirúrgicas

Un campo que despierta un interés creciente dentro del ámbito de los biomateriales para la regeneración osea es el de los cementos de fosfatos de calcio. Un cemento de fosfato de calcio consiste en una fase sólida formada por polvos de fosfatos de calcio, y una fase liquida, que al ser mezcladas fraguan a temperatura ambiente o fisiológica, dando lugar a un cuerpo sólido. Junto a la biocompatibilidad y bioactividad de las cerámicas de fosfatos de calcio, los cementos presentan una serie de ventajas especificas, derivadas de su proceso de obtención, como la moldeabilidad y la capacidad de fraguar "in situ", que pueden resolver los problemas de fijación y adaptación planteados por las cerámicas de fosfatos de calcio.En este trabajo se desarrolla y caracteriza un cemento basado en la hidrólisis del fosfato tricalcico alfa. El fraguado del cemento se produce a partir de la disolución de las partículas de fosfato tricalcico y la precipitación de hidroxiapatita deficiente en calcio de baja cristalinidad, similar a las biológicas, según la reacción:3ALFA-CA3(PO4)2+H2O-CA9(HPO4)(PO4)5(OH) existe una relación lineal directa entre la resistencia mecánica del cemento y el porcentaje de fosfato tricalcico que ha reaccionado. Los estudios microestructurales muestran que la resistencia a la comprensión se puede atribuir al entrelazamiento entre los cristales que precipitan.Existen distintas variables de procesado que afectan de forma significativa a las propiedades de fraguado y endurecimiento del cemento. Entre estas cabe destacar el tamaño de partícula del polvo del cemento, la adición de semillas, la utilización de soluciones de Na2HPO4, la relación liquido/polvo empleada y la temperatura. Tras estudiar el efecto de estas variables sobre algunos parámetros del cemento, como los tiempos de fraguado, el tiempo de cohesión, y la velocidad de endurecimiento se investigan los mecanismos a través de los cuales tienen lugar los efectosCalcium phosphate cements have attracted much attention in recent years as bone regeneration materials. A calcium phosphate cement consists of a solid phase formed by calcium phosphate powders and a liquid phase, which after mixing are able to set at room or body temperature, producing a solid body Together with the biocompatibility and bioactivity of the calcium phosphate ceramics, calcium phosphate cements have specific advantages derived from their processing route, such as their mouldability, in situ setting ability, which can solve the fixation and adaptation problems inherent to calcium phosphate ceramics.This PhD Thesis is focused on the development and characterisation of a calcium phosphate cement based on the alpha-tricalcium phosphate (alfa-TCP) hydrolysis. The setting of the cement is produced by the dissolution of the alfa-TCP particles and the precipitation of a low crystallinity calcium deficient hydroxyapatite, similar to the biological hydroxyapatite, according to the following reaction: 3ALFA-CA3(PO4)2+H2O-CA9(HPO4)(PO4)5(OH) Both the degree of reaction and the compressive strength increase initially linearly with time, reaching subsequently a saturation level. A direct relation exists between the mechanical strength and the amount of reacted alfa-TCP. The microstructural analysis show that the cement hardening is caused by the entanglement between the precipitated crystals. The reaction mechanisms which control the reaction kinetics in the different stages of reaction are identified based on X-ray diffraction and electron microscopy studies.The contact of the cement with aqueous solutions which simulate body fluids does not affect its properties, and its setting and hardening behaviour at 37ºC fulfil the clinical requirements.Several processing parameters have been identified which affect significantly the setting and hardening properties of the cement. Among them, the particle size distribution of the starting powder, the addition of seed materials, the use Na2HPO4 solutions and the liquid to powder ratio used, are especially relevant. The effect of these parameters on several cement properties, such as the setting and cohesión times, the hardening rate, the maximum strength, the reaction kinetics and the final microstructure is analysed, and the underlying mechanisms are discussed

Multiple characterization study on porosity and pore structure of calcium phosphate cements

Characterization of the intricate pore structure of calcium phosphate cements is a key step to successfully link the structural properties of these synthetic bone grafts with their most relevant properties, such as in vitro or in vivo behaviour, drug loading and release properties, or degradation over time. This is a challenging task due to the wide range of pore sizes in calcium phosphate cements, compared to most other ceramic biomaterials. This work provides a critical assessment of three different techniques based on different physical phenomena, namely mercury intrusion porosimetry (MIP), Nitrogen sorption, and thermoporometry (TPM) for the detailed characterization of four calcium phosphate cements with different textural properties in terms of total porosity, pore size distribution (PSD), and pore entrance size distribution (PESD). MIP covers a much wider size range than TPM and Nitrogen sorption, offering more comprehensive information at the micrometer level. TPM, and especially Nitrogen sorption, are non-destructive techniques and, although they cover a limited size range, provide complementary information regarding pore structure associated with crystal shape at the nanoscale, recording both PSD and PESD in a single experiment. MIP tended to register smaller sizes, especially at low L/P ratios, due to the network effect, which has a strong influence on the outcome of this technique. Statement of significance The detailed characterisation of the porosity of calcium phosphate cements is of paramount importance, since it is a key parameter influencing some of the most relevant features, like mechanical properties, degradation rate or drug loading and release kinetics. However, this is a challenging task because, once hardened, calcium phosphate cements present an intricate morphology, consisting of a network of precipitated crystals, which generate a high intrinsic micro/nano porosity, with pore sizes covering six orders of magnitude. This work provides for the first time a critical assessment of the advantages and limitations of three different techniques, namely mercury intrusion porosimetry, Nitrogen sorption and Thermoporometry, for the characterisation of the porosity of four calcium phosphate cements with different textural propertiePeer ReviewedPostprint (author's final draft

Heparinization of beta tricalcium phosphate: osteo-immunomodulatory effects

"This is the pre-peer reviewed version of the following article: A. Diez-Escudero, M. Espanol, M. Bonany, X. Lu, C. Persson, M.-P. Ginebra, Adv. Healthcare Mater. 2018, 7, 1700867, which has been published in final form at https://doi.org/10.1002/adhm.201700867. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."Immune cells play a vital role in regulating bone dynamics. This has boosted the interest in developing biomaterials that can modulate both the immune and skeletal systems. In this study, calcium phosphates discs (i.e., beta-tricalcium phosphate, ß-TCP) are functionalized with heparin to investigate the effects on immune and stem cell responses. The results show that the functionalized surfaces downregulate the release of hydrogen peroxide and proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 beta) from human monocytes and neutrophils, compared to nonfunctionalized discs. The macrophages show both elongated and round shapes on the two ceramic substrates, but the morphology of cells on heparinized ß-TCP tends toward a higher elongation after 72 h. The heparinized substrates support rat mesenchymal stem cell (MSC) adhesion and proliferation, and anticipate the differentiation toward the osteoblastic lineage as compared to ß-TCP and control. The coupling between the inflammatory response and osteogenesis is assessed by culturing MSCs with the macrophage supernatants. The downregulation of inflammation in contact with the heparinized substrates induces higher expression of bone-related markers by MSCsPeer ReviewedPostprint (author's final draft

Modulation of release kinetics by plasma polymerization of ampicillin-loaded ß-TCP ceramics

Beta-tricalcium phosphate (ß-TCP) bioceramics are employed in bone repair surgery. Their local implantation in bone defects puts them in the limelight as potential materials for local drug delivery. However, obtaining suitable release patterns fitting the required therapeutics is a challenge. Here, plasma polymerization of ampicillin-loaded ß-TCP is studied for the design of a novel antibiotic delivery system. Polyethylene glycol-like (PEG-like) coating of ß-TCP by low pressure plasma polymerization was performed using diglyme as precursor, and nanometric PEG-like layers were obtained by simple and double plasma polymerization processes. A significant increase in hydrophobicity, and the presence of plasma polymer was visible on the surface by SEM and quantified by XPS. As a main consequence of the plasma polymerisation, the release kinetics were successfully modified, avoiding burst release, and slowing down the initial rate of release leading to a 4.5¿h delay in reaching the same antibiotic release percentage, whilst conservation of the activity of the antibiotic was simultaneously maintained. Thus, plasma polymerisation on the surface of bioceramics may be a good strategy to design controlled drug delivery matrices for local bone therapiesPeer ReviewedPostprint (author's final draft

In vivo resorption of biomimetic hydroxyapatite/collagen composites: injectable cements versus pre-set microspheres

Postprint (published version

In vitro degradation of calcium phosphates: Effect of multiscale porosity, textural properties and composition

The capacity of calcium phosphates to be replaced by bone is tightly linked to their resorbability. However, the relative importance of some textural parameters on their degradation behavior is still unclear. The present study aims to quantify the effect of composition, specific surface area (SSA), and porosity at various length scales (nano-, micro- and macroporosity) on the in vitro degradation of different calcium phosphates. Degradation studies were performed in an acidic medium to mimic the osteoclastic environment. Small degradations were found in samples with interconnected nano- and micropores with sizes below 3 µm although they were highly porous (35–65%), with maximum weight loss of 8 wt%. Biomimetic calcium deficient hydroxyapatite, with high SSA and low crystallinity, presented the highest degradation rates exceeding even the more soluble ß-TCP. A dependence of degradation on SSA was indisputable when porosity and pore sizes were increased. The introduction of additional macroporosity with pore interconnections above 20 µm significantly impacted degradation, more markedly in the substrates with high SSA (>15 m2/g), whereas in sintered substrates with low SSA (<1 m2/g) it resulted just in a linear increase of degradation. Up to 30 % of degradation was registered in biomimetic substrates, compared to 15 % in ß-TCP or 8 % in sintered hydroxyapatite. The incorporation of carbonate in calcium deficient hydroxyapatite did not increase its degradation rate. Overall, the study highlights the importance of textural properties, which can modulate or even outweigh the effect of other features such as the solubility of the compounds. Statement of Significance The physicochemical features of calcium phosphates are crucial to tune biological events like resorption during bone remodeling. Understanding in vitro resorption can help to predict the in vivo behavior. Besides chemical composition, other parameters such as porosity and specific surface area have a strong influence on resorption. The complexity of isolating the contribution of each parameter lies in the close interrelation between them. In this work, a multiscale study was proposed to discern the extent to which each parameter influences degradation in a variety of calcium phosphates, using an acidic medium to resemble the osteoclastic environment. The results emphasize the importance of textural properties, which can modulate or even outweigh the effect of the intrinsic solubility of the compounds.Peer ReviewedPostprint (author's final draft

Modification of hydrogel-based biomaterials by atmospheric pressure plasma to enhance tissue regeneration

Postprint (published version

Rheological characterisation of ceramic inks for 3D direct ink writing: A review

3D printing is a competitive manufacturing technology, which has opened up new possibilities for the fabrication of complex ceramic structures and customised parts. Extrusion-based technologies, also known as direct ink writing (DIW) or robocasting, are amongst the most used for ceramic materials. In them, the rheological properties of the ink play a crucial role, determining both the extrudability of the paste and the shape fidelity of the printed parts. However, comprehensive rheological studies of printable ceramic inks are scarce and may be difficult to understand for non-specialists. The aim of this review is to provide an overview of the main types of ceramic ink formulations developed for DIW and a detailed description of the more relevant rheological tests for assessing the printability of ceramic pastes. Moreover, the key rheological parameters are identified and linked to printability aspects, including the values reported in the literature for different ink compositions.Peer ReviewedPostprint (published version

Correlating rheological properties with direct ink writing printability in hydrogel – calcium phosphate slurries: effect of polymeric and ceramic content

Direct Ink Writing enables the fabrication of personalized scaffolds for bone tissue engineering. The ink must be smoothly extruded through a narrow nozzle without clogging to form continuous filaments, which must retain the nozzle shape and be capable of supporting its own weight during the assembly. This is linked to the viscoelastic properties of the ink, which should have a shear-thinning viscous behaviour at high shear rates, a high storage modulus at rest and a fast elastic recovery when flow stops [1]. The aim of this study was to develop a reliable method that allows linking the rheological properties of a calcium phosphate/hydrogel paste with its printability, analysing the effect of polymeric and ceramic contents. CaP pastes were obtained by mixing Pluronic hydrogels (polymeric content: 20-35 wt.%) with β-TCP powder (ceramic concentration: 50-70 wt.%). The rheological characterisation was carried out in a rotational rheometer, using a 20 mm rough parallel plate geometry and a 500 μm gap. All slurries showed a viscoelastic behaviour with a strong shear- thinning and a fast elastic recovery, both provided by the hydrogel network. The ceramic content affected significantly the properties at low shear rates: the elastic stiffness at rest, the percentage of elastic strength recovery and, above a 60 wt.%, the yield stress. Regarding printability, the filament shape-retention of extrudable pastes was assessed by image analysis of the sagging of a single filament printed over a row of pillars with increasing separation, whereas shape fidelity was evaluated by comparing 3D-printed scaffolds with the virtual model