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.

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.

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.

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.

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.

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.

Synthesis of tricyclic aromatic compounds by the intramolecular pauson-khand reaction promoted by molecular sieves(,)(1)

Pauson-Khand reactions are carried out with different substituted aromatic enynes, yielding tricyclic cyclopentenones related to natural products such as chromenes. Enynes are easily obtained in a two-step approximation from the corresponding salicylaldehydes. The reaction is promoted by dissolved TMANO (trimethylamine N-oxide) and/or 4 A molecular sieves. This new way of induction for the Pauson-Khand reaction increases yields remarkably, allowing the reaction of some substituted alkenes which fail to react in the absence of the zeolite. Isomerization of the double bond of the cyclopentenone ring is observed except when nonterminal triple bonds are used. For trisubstituted alkenes, an interrupted Pauson-Khand process is observed with moderate yields.