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.

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.

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.

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.

Thermoseeds for interstitial magnetic hyperthermia: from bioceramics to nanoparticles.

The development of magnetic materials for interstitial hyperthermia treatment of cancer is an ever evolving research field which provides new alternatives to antitumoral therapies. The development of biocompatible magnetic materials has resulted in new biomaterials with multifunctional properties, which are able to adapt to the complex scenario of tumoral processes. Once implanted or injected in the body, magnetic materials can behave as thermoseeds under the effect of AC magnetic fields. Magnetic bioceramics aimed to treat bone tumors and magnetic nanoparticles are among the most studied thermoseeds, and supply different solutions for the different scenarios in cancerous processes. This paper reviews some of the biomaterials used for bone cancer treatment and skeletal reinforcing, as well as the more complex topic of magnetic nanoparticles for intracellular targeting and hyperthermia.

Magnetic mesoporous silica spheres for hyperthermia therapy.

Magnetic nanoparticles coated with materials having unique properties, such as ordered pore structures and large surface areas, hold great potential for multimodal therapies. This study reports on the biocompatibility of composites of maghemite nanoparticles embedded in an ordered mesoporous silica-matrix to form magnetic microspheres (MMS), and on their ability to conduct magnetic hyperthermia upon exposure to a low-frequency alternating magnetic field (AMF). MMS particles were efficiently internalized by human A549, Saos-2 and HepG2 cells, and were excluded from the nuclear compartment. MMS treatment did not interfere with morphological features or metabolic activities of the cells, indicating good biocompatibility of the material. MMS did not affect the endogenous heat-shock response of a HeLa-derived cell line that precisely reports the intensity of thermal stresses through changes in the activities of a stably integrated hsp70B promoter and a constitutive viral promoter. Maximum temperature in MMS suspensions increased to a range above 42°C as a function of the amounts of particles exposed to AMF. Cell culture experiments showed that, by adjusting the amount of MMS and the time of exposure to AMF, heat treatments of mild to very high intensities could be achieved. Cell viability dropped as a function of the intensity of the heat treatment achieved by MMS and AMF exposures. The possibility of fine-tuning the heating power output, together with efficient uptake by tumor cells in vitro, makes MMS a promising agent by which to provide hyperthermia treatments aimed toward remission of solid tumors.

Glass-glass ceramic thermoseeds for hyperthermic treatment of bone tumors.

Implantable thermoseeds are synthesised from mixtures of a melt-derived glass with composition SiO(2) (40)-CaO(40)-Fe(2)O(3)(20) (mol%) and a sol-gel glass with composition SiO(2)(58)-P(2)O(5)(6)-CaO(36) (mol%). Structural, textural and magnetic properties of the samples are evaluated. In vitro bioactivity is assessed in order to determine the potential capability to bond to living bone. In spite of the low textural properties of the material, a bioactive behavior is observed as a result of the sol-gel glass content. Although the crystallization of the glass ceramic provides the magnetic phase, the presence of sol-gel glass modifies the magnetic properties, improving the heating power. For the first time, hyperthermia heating experiments as well as preliminary biocompatibility assays have been carried out for this kind of material. The ability to reach hyperthermic temperature range together with the bioactive behavior makes this biomaterial a very promising candidate for bone cancer treatment.

A bioactive sol-gel glass implant for in vivo gentamicin release. Experimental model in Rabbit.

Biomaterial pieces with osteogenic properties, suitable for use in the treatment of bone defects, were synthesized. The materials, which avoid bone infections, are exclusively composed of gentamicin sulfate and bioactive SiO2-CaO-P2O5 sol-gel glass (synthesized previously), and were manufactured by means of uniaxial and isostatic pressure of the mixed components. After implanting the pieces into rabbit femur, we studied (1) antibiotic release, determining the concentration in proximal and distal bone, liver, kidney, and lung as a function of time, and (2) bone growth as a consequence of the glass reactivity in the biological environment. The results demonstrated that the implants are good carriers for local gentamicin release into the local osseous tissue, where they show excellent biocompatibility and bone integration. Moreover, these implants are able to promote bone growth during the resorption process.

Biphasic materials for bone grafting and hyperthermia treatment of cancer.

Three biphasic materials have been synthesized from a magnetic glass-ceramic (Si-Ca-Fe) and a bioactive sol-gel glass (Si-P-Ca). The ratios of glass-ceramic:sol-gel glass used in this work were 1:1, 2:1, and 5:1. These materials show bioactive and magnetic properties and can be used as thermoseeds for hyperthermia treatment of bone tumors. The sol-gel glass content affects the textural properties of the glass-ceramic, giving rise to porosity, which plays a fundamental role in the formation of an apatite-like layer on the surface. On the other hand, as the sol-gel glass content increases, the magnetic properties change due to the diffusion of Fe ions to the glassy phases of the biphasic materials. The biphasic nature of these materials allows the changing of both properties, depending on the requirements of the patient.

Biocompatibility and in vivo gentamicin release from bioactive sol-gel glass implants.

Biomaterial pieces, with suitable osteogenic properties for use in the treatment of bone defects and the capability to avoid bone infections, have been synthesized. These materials are composed exclusively of gentamicin sulfate and bioactive SiO(2)-CaO-P(2)O(5) sol-gel glass (previously synthesized). Implant processing was achieved by uniaxial and isostatic pressure of the components mixture. After implanting the pieces into rabbit femur, we studied (i) the antibiotic release, determining the concentration in proximal and distal bone, liver, kidney, and lung as a function of time; and (ii) the bone growth resulting from the glass reactivity in the biologic environment. The results indicate that the implants are good carriers for local gentamicin release in the osseous tissue, exhibiting excellent biocompatibility and bone integration. Moreover, these implants are able to promote bone growth during their resorption process.

Ibuprofen release from hydrophilic ceramic-polymer composites.

Two composite systems composed of alpha-Al2O3/poly(methyl methacrylate) (PMMA)/poly(vinyl pyrrolidone) (PVP)/ibuprofen or alpha-Al2O3/PMMA/co-vinyl pyrrolidone-methyl methacrylate/ibuprofen were prepared by free radical polymerization. These systems were characterized by spectroscopic techniques and thermogravimetric and differential thermal analyses. The hydration behaviour of composites with different hydrophilic characters was analysed after the immersion of the composites in buffered solution at pH 7.4 and 37 degrees C. The swelling of the composites depends strongly on the content of the hydrophilic component and is controlled by the presence of the ceramic component. The release of the anti-inflammatory drug, ibuprofen, from the composites in buffered solution was followed by UV spectroscopy and the results obtained indicated that the components of the composites influenced the rate of release of the drug, without the classical 'burst' effect observed frequently with hydrophilic systems.