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Human mesenchymal stromal cells (hMSCs) seeded on calcium phosphate (CaP) bioceramics are extensively explored in bone tissue engineering and have recently shown effective clinical outcomes. In previous pre-clinical studies, hMSCs-CaP-mediated bone formation was preceded by osteoclastogenesis at the implantation site. The current study evaluates to what extent phase composition of CaPs affects the osteoclast response and ultimately influence bone formation. To this end, four different CaP bioceramics were used, hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP) and two biphasic composites of HA/ß-TCP ratios of 60/40 and 20/80 respectively, for in vitro osteoclast differentiation and correlation with in vivo osteoclastogenesis and bone formation. All ceramics allowed osteoclast formation in vitro from mouse and human precursors, except for pure HA, which significantly impaired their maturation. Ectopic implantation alongside hMSCs in subcutis sites of nude mice revealed new bone formation at 8 weeks in all conditions with relative amounts for ß-TCP > biphasic CaPs > HA. Surprisingly, while hMSCs were essential for osteoinduction, their survival did not correlate with bone formation. By contrast, the degree of early osteoclastogenesis (2 weeks) seemed to define the extent of subsequent bone formation. Together, our findings suggest that the osteoclastic response could be used as a predictive marker in hMSC-CaP-based bone regeneration and strengthens the need to understand the underlying mechanisms for future biomaterial development., This work was financially supported by the European Commission through the H2020 project ORTHOUNION (Grant Agreement: 733288) and by Campus France and the Austrian`s Agency for Education and Internationalisation through the PHC Amadeus 2018 program and the scientific and technological cooperation program “Amadée”. PH received a PhD fellowship from the Regional Council Pays de la Loire and the ORTHOUNION project. MAB received fundings from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Individual Fellowship, Grant Agreement No 708711. MPG and CC acknowledge PID2019-103892RB-I00/AEI/10.13039/501100011033 project from the Spanish Ministry of Science and Innovation and the SGR2017-1165 and the ICREA Academia Awards for Excellence in Research from the Generalitat de Catalunya., Peer Reviewed, Objectius de Desenvolupament Sostenible::3 - Salut i Benestar, Postprint (published version)

The research has demonstrated the antimicrobial properties of plasma urging the incorporation of cold atmospheric plasma (CAP) decontamination in current clinical therapies with the aim to improve the benefits on the patients and on society., Postprint (published version)

Since the discovery that nanostructured surfaces were able to kill bacteria, many works have been published focusing on the design of nanopatterned surfaces with antimicrobial properties. Synthetic bone grafts, based on calcium phosphate (CaP) formulations, can greatly benefit from this discovery if adequate nanotopographies can be developed. However, CaP are reactive materials and experience ionic exchanges when placed into aqueous solutions which may in turn affect cell behaviour and complicate the interpretation of the bactericidal results. The present study explores the bactericidal potential of two nanopillared CaP prepared by hydrolysis of two different sizes of a-tricalcium phosphate (a-TCP) powders under biomimetic or hydrothermal conditions. A more lethal bactericidal response toward Pseudomonas aeruginosa (~75% killing efficiency of adhered bacteria) was obtained from the hydrothermally treated CaP which consisted in a more irregular topography in terms of pillar size (radius: 20–60 nm), interpillar distances (100–1500 nm) and pillar distribution (pillar groups forming bouquets) than the biomimetically treated one (radius: 20–40 nm and interpillar distances: 50–200 nm with a homogeneous pillar distribution). The material reactivity was greatly influenced by the type of medium (nutrient-rich versus nutrient-free) and the presence or not of bacteria. A lower reactivity and superior bacterial attachment were observed in the nutrient-free medium while a lower attachment was observed for the nutrient rich medium which was explained by a superior reactivity of the material paired with the lower tendency of planktonic bacteria to adhere on surfaces in the presence of nutrients. Importantly, the ionic exchanges produced by the presence of materials were not toxic to planktonic cells. Thus, we can conclude that topography was the main contributor to mortality in the bacterial adhesion tests., Peer Reviewed, Postprint (published version)

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation., Peer Reviewed, Postprint (author's final draft)

Biomaterial implantation triggers inflammatory reactions. Understanding the effect of physicochemical features of biomaterials on the release of inflammatory cytokines from immune cells would be of great interest in view of designing bone graft materials to enhance the healing of bone defects. The present work investigated the interactions of two chemically and texturally different calcium phosphate (CaPs) substrates with macrophages, one of the main innate immune cells, and its further impact on osteogenic differentiation of bone forming cells. The behaviour of macrophages seeded on biomimetic calcium deficient hydroxyapatite (CDHA) and sintered ß-tricalcium phosphate (ß-TCP) was assessed in terms of the release of inflammatory cytokines and osteoclastogenic factors. The osteogenic differentiation of bone progenitor cells (bone marrow stromal cells (BMSCs) and osteoblastic cell line (SaOS-2)) were subsequently studied by incubating with the conditioned medium induced by macrophage-CaPs interaction in order to reveal the effect of immune cell reaction to CaPs on osteogenic differentiation. It was found that the incubation of macrophages with CaPs substrates caused a decrease of pro-inflammatory cytokines, more pronounced for ß-TCP compared with CDHA showing significantly decreased IL-6, TNF-a, and iNOS. However, the macrophage-CDHA interaction resulted in a more favourable environment for osteogenic differentiation of osteoblasts with more collagen type I production and osteogenic genes (Runx2, BSP) expression, suggesting that osteogenic differentiation of bone cells is not only determined by the nature of biomaterials, but also significantly influenced by the inflammatory environment generated by the interaction of immune cells and biomaterials., Peer Reviewed, Postprint (author's final draft)

Calcium phosphate (CaP) substrates are successfully used as bone grafts due to their osteogenic properties. However, the influence of the physicochemical features of CaPs in angiogenesis is frequently neglected despite it being a crucial process for bone regeneration. The present work focuses on analyzing the effects of textural parameters of biomimetic calcium deficient hydroxyapatite (CDHA) and sintered beta-tricalcium phosphate (ß-TCP), such as specific surface area, surface roughness, and microstructure, on the behavior of rat endothelial progenitor cells (rEPCs) and their crosstalk with rat mesenchymal stem cells (rMSCs). The higher reactivity of CDHA results in low proliferation rates in monocultured and cocultured systems. This effect is especially pronounced for rMSCs alone, and for CDHA with a fine microstructure. In terms of angiogenic and osteogenic gene expressions, the upregulation of particular genes is especially enhanced for needle-like CDHA compared to plate-like CDHA and ß-TCP, suggesting the importance not only of the chemistry of the substrate, but also of its textural features. Moreover, the coculture of rEPCs and rMSCs on needle-like CDHA results in early upregulation of osteogenic modulator, i.e., protein deglycase 1 might be a possible cause of overexpression of osteogenic-related genes on the same substrate., Peer Reviewed, Postprint (published version)

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7¿cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines., Peer Reviewed, Postprint (author's final draft)

Aplicat embargament des de la data de defensa fins al 31 de juliol de 2019, Bone has a natural ability to regenerate. However, it cannot bridge large defects without the additional help of a supporting material. Among the different synthetic bone grafts, calcium phosphates (CaPs) are excellent candidates for bone regeneration due to their close resemblance to the mineral phase of bone, as well as the bioactive and osteoinductive potential. The present thesis explores the effect of the physicochemical features of CaPs on the interaction with the cells involved in the different stages of the bone healing process: inflammation, angiogenesis and osteogenesis, paying special attention to biomimetic calcium deficient hydroxyapatite (CDHA). This is analysed through in vitro cell cultures with immune, endothelial and bone forming cells, respectively. An overview of bone biology and bone healing process is presented in Chapter 1. Moreover, the Chapter 1 describes synthetic bone grafts based on CaPs and the impact of their physicochemical features on cellular behaviour in vitro. Chapter 2 and Chapter 3 study the response of immune cells to calcium phosphates and its outcome on osteogenic differentiation of bone forming cells. Specifically, Chapter 2 explores the link between nanotopography, specific surface area and porosity of biomimetic CDHA and its immunomodulatory, osteoimmunomodulatory and antinflammatory features, demonstrating that neddle- like topography of CDHA stimulated the osteogenic activity of bone forming cells, whilst a reduction of porosity to CDHA substrates decreased the inflammatory state. Chapter 3 is devoted to study the interaction of two chemically and texturally different calcium phosphates i.e. biomimetic CDHA and sintered ß-tricalcium phosphate (ß-TCP) with macrophages under inflammatory environment and its further impact on osteogenic differentiation of mesenchymal (MSCs) and osteoblastic cells. Angiogenesis ensures the availability of oxygen and nutrients and controls the recruitment and osteogenic differentiation of bone, El hueso tiene una capacidad natural para regenerarse. Sin embargo, no puede restaurar grandes defectos sin la ayuda adicional de un material de soporte. Entre los distintos injertos óseos sintéticos, los fosfatos de calcio son excelentes candidatos para la regeneración ósea debido a su gran parecido con la fase mineral del hueso, así como también a su potencial bioactivo y osteoinductor. La presente tesis explora el efecto de las características fisicoquímicas de los fosfatos de calcio en la interacción con las células involucradas en las diferentes etapas del proceso de regeneración ósea, es decir, inflamación, angiogénesis y osteogénesis, prestando atención especial a la hidroxiapatita biomimética deficiente en calcio (CDHA). Dicho efecto se analiza mediante el cultivo celular in vitro de células inmunológicas, endoteliales y formadoras del hueso, respectivamente. En el Capítulo 1 se presenta una descripción general de la biología ósea y el proceso de regeneración ósea. Además, el Capítulo 1 describe el estado del arte respecto a los injertos óseos basados en fosfatos de calcio y el impacto de sus características fisicoquímicas en el comportamiento celular in vitro. En el Capítulo 2 y el Capítulo 3 se estudia la respuesta de las células inmunológicas a los fosfatos de calcio y su implicación en la diferenciación osteogénica de las células formadoras de hueso. Específicamente, el Capítulo 2 explora el vínculo entre la nanotopografía, la superficie específica (SSA) y la porosidad de la CDHA biomimética y sus características inmunomoduladoras, osteoinmunomoduladoras y antiinflamatorias, demostrando que la CDHA con topografía en forma de aguja estimuló la actividad osteogénica de las células formadoras de hueso, mientras que una reducción en la porosidad de los sustratos CDHA resultó en una disminución del estado inflamatorio. El Capítulo 3 está dedicado a estudiar la interacción de dos fosfatos de calcio químicamente y texturalmente diferentes, es decir, la CDHA bio, Postprint (published version)

Biomaterials can interact with cells directly, that is, by direct contact of the cells with the material surface, or indirectly, through soluble species that can be released to or uptaken from the surrounding fluids. However, it is difficult to characterise the relevance of this fluid-mediated interaction separately from the topography and composition of the substrate, because they are coupled variables. These fluid-mediated interactions are amplified in the case of highly reactive calcium phosphates (CaPs) such as biomimetic calcium deficient hydroxyapatite (CDHA), particularly in static in vitro cultures. The present work proposes a strategy to decouple the effect of ion exchange from topographical features by adjusting the volume ratio between the cell culture medium and biomaterial (VCM/VB). Increasing this ratio allowed mitigating the drastic ionic exchanges associated to the compositional changes experienced by the material exposed to the cell culture medium. This strategy was validated using rat mesenchymal stem cells (rMSCs) cultured on CDHA and beta-tricalcium phosphate (ß-TCP) discs using different VCM/VB ratios. Whereas in the case of ß-TCP the cell response was not affected by this ratio, a significant effect on cell adhesion and proliferation was found for the more reactive CDHA. The ionic exchange, produced by CDHA at low VCM/VB, altered cell adhesion due to the reduced number of focal adhesions, caused cell shrinkage and further rMCSs apoptosis. This was mitigated when using a high VCM/VB, which attenuated the changes of calcium and phosphate concentrations in the cell culture medium, resulting in rMSCs spreading and a viability over time. Moreover, rMSCs showed an earlier expression of osteogenic genes on CDHA compared to sintered ß-TCP when extracellular calcium fluctuations were reduced. Statement of Significance Fluid mediated interactions play a significant role in the bioactivity of calcium phosphates. Ionic exchange is amplified in the case of, Peer Reviewed, Postprint (author's final draft)

The fabrication of calcium phosphates using biomimetic routes, namely, precipitation processes at body temperature, results in distinct features compared to conventional sintered calcium phosphate ceramics, such as a high specific surface area (SSA) and micro- or nanometric crystal size. The aim of this article is to analyze the effects of these parameters on cell response, focusing on two bone cell types: rat mesenchymal stem cells (rMSCs) and human osteoblastic cells (SaOS-2). Biomimetic calcium-deficient hydroxyapatite (CDHA) was obtained by a low temperature setting reaction, and -tricalcium phosphate (-TCP) and -tricalcium phosphate were subsequently obtained by sintering CDHA either at 1400 degrees C or 1100 degrees C. Sintered stoichiometric hydroxyapatite (HA) was also prepared using ceramic routes. The materials were characterized in terms of SSA, skeletal density, porosity, and pore size distribution. SaOS-2 cells and rMSCs were seeded either directly on the surfaces of the materials or on glass coverslips subsequently placed on top of the materials to expose the cells to the CaP-induced ionic changes in the culture medium, while avoiding any topography-related effects. CDHA produced higher ionic fluctuations in both cell culture media than sintered ceramics, with a strong decrease of calcium and a release of phosphate. Indirect contact cell cultures revealed that both cell types were sensitive to these ionic modifications, resulting in a decrease in proliferation rate, more marked for CDHA, this effect being more pronounced for rMSCs. In direct contact cultures, good cell adhesion was found on all materials, but, while cells were able to proliferate on the sintered calcium phosphates, cell number was significantly reduced with time on biomimetic CDHA, which was associated to a higher percentage of apoptotic cells. Direct contact of the cells with biomimetic CDHA resulted also in a higher alkaline phosphatase activity for both cell types compared to sinter

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics., Peer Reviewed, Postprint (author's final draft)

© Copyright 2017, Mary Ann Liebert, Inc.The fabrication of calcium phosphates using biomimetic routes, namely, precipitation processes at body temperature, results in distinct features compared to conventional sintered calcium phosphate ceramics, such as a high specific surface area (SSA) and micro-or nanometric crystal size. The aim of this article is to analyze the effects of these parameters on cell response, focusing on two bone cell types: rat mesenchymal stem cells (rMSCs) and human osteoblastic cells (SaOS-2). Biomimetic calcium-deficient hydroxyapatite (CDHA) was obtained by a low temperature setting reaction, and a-Tricalcium phosphate (a-TCP) and ß-Tricalcium phosphate were subsequently obtained by sintering CDHA either at 1400°C or 1100°C. Sintered stoichiometric hydroxyapatite (HA) was also prepared using ceramic routes. The materials were characterized in terms of SSA, skeletal density, porosity, and pore size distribution. SaOS-2 cells and rMSCs were seeded either directly on the surfaces of the materials or on glass coverslips subsequently placed on top of the materials to expose the cells to the CaP-induced ionic changes in the culture medium, while avoiding any topography-related effects. CDHA produced higher ionic fluctuations in both cell culture media than sintered ceramics, with a strong decrease of calcium and a release of phosphate. Indirect contact cell cultures revealed that both cell types were sensitive to these ionic modifications, resulting in a decrease in proliferation rate, more marked for CDHA, this effect being more pronounced for rMSCs. In direct contact cultures, good cell adhesion was found on all materials, but, while cells were able to proliferate on the sintered calcium phosphates, cell number was significantly reduced with time on biomimetic CDHA, which was associated to a higher percentage of apoptotic cells. Direct contact of the cells with biomimetic CDHA resulted also in a higher alkaline phosphatase activity for both cel, Peer Reviewed, Postprint (author's final draft)