Antibiotic delivery from bone-targeted mesoporous silica nanoparticles for the treatment of osteomyelitis caused by methicillin-resistant Staphylococcus aureus.

Osteomyelitis is a hard-to-treat infection of the bone and bone marrow that is mainly caused by Staphylococcus aureus, with an increasing incidence of methicillin-resistant S. aureus (MRSA). Owing to the aggressiveness of these bacteria in colonizing and destroying the bone, systemic antibiotic treatments fail to eradicate the infection. Instead, it normally entails surgery to remove the dead or infected bone. In this work, we report bone-targeted mesoporous silica nanoparticles for the treatment of osteomyelitis. The nanoparticles have been engineered with a functional gelatine/colistin coating able to hamper premature release from the mesopores while effectively disaggregating the bacterial biofilm. Because antibiotic resistance is a global emergency, we have designed two sets of identical nanoparticles, carrying each of them a clinically relevant antibiotic, that have demonstrated to have synergistic effect. The bone-targeted nanoparticles have been thoroughly evaluated in vitro and in vivo, obtaining a notable reduction of the amount of bacteria in the bone in just 24 h after only one dose, and paving the way for localized, nanoparticle-mediated treatment of MRSA-caused osteomyelitis. STATEMENT OF SIGNIFICANCE: In this work, we propose the use of bone-targeted mesoporous silica nanoparticles to address S. aureus-caused osteomyelitis that render synergistic therapeutic effect via multidrug delivery. Because the bacterial biofilm is responsible for an aggressive surgical approach and prolonged antibiotic treatment, the nanoparticles have been functionalized with a functional coating able to both disaggregate the biofilm, hamper premature antibiotic release and protect the intact bone. These engineered nanoparticles are able to effectively target bone tissue both in vitro and in vivo, showing high biocompatibility and elevated antibacterial effect.

Injectable mesoporous bioactive nanoparticles regenerate bone tissue under osteoporosis conditions.

The osteogenic capability of mesoporous bioactive nanoparticles (MBNPs) in the SiO2CaO system has been assessed in vivo using an osteoporotic rabbit model. MBNPs have been prepared using a double template method, resulting in spherical nanoparticles with a porous core-shell structure that has a high surface area and the ability to incorporate the anti-osteoporotic drug ipriflavone. In vitro expression of the pro-inflammatory genes NF-κB1, IL-6, TNF-α, P38 and NOS2 in RAW-264.7 macrophages, indicates that these nanoparticles do not show adverse inflammatory effects. An injectable system has been prepared by suspending MBNPs in a hyaluronic acid-based hydrogel, which has been injected intraosseously into cavitary bone defects in osteoporotic rabbits. The histological analyses evidenced that MBNPs promote bone regeneration with a moderate inflammatory response. The incorporation of ipriflavone into these nanoparticles resulted in a higher presence of osteoblasts and enhanced angiogenesis at the defect site, but without showing significant differences in terms of new bone formation. STATEMENT OF SIGNIFICANCE: Mesoporous bioactive glass nanoparticles have emerged as one of the most interesting materials in the field of bone regeneration therapies. For the first time, injectable mesoporous bioactive nanoparticles have been tested in vivo using an osteoporotic animal model. Our findings evidence that MBG nanoparticles can be loaded with an antiosteoporotic drug, ipriflavone, and incorporated in hyaluronic acid to make up an injectable hydrogel. The incorporation of MBG nanoparticles promotes bone regeneration even under osteoporotic conditions, whereas the presence of IP enhances angiogenesis as well as the presence of osteoblast cells lining in the newly formed bone. The injectable device presented in this work opens new possibilities for the intraosseous treatment of osteoporotic bone using minimally invasive surgery.

Arabic gum plus colistin coated moxifloxacin-loaded nanoparticles for the treatment of bone infection caused by Escherichia coli.

Osteomyelitis is an inflammatory process of bone and bone marrow that may even lead to patient death. Even though this disease is mainly caused by Gram-positive organisms, the proportion of bone infections caused by Gram-negative bacteria, such as Escherichia coli, has significantly increased in recent years. In this work, mesoporous silica nanoparticles have been employed as platform to engineer a nanomedicine able to eradicate E. coli- related bone infections. For that purpose, the nanoparticles have been loaded with moxifloxacin and further functionalized with Arabic gum and colistin (AG+CO-coated MX-loaded MSNs). The nanosystem demonstrated high affinity toward E. coli biofilm matrix, thanks to AG coating, and marked antibacterial effect because of the bactericidal effect of moxifloxacin and the disaggregating effect of colistin. AG+CO-coated MX-loaded MSNs were able to eradicate the infection developed on a trabecular bone in vitro and showed pronounced antibacterial efficacy in vivo against an osteomyelitis provoked by E. coli. Furthermore, AG+CO-coated MX-loaded MSNs were shown to be essentially non-cytotoxic with only slight effect on cell proliferation and mild hepatotoxicity, which might be attributed to the nature of both antibiotics. In view of these results, these nanoparticles may be considered as a promising treatment for bone infections caused by enterobacteria, such as E. coli, and introduce a general strategy against bone infections based on the implementation of antibiotics with different but complementary activity into a single nanocarrier. STATEMENT OF SIGNIFICANCE: In this work, we propose a methodology to address E.coli bone infections by using moxifloxacin-loaded mesoporous silica nanoparticles coated with Arabic gum containing colistin (AG+CO-coated MX-loaded MSNs). The in vitro evaluation of this nanosystem demonstrated high affinity toward E. coli biofilm matrix thanks to the Arabic gum coating, a disaggregating and antibacterial effect of colistin, and a remarkable antibiofilm action because of the bactericidal ability of moxifloxacin and colistin. This anti-E. coli capacity of AG+CO-coated MX-loaded MSNs was brought out in an in vivo rabbit model of osteomyelitis where the nanosystem was able to eradicate more than 90% of the bacterial load within the infected bone.

Response of RAW 264.7 and J774A.1 macrophages to particles and nanoparticles of a mesoporous bioactive glass: A comparative study.

Mesoporous bioactive glasses (MBGs) are bioceramics designed to induce bone tissue regeneration and very useful materials with the ability to act as drug delivery systems. MBGs can be implanted in contact with bone tissue in different ways, as particulate material, in 3D scaffolds or as nanospheres. In this work, we assessed the effects of particles of mesoporous bioactive glass MBG-75S and mesoporous nanospheres NanoMBG-75S on RAW 264.7 and J774A.1 macrophages, which present different sensitivity and are considered as ideal models for the study of innate immune response. After evaluating several cellular parameters (morphology, size, complexity, proliferation, cell cycle and intracellular content of reactive oxygen species), the action of MBG-75S particles and NanoMBG-75S on the polarization of these macrophages towards the pro-inflammatory (M1) or reparative (M2) phenotype was determined by the expression of specific M1 (CD80) and M2 (CD206, CD163) markers. We previously measured the adsorption of albumin and fibrinogen on MBG-75S particles and the production of pro-inflammatory cytokines as TNF-α and IL-6 by macrophages in response to these particles. This comparative study demonstrates that particles of mesoporous bioactive glass MBG-75S and mesoporous nanospheres NanoMBG-75S allow the appropriated development and function of RAW 264.7 and J774A.1 macrophages and do not induce polarization towards the M1 pro-inflammatory phenotype. Therefore, considering that these mesoporous biomaterials offer the possibility of loading drugs into their pores, the results obtained indicate their high potential for use as drug-delivery systems in bone repair and osteoporosis treatments without triggering an adverse inflammatory response.

Mesoporous bioactive glasses for regenerative medicine.

Stem cells are the central element of regenerative medicine (RM). However, in many clinical applications, the use of scaffolds fabricated with biomaterials is required. In this sense, mesoporous bioactive glasses (MBGs) are going to play an important role in bone regeneration because of their striking textural properties, quick bioactive response, and biocompatibility. As other bioactive glasses, MBGs are mainly formed by silicon, calcium, and phosphorus oxides whose ions play an important role in cell proliferation as well as in homeostasis and bone remodeling process. A common improvement of bioactive glasses for RM is by adding small amounts of oxides of elements that confer them additional biological capacities, including osteogenic, angiogenic, antibacterial, anti-inflammatory, hemostatic, or anticancer properties. Moreover, MBGs are versatile in terms of the different ways in which they can be processed, such as scaffolds, fibers, coatings, or nanoparticles. MBGs are unique because their textural properties are so high that they still exhibit outstanding bioactive responses even after adding extra inorganic ions or being processed as scaffolds or nanoparticles. Moreover, they can be further improved by loading with biomolecules, drugs, and stem cells. This article reviews the state of the art and future perspectives of MBGs in the field of RM of hard tissues.

Effects of mesoporous SiO2-CaO nanospheres on the murine peritoneal macrophages/Candidaalbicans interface.

The use of nanoparticles for intracellular drug delivery could reduce the toxicity and side effects of the drug but, the uptake of these nanocarriers could induce adverse effects on cells and tissues after their incorporation. Macrophages play a central role in host defense and are responsible for in vivo nanoparticle trafficking. Assessment of their defense capacity against pathogenic micro-organisms after nanoparticle uptake, is necessary to prevent infections associated with nanoparticle therapies. In this study, the effects of hollow mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) on the function of peritoneal macrophages was assessed by measuring their ability to phagocytize Candidaalbicans expressing a red fluorescent protein. Two macrophage/fungus ratios (MOI1 and MOI5) were used and two experimental strategies were carried out: a) pretreatment of macrophages with FITC-NanoMBGs and subsequent fungal infection; b) competition assays after simultaneous addition of fungus and nanospheres. Macrophage pro-inflammatory phenotype markers (CD80 expression and interleukin 6 secretion) were also evaluated. Significant decreases of CD80+ macrophage percentage and interleukin 6 secretion were observed after 30 min, indicating that the simultaneous incorporation of NanoMBG and fungus favors the macrophage non-inflammatory phenotype. The present study evidences that the uptake of these nanospheres in all the studied conditions does not alter the macrophage function. Moreover, intracellular FITC-NanoMBGs induce a transitory increase of the fungal phagocytosis by macrophages at MOI 1 and after a short time of interaction. In the competition assays, as the intracellular fungus quantity increased, the intracellular FITC-NanoMBG content decreased in a MOI- and time-dependent manner. These results have confirmed that macrophages clearly distinguish between inert material and the live yeast in a dynamic intracellular incorporation. Furthermore, macrophage phagocytosis is a critical determinant to know their functional state and a valuable parameter to study the nanomaterial / macrophages / Candida albicans interface.

ZnO-mesoporous glass scaffolds loaded with osteostatin and mesenchymal cells improve bone healing in a rabbit bone defect.

The use of 3D scaffolds based on mesoporous bioactive glasses (MBG) enhanced with therapeutic ions, biomolecules and cells is emerging as a strategy to improve bone healing. In this paper, the osteogenic capability of ZnO-enriched MBG scaffolds loaded or not with osteostatin (OST) and human mesenchymal stem cells (MSC) was evaluated after implantation in New Zealand rabbits. Cylindrical meso-macroporous scaffolds with composition (mol %) 82.2SiO2-10.3CaO-3.3P2O5-4.2ZnO (4ZN) were obtained by rapid prototyping and then, coated with gelatin for easy handling and potentiating the release of inorganic ions and OST. Bone defects (7.5 mm diameter, 12 mm depth) were drilled in the distal femoral epiphysis and filled with 4ZN, 4ZN + MSC, 4ZN + OST or 4ZN + MSC + OST materials to evaluate and compare their osteogenic features. Rabbits were sacrificed at 3 months extracting the distal third of bone specimens for necropsy, histological, and microtomography (µCT) evaluations. Systems investigated exhibited bone regeneration capability. Thus, trabecular bone volume density (BV/TV) values obtained from µCT showed that the good bone healing capability of 4ZN was significantly improved by the scaffolds coated with OST and MSC. Our findings in vivo suggest the interest of these MBG complete systems to improve bone repair in the clinical practice.

Development and evaluation of copper-containing mesoporous bioactive glasses for bone defects therapy.

Mesoporous bioactive glasses (MBGs) are gaining increasing interest in the design of new biomaterials for bone defects treatment. An important research trend to enhance their biological behavior is the inclusion of moderate amounts of oxides with therapeutical action such as CuO. MBGs with composition (85-x)SiO2-10-CaO-5P2O5-xCuO (x = 0, 2.5 or 5 mol-%) were synthesized, investigating the influence of the CuO content and some synthesis parameters in their properties. Two series were developed; first one used HCl as catalyst and chlorides as CaO and CuO precursors, second one, used HNO3 and nitrates. MBGs of chlorides family exhibited calcium/copper phosphate nanoparticles between 10 and 20 nm in size. Nevertheless, CuO-containing MBGs of nitrates family showed metallic copper nanoparticles larger than 50 nm as well as quicker in vitro bioactive responses. Thus, MBGs of the nitrate series were coated by an apatite-like layer after 24 h soaked in simulated body fluid (SBF) a remarkably short period for a MBG containing 5% of CuO. A model, focused in the location of copper in the glass network, was proposed to relate nanostructure and in vitro behaviour. Moreover, after 24 h soaked in MEM or THB culture media, all the MBGs released therapeutic amounts of Ca2+ and Cu2+ ions. Because the quick bioactive response in SBF, the capacity to host biomolecules in their pores and to release therapeutic concentrations of Ca2+ and Cu2+ ions, MBGs of the nitrate families are proposed as excellent biomaterials for bone regeneration.

Multifunctional antibiotic- and zinc-containing mesoporous bioactive glass scaffolds to fight bone infection.

Bone regeneration is a clinical challenge which requires multiple approaches. Sometimes, it also includes the development of osteogenic and antibacterial biomaterials to treat the emergence of possible infection processes arising from surgery. This study evaluates the antibacterial properties of gelatin-coated meso-macroporous scaffolds based on the bioactive glass 80%SiO2-15%CaO-5%P2O5 (mol-%) before (BL-GE) and after being doped with 4% of ZnO (4ZN-GE) and loaded with both saturated and the minimal inhibitory concentrations of one of the antibiotics: levofloxacin (LEVO), vancomycin (VANCO), rifampicin (RIFAM) or gentamicin (GENTA). After physical-chemical characterization of materials, release studies of inorganic ions and antibiotics from the scaffolds were carried out. Moreover, molecular modelling allowed determining the electrostatic potential density maps and the hydrogen bonds of antibiotics and the glass matrix. Antibacterial in vitro studies (in planktonic, inhibition halos and biofilm destruction) with S. aureus and E. coli as bacteria models showed a synergistic effect of zinc ions and antibiotics. The effect was especially noticeable in planktonic cultures of S. aureus with 4ZN-GE scaffolds loaded with VANCO, LEVO or RIFAM and in E. coli cultures with LEVO or GENTA. Moreover, S. aureus biofilms were completely destroyed by 4ZN-GE scaffolds loaded with VANCO, LEVO or RIFAM and the E. coli biofilm total destruction was accomplished with 4ZN-GE scaffolds loaded with GENTA or LEVO. This approach could be an important step in the fight against microbial resistance and provide needed options for bone infection treatment. STATEMENT OF SIGNIFICANCE: Antibacterial capabilities of scaffolds based on mesoporous bioactive glasses before and after adding a 4% ZnO and loading with saturated and minimal inhibitory concentrations of levofloxacin, vancomycin, gentamicin or rifampicin were evaluated. Staphylococcus aureus and Escherichia coli were the infection model strains for the performed assays of inhibition zone, planktonic growth and biofilm. Good inhibition results and a synergistic effect of zinc ions released from scaffolds and antibiotics were observed. Thus, the amount of antibiotic required to inhibit the bacterial planktonic growth was substantially reduced with the ZnO inclusion in the scaffold. This study shows that the ZnO-MBG osteogenic scaffolds are multifunctional tools in bone tissue engineering because they are able to fight bacterial infections with lower antibiotic dosage.

Silicon substituted hydroxyapatite/VEGF scaffolds stimulate bone regeneration in osteoporotic sheep.

Silicon-substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to optimize their bone regeneration properties, we have manufactured these scaffolds presenting different microstructures: nanocrystalline and crystalline. Moreover, their surfaces have been decorated with vascular endothelial growth factor (VEGF) to evaluate the potential coupling between vascularization and bone regeneration. In vitro cell culture tests evidence that nanocrystalline SiHA hinders pre-osteblast proliferation, whereas the presence of VEGF enhances the biological functions of both endothelial cells and pre-osteoblasts. The bone regeneration capability has been evaluated using an osteoporotic sheep model. In vivo observations strongly correlate with in vitro cell culture tests. Those scaffolds made of nanocrystalline SiHA were colonized by fibrous tissue, promoted inflammatory response and fostered osteoclast recruitment. These observations discard nanocystalline SiHA as a suitable material for bone regeneration purposes. On the contrary, those scaffolds made of crystalline SiHA and decorated with VEGF exhibited bone regeneration properties, with high ossification degree, thicker trabeculae and higher presence of osteoblasts and blood vessels. Considering these results, macroporous scaffolds made of SiHA and decorated with VEGF are suitable bone grafts for regeneration purposes, even in adverse pathological scenarios such as osteoporosis. STATEMENT OF SIGNIFICANCE: For the first time, the in vivo behavior of scaffolds made of silicon substituted hydroxyapatites (SiHA) has been evaluated under osteoporosis conditions. In order to optimize the bone regeneration properties of these bioceramics, 3D macroporous scaffolds have been manufactured by robocasting and implanted in osteoporotic sheep. Our experimental design shed light on the important issue of the biological response of nano-sized bioceramics vs highly crystalline bioceramics, as well as on the importance of coupling vascularization and bone growth processes by decorating SiHA scaffolds with vascular endothelial growth factor.

Cerium (III) and (IV) containing mesoporous glasses/alginate beads for bone regeneration: Bioactivity, biocompatibility and reactive oxygen species activity.

A very small number of biomaterials investigated for bone regeneration were reported as able to prevent the oxidative stress. In this study beads based on alginate hydrogel and mesoporous glasses (MG) containing different amounts of cerium oxides (Ce3+/Ce4+) exhibiting antioxidant properties were investigated as a good approach to mimic the action of antioxidant enzymes in our organism. The effect of cerium contents on the bioactivity and biocompatibility of beads were investigated. Moreover, the potential capability of Ce-containing MG to prevent the oxidative stress caused by the activity of reactive oxygen species (ROS) was here investigated for the first time. The increment of cerium oxide from 1.2, to 3.6 and 5.3 mol% decreases the surface area and porosity of MG and increases the catalase mimetic activity after 168 h. Swelling tests in different cell culture media (D- and α-MEM) demonstrated the rehydration capability of beads. The presence of beads with the highest Ce-contents (3.6 and 5.3%) improved the proliferation of pre-osteoblastic cells MC3T3-C1 cells. However, the cell differentiation decreased when increased the cerium content. Lactate dehydrogenase assays showed beads are cytocompatible materials. Moreover, oxidative stress tests with H2O2 showed a better response related to cell viability and the elimination of oxidant species when increased cerium content. Beads of glasses with 1.2 and 3.6% of CeO2 are excellent candidates as bioactive scaffolds for bone regeneration capable of counteract the oxidative stress.

Cerium (III) and (IV) containing mesoporous glasses/alginate beads for bone regeneration: bioactivity, biocompatibility and reactive oxygen species activity.

A very small number of biomaterials investigated for bone regeneration was reported as able to prevent the oxidative stress. In this study beads based on alginate hydrogel and mesoporous glasses (MG) containing different amounts of cerium oxides (Ce3+/Ce4+) exhibiting antioxidant properties were investigated as a good approach to mimic the action of antioxidant enzymes in our organism. The effect of cerium contents on the bioactivity and biocompatibility of beads were investigated. Moreover, the potential capability of Ce-containing MG to prevent the oxidative stress caused by the activity of reactive oxygen species (ROS) was here investigated for the first time. The increment of cerium oxide from 1.2, to 3.6 and 5.3 mol-% decreases the surface area and porosity of MG and increases the catalase mimetic activity after 168 h. Swelling tests in different cell culture media (D- and α-MEM) demonstrated the rehydration capability of beads. The presence of beads with the highest Ce-contents (3.6 and 5.3 %) improved the proliferation of pre-osteoblastic cells MC3T3-Cl cells. However, the cell differentiation decreased when increased the cerium content. Lactate dehydrogenase assays showed beads are cytocompatible materials. Moreover, oxidative stress tests with H2O2 showed a better response related to cell viability and the elimination of oxidant species when increased cerium content. Beads of glasses with 1.2 and 3.6 % of CeO2 are excellent candidates as bioactive scaffolds for bone regeneration capable of counteract the oxidative stress.

Mesoporous bioactive glass/ɛ-polycaprolactone scaffolds promote bone regeneration in osteoporotic sheep.

Macroporous scaffolds made of a SiO2-CaO-P2O5 mesoporous bioactive glass (MBG) and ɛ-polycaprolactone (PCL) have been prepared by robocasting. These scaffolds showed an excellent in vitro biocompatibility in contact with osteoblast like cells (Saos 2) and osteoclasts derived from RAW 264.7 macrophages. In vivo studies were carried out by implantation into cavitary defects drilled in osteoporotic sheep. The scaffolds evidenced excellent bone regeneration properties, promoting new bone formation at both the peripheral and the inner parts of the scaffolds, thick trabeculae, high vascularization and high presence of osteoblasts and osteoclasts. In order to evaluate the effects of the local release of an antiosteoporotic drug, 1% (%wt) of zoledronic acid was incorporated to the scaffolds. The scaffolds loaded with zoledronic acid induced apoptosis in Saos 2 cells, impeded osteoclast differentiation in a time dependent manner and inhibited bone healing, promoting an intense inflammatory response in osteoporotic sheep. STATEMENT OF SIGNIFICANCE: In addition to an increase in bone fragility and susceptibility to fracture, osteoporosis also hinders the clinical success of endosseous implants and grafting materials for the treatment of bone defects. For the first time, macroporous scaffolds made of mesoporous bioactive glass and ε-caprolactone have been evaluated in a sheep model that mimics the osteoporosis conditions in humans. These implants fostered bone regeneration, promoting new bone formation at both the peripheral and the inner parts of the scaffolds, showing thick trabeculae and a high vascularization degree. Our results indicate that macroporous structures containing highly bioactive mesoporous glasses could be excellent candidates for the regenerative treatment of bone defects in osteoporotic patients.

Osteostatin potentiates the bioactivity of mesoporous glass scaffolds containing Zn2+ ions in human mesenchymal stem cells.

There is an urgent need of biosynthetic bone grafts with enhanced osteogenic capacity. In this study, we describe the design of hierarchical meso-macroporous 3D-scaffolds based on mesoporous bioactive glasses (MBGs), enriched with the peptide osteostatin and Zn2+ ions, and their osteogenic effect on human mesenchymal stem cells (hMSCs) as a preclinical strategy in bone regeneration. The MBG compositions investigated were 80%SiO2-15%CaO-5%P2O5 (in mol-%) Blank (BL), and two analogous glasses containing 4% ZnO (4ZN) and 5% ZnO (5ZN). By using additive fabrication techniques, scaffolds exhibiting hierarchical porosity: mesopores (around 4 nm), macropores (1-600 µm) and big channels (∼1000 µm), were prepared. These MBG scaffolds with or without osteostatin were evaluated in hMCSs cultures. Zinc promoted hMSCs colonization (both the surface and inside) of MBG scaffolds. Moreover, Zn2+ ions and osteostatin together, but not independently, in the scaffolds were found to induce the osteoblast differentiation genes runt related transcription factor-2 (RUNX2) and alkaline phosphatase (ALP) in hMSCs after 7 d of culture in the absence of an osteogenic differentiation-promoting medium. These results add credence to the combined use of zinc and osteostatin as an effective strategy for bone regeneration applications. STATEMENT OF SIGNIFICANCE: Mesoporous bioactive glasses (MBGs) are bioceramics whose unique properties make them excellent materials for bone tissue engineering. Physico-chemical characterization of MBGs as scaffolds made by rapid prototyping, doped with zinc (potential osteogenic, angiogenic and bactericidal ion) and loaded with osteostatin (osteogenic peptide) are described. These Zn-MBGs scaffolds showed 3D hierarchical meso-macroporous structure that enables to host and release osteostatin. When decorated with human mesenchymal stem cells (hMSCs), MBGs scaffoldsenriched with both zinc and osteostatin exhibited a synergistic effect to enhance hMSCs growth, and also hMSCs osteogenic differentiationwithout addition of other osteoblastic differentiation factors to the culture medium. This novel strategy has a great potential for use in bone tissue engineering.

Synergistic effect of Si-hydroxyapatite coating and VEGF adsorption on Ti6Al4V-ELI scaffolds for bone regeneration in an osteoporotic bone environment.

The osteogenic and angiogenic responses to metal macroporous scaffolds coated with silicon substituted hydroxyapatite (SiHA) and decorated with vascular endothelial growth factor (VEGF) have been evaluated in vitro and in vivo. Ti6Al4V-ELI scaffolds were prepared by electron beam melting and subsequently coated with Ca10(PO4)5.6(SiO4)0.4(OH)1.6 following a dip coating method. In vitro studies demonstrated that SiHA stimulates the proliferation of MC3T3-E1 pre-osteoblastic cells, whereas the adsorption of VEGF stimulates the proliferation of EC2 mature endothelial cells. In vivo studies were carried out in an osteoporotic sheep model, evidencing that only the simultaneous presence of both components led to a significant increase of new tissue formation in osteoporotic bone. STATEMENT OF SIGNIFICANCE: Reconstruction of bones after severe trauma or tumors extirpation is one of the most challenging tasks in the field of orthopedic surgery. This scenario is even more complicated in the case of osteoporotic patients, since their bone regeneration capability is decreased. In this work we present a porous implant that promotes bone regeneration even in osteoporotic bone. By coating the implant with osteogenic bioceramics such as silicon substituted hydroxyapatite and subsequent adsorption of vascular endothelial growth factor, these implants stimulate the bone ingrowth when they are implanted in osteoporotic sheep.

The response of pre-osteoblasts and osteoclasts to gallium containing mesoporous bioactive glasses.

Mesoporous bioactive glasses (MBGs) in the system SiO2-CaO-P2O5-Ga2O3 have been synthesized by the evaporation induced self-assembly method and subsequent impregnation with Ga cations. Two different compositions have been prepared and the local environment of Ga(III) has been characterized using 29Si, 71Ga and 31P NMR analysis, demonstrating that Ga(III) is efficiently incorporated as both, network former (GaO4 units) and network modifier (GaO6 units). In vitro bioactivity tests evidenced that Ga-containing MBGs retain their capability for nucleation and growth of an apatite-like layer in contact with a simulated body fluid with ion concentrations nearly equal to those of human blood plasma. Finally, in vitro cell culture tests evidenced that Ga incorporation results in a selective effect on osteoblasts and osteoclasts. Indeed, the presence of this element enhances the early differentiation towards osteoblast phenotype while disturbing osteoclastogenesis. Considering these results, Ga-doped MBGs might be proposed as bone substitutes, especially in osteoporosis scenarios. STATEMENT OF SIGNIFCANCE: Osteoporosis is the most prevalent bone disease affecting millions of patients every year. However, there is a lack of bone grafts specifically designed for the treatment of bone defects occurred because of osteoporotic fractures. The consequence is that osteoporotic bone defects are commonly treated with the same biomaterials intended for high quality bone tissue. In this work we have prepared mesoporous bioactive glasses doped with gallium, demonstrating osteoinductive capability by promoting the differentiation of pre-osteoblast toward osteoblasts and partial inhibition of osteoclastogenesis. Through a deep study of the local environment of gallium within the mesoporous matrix, this work shows that gallium release is not required to produce this effect on osteoblasts and osteoclasts. In this sense, the presence of this element at the surface of the mesoporous bioactive glasses would be enough to locally promote bone formation while reducing bone resorption.

Effects of a mesoporous bioactive glass on osteoblasts, osteoclasts and macrophages.

A mesoporous bioactive glass (MBG) of molar composition 75SiO2-20CaO-5P2O5 (MBG-75S) has been synthetized as a potential bioceramic for bone regeneration purposes. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption studies and transmission electron microscopy (TEM) demonstrated that MBG-75S possess a highly ordered mesoporous structure with high surface area and porosity, which would explain the high ionic exchange rate (mainly calcium and silicon soluble species) with the surrounded media. MBG-75S showed high biocompatibility in contact with Saos-2 osteoblast-like cells. Concentrations up to 1 mg/ml did not lead to significant alterations on either morphology or cell cycle. Regarding the effects on osteoclasts, MBG-75S allowed the differentiation of RAW-264.7 macrophages into osteoclast-like cells but exhibiting a decreased resorptive activity. These results point out that MBG-75S does not inhibit osteoclastogenesis but reduces the osteoclast bone-resorbing capability. Finally, in vitro studies focused on the innate immune response, evidenced that MBG-75S allows the proliferation of macrophages without inducing their polarization towards the M1 pro-inflammatory phenotype. This in vitro behavior is indicative that MBG-75S would just induce the required innate immune response without further inflammatory complications under in vivo conditions. The overall behavior respect to osteoblasts, osteoclasts and macrophages, makes this MBG a very interesting candidate for bone grafting applications in osteoporotic patients.

A novel visible light responsive nanosystem for cancer treatment.

A novel singlet-oxygen sensitive drug delivery nanocarrier able to release its cargo after exposure to visible (Vis) light from a common lamp is presented. This nanodevice is based on mesoporous silica nanoparticles (MSN) decorated with porphyrin-caps grafted via reactive oxygen species (ROS)-cleavable linkages. In the presence of Vis light porphyrin-nanocaps produce singlet oxygen molecules that break the sensitive-linker, which triggers pore uncapping and therefore allows the release of the entrapped cargo (topotecan, TOP). This new system takes advantage of the non-toxicity and greater penetration capacity of Vis radiation and a double antitumor effect due to the drug release and the ROS production. In vitro tests with HOS osteosarcoma cancer cells reveal that TOP is able to be released in a controlled fashion inside the tumor cells. This research work constitutes a proof of concept that opens up promising expectations in the search for new alternatives for the treatment of cancer.

Electron microscopy for inorganic-type drug delivery nanocarriers for antitumoral applications: what does it reveal?

The use of nanoparticles with the ability to transport drugs in a selective and controllable manner directly to diseased tissues and cells has improved the therapeutic arsenal for addressing unmet clinical situations. In recent years, a vast number of nanocarriers with inorganic, organic, hybrid and even biological nature have been developed especially for their application in the oncology field. The exponential growth in the field of nanomedicine would not have been possible without the also-rapid expansion of electron microscopy techniques, which allow a more precise observation of nanometric objects. The use of these techniques provides a better understanding of the key parameters which rule the nanoparticles' synthesis and behavior. In this review, the recent advances made in the application of inorganic nanoparticles to clinical uses and the role which electron microscopy has played are presented.