Effect of Washing Treatment on the Textural Properties and Bioactivity of Silica/Chitosan/TCP Xerogels for Bone Regeneration.

Silica/biopolymer hydrogel-based materials constitute very attractive platforms for various emerging biomedical applications, particularly for bone repair. The incorporation of calcium phosphates in the hybrid network allows for designing implants with interesting biological properties. Here, we introduce a synthesis procedure for obtaining silica-chitosan (CS)-tricalcium phosphate (TCP) xerogels, with CS nominal content varying from 4 to 40 wt.% and 10 to 20 wt.% TCP. Samples were obtained using the sol-gel process assisted with ultrasound probe, and the influence of ethanol or water as washing solvents on surface area, micro- and mesopore volume, and average pore size were examined in order to optimize their textural properties. Three washing solutions with different soaking conditions were tested: 1 or 7 days in absolute ethanol and 30 days in distilled water, resulting in E1, E7, and W30 washing series, respectively. Soaked samples were eventually dried by evaporative drying at air ambient pressure, and the formation of interpenetrated hybrid structures was suggested by Fourier transformed infrared (FTIR) spectroscopy. In addition the impact that both washing solvent and TCP content have on the biodegradation, in vitro bioactivity and osteoconduction of xerogels were explored. It was found that calcium and phosphate-containing ethanol-washed xerogels presented in vitro release of calcium (2-12 mg/L) and silicon ions (~60-75 mg/L) after one week of soaking in phosphate-buffered saline (PBS), as revealed by inductive coupled plasma (ICP) spectroscopy analysis. However, only the release of silicon was detected for water-washed samples. Besides, all the samples exhibited in vitro bioactivity in simulated body fluid (SBF), as well as enhanced in vitro cell growth and also significant focal adhesion development and maturation.

Intracellular Elemental Patterns of Apoptosis Resistance in Transdifferentiated Androgen-Dependent Prostatic Carcinoma Cells.

The acquisition of neuroendocrine (NE) characteristics by prostate cancer (PC) cells relates to tumor progression and hormone resistance. PC cells may survive and function in androgen-deprived environments, where they could establish paracrine signaling networks, providing stimuli for the propagation of local carcinoma cells. We previously demonstrated, using electron probe X-ray microanalysis (EPXMA), in LNCaP, PC-3, and Du 145 cell lines that apoptosis is associated with intracellular elemental changes, and that the NE secretory products, bombesin and calcitonin, inhibit etoposide-induced apoptosis, as well as some of these elemental changes. In this study, LNCaP cells were induced in vitro to transdifferentiate under androgen deprivation, to mimic the role of NE cells in the apoptotic activity of transdifferentiated androgen-dependent PC cells. Changes in intracellular ion content associated with apoptosis, assessed by EPXMA, demonstrate that the transdifferentiated LNCaP cells are resistant to etoposide-induced apoptosis and also to the etoposide-induced elemental changes. The aggressive malignant potential of PC with neuroendocrine differentiation, associated with hormonal independence, is partly because of the ability that most NE tumor cells have to escape apoptosis, which can enhance the malignant properties of tumor cells and may have therapeutic implications as tumor cells are usually resistant to cytotoxic drugs as etoposide.

Chitosan-Silica Hybrid Biomaterials for Bone Tissue Engineering: A Comparative Study of Xerogels and Aerogels.

Chitosan (CS) is a natural biopolymer that shows promise as a biomaterial for bone-tissue regeneration. However, because of their limited ability to induce cell differentiation and high degradation rate, among other drawbacks associated with its use, the creation of CS-based biomaterials remains a problem in bone tissue engineering research. Here we aimed to reduce these disadvantages while retaining the benefits of potential CS biomaterial by combining it with silica to provide sufficient additional structural support for bone regeneration. In this work, CS-silica xerogel and aerogel hybrids with 8 wt.% CS content, designated SCS8X and SCS8A, respectively, were prepared by sol-gel method, either by direct solvent evaporation at the atmospheric pressure or by supercritical drying in CO2, respectively. As reported in previous studies, it was confirmed that both types of mesoporous materials exhibited large surface areas (821 m2g-1-858 m2g-1) and outstanding bioactivity, as well as osteoconductive properties. In addition to silica and chitosan, the inclusion of 10 wt.% of tricalcium phosphate (TCP), designated SCS8T10X, was also considered, which stimulates a fast bioactive response of the xerogel surface. The results here obtained also demonstrate that xerogels induced earlier cell differentiation than the aerogels with identical composition. In conclusion, our study shows that the sol-gel synthesis of CS-silica xerogels and aerogels enhances not only their bioactive response, but also osteoconduction and cell differentiation properties. Therefore, these new biomaterials should provide adequate secretion of the osteoid for a fast bone regeneration.

Structure-Related Mechanical Properties and Bioactivity of Silica-Gelatin Hybrid Aerogels for Bone Regeneration.

We report the synthesis of mesoporous silica-gelatin hybrid aerogels with 15, 25, and 30 wt. % gelatin contents, using 3-glycidoxypropyl trimethoxysilane (GPTMS) as a coupling agent, for tissue-engineering applications. Aerogels were obtained using a one-step sol-gel process followed by CO2 supercritical drying, resulting in crack-free monolith samples with bulk densities ranging from 0.41 g cm-3 to 0.66 g cm-3. Nitrogen adsorption measurements revealed an interconnected mesopore network and a general decrease in the textural parameters: specific surface areas (651-361 m2 g-1), pore volume (1.98-0.89 cm3 g-1), and pore sizes (10.8-8.6 nm), by increasing gelatin content. Thermogravimetric analysis (TGA), Fourier-transform infrared (FTIR) spectroscopy and uniaxial compression experiments confirmed that the structure, thermal properties and mechanical behavior of these aerogels changed significantly when the concentration of gelatin reached 25 wt.%, suggesting that this composition corresponds to the percolation threshold of the organic phase. In addition, the samples exhibited hydrophilic behavior and extremely fast swelling in phosphate-buffered saline (PBS), with swelling ratios from 2.32 to 3.32. Furthermore, in vitro bioactivity studies revealed a strong relationship between the kinetics of the nucleation and growth processes of hydroxyapatite in simulated body fluid (SBF) and the gelatin content. The live/dead assay revealed no cytotoxicity in HOB® osteoblasts in vitro and a positive influence on cell growth, focal adhesion development, and cytoskeletal arrangement for cell adhesion. Mineralization assays confirmed the positive effects of the samples on osteoblast differentiation. The biomaterials described are versatile, can be easily sterilized and are suitable for a wide range of applications in bone tissue-engineering, either alone or in combination with bioactive-reinforced phases.

Robocasting and Laser Micromachining of Sol-Gel Derived 3D Silica/Gelatin/ß-TCP Scaffolds for Bone Tissue Regeneration.

The design and synthesis of sol-gel silica-based hybrid materials and composites offer significant benefits to obtain innovative biomaterials with controlled porosity at the nanostructure level for applications in bone tissue engineering. In this work, the combination of robocasting with sol-gel ink of suitable viscosity prepared by mixing tetraethoxysilane (TEOS), gelatin and ß-tricalcium phosphate (ß-TCP) allowed for the manufacture of 3D scaffolds consisting of a 3D square mesh of interpenetrating rods, with macropore size of 354.0 ± 17.0 µm, without the use of chemical additives at room temperature. The silica/gelatin/ß-TCP system underwent irreversible gelation, and the resulting gels were also used to fabricate different 3D structures by means of an alternative scaffolding method, involving high-resolution laser micromachining by laser ablation. By this way, 3D scaffolds made of 2 mm thick rectangular prisms presenting a parallel macropore system drilled through the whole thickness and consisting of laser micromachined holes of 350.8 ± 16.6-micrometer diameter, whose centers were spaced 1312.0 ± 23.0 µm, were created. Both sol-gel based 3D scaffold configurations combined compressive strength in the range of 2-3 MPa and the biocompatibility of the hybrid material. In addition, the observed Si, Ca and P biodegradation provided a suitable microenvironment with significant focal adhesion development, maturation and also enhanced in vitro cell growth. In conclusion, this work successfully confirmed the feasibility of both strategies for the fabrication of new sol-gel-based hybrid scaffolds with osteoconductive properties.

Hydroxyl Groups Induce Bioactivity in Silica/Chitosan Aerogels Designed for Bone Tissue Engineering. In Vitro Model for the Assessment of Osteoblasts Behavior.

Silica (SiO2)/chitosan (CS) composite aerogels are bioactive when they are submerged in simulated body fluid (SBF), causing the formation of bone-like hydroxyapatite (HAp) layer. Silica-based hybrid aerogels improve the elastic behavior, and the combined CS modifies the network entanglement as a crosslinking biopolymer. Tetraethoxysilane (TEOS)/CS is used as network precursors by employing a sol-gel method assisted with high power ultrasound (600 W). Upon gelation and aging, gels are dried in supercritical CO2 to obtain monoliths. Thermograms provide information about the condensation of the remaining hydroxyl groups (400-700 °C). This step permits the evaluation of the hydroxyl group's content of 2 to 5 OH nm-2. The formed Si-OH groups act as the inductor of apatite crystal nucleation in SBF. The N2 physisorption isotherms show a hysteresis loop of type H3, characteristic to good interconnected porosity, which facilitates both the bioactivity and the adhesion of osteoblasts cells. After two weeks of immersion in SBF, a layer of HAp microcrystals develops on the surface with a stoichiometric Ca/P molar ratio of 1.67 with spherulite morphology and uniform sizes of 6 µm. This fact asserts the bioactive behavior of these hybrid aerogels. Osteoblasts are cultured on the selected samples and immunolabeled for cytoskeletal and focal adhesion expression related to scaffold nanostructure and composition. The initial osteoconductive response observes points to a great potential of tissue engineering for the designed composite aerogels.

Chitosan-GPTMS-Silica Hybrid Mesoporous Aerogels for Bone Tissue Engineering.

This study introduces a new synthesis route for obtaining homogeneous chitosan (CS)-silica hybrid aerogels with CS contents up to 10 wt%, using 3-glycidoxypropyl trimethoxysilane (GPTMS) as coupling agent, for tissue engineering applications. Aerogels were obtained using the sol-gel process followed by CO2 supercritical drying, resulting in samples with bulk densities ranging from 0.17 g/cm3 to 0.38 g/cm3. The textural analysis by N2-physisorption revealed an interconnected mesopore network with decreasing specific surface areas (1230-700 m2/g) and pore sizes (11.1-8.7 nm) by increasing GPTMS content (2-4 molar ratio GPTMS:CS monomer). In addition, samples exhibited extremely fast swelling by spontaneous capillary imbibition in PBS solution, presenting swelling capacities from 1.75 to 3.75. The formation of a covalent crosslinked hybrid structure was suggested by FTIR and confirmed by an increase of four hundred fold or more in the compressive strength up to 96 MPa. Instead, samples synthesized without GPTMS fractured at only 0.10-0.26 MPa, revealing a week structure consisted in interpenetrated polymer networks. The aerogels presented bioactivity in simulated body fluid (SBF), as confirmed by the in vitro formation of hydroxyapatite (HAp) layer with crystal size of approximately 2 µm size in diameter. In vitro studies revealed also non cytotoxic effect on HOB® osteoblasts and also a mechanosensitive response. Additionally, control cells grown on glass developed scarce or no stress fibers, while cells grown on hybrid samples showed a significant (p < 0.05) increase in well-developed stress fibers and mature focal adhesion complexes.

Loss of mitochondrial membrane potential is inhibited by bombesin in etoposide-induced apoptosis in PC-3 prostate carcinoma cells.

Neuroendocrine secretory products and their interactions with epithelial prostate cells are currently under investigation in order to understand their significance in the pathogenesis, prognosis, and therapy of prostate carcinoma. These neuropeptides have the potential to disrupt the balance between cell death and cell growth in the tumor. Our research was based on the role of bombesin in modulating the mitochondrial membrane potential (Delta psi(m)) in cell death induced by etoposide on PC-3 cells. Cells were cultured and stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). At low membrane potentials, JC-1 produces a green fluorescence, and at high membrane potentials, it forms "J aggregates" with red fluorescence. Cells were examined in a confocal microscope. For quantitative analyses, regions of interest were selected. The size, number of pixels, and ratios between fluorescence intensity in the red and green channels in each region of interest were calculated. The loss of Delta psi(m) in etoposide-treated PC-3 cells was prevented by bombesin. The quantitative analysis of JC-1-stained cells revealed a significant decrease in the red (high Delta psi(m)) to green (low Delta psi(m)) ratio in etoposide-treated cells when compared with control cells, which was restored in the presence of bombesin (P < 0.00001). The interaction between treatments and area (P = 0.0002) was highly significant, and confirms that PC-3 cells keep their apoptosis machinery, showing an apoptotic volume decrease in response to etoposide. The protection by bombesin occurs by inhibition of apoptosis and maintenance of mitochondrial integrity. New therapeutic protocols and trials need to be developed to test drugs acting through the neutralization of antiapoptotic intracellular pathways mediated by neuroendocrine hormones.

Osteoconductive potential of barrier nanoSiO2 PLGA membranes functionalized by plasma enhanced chemical vapour deposition.

The possibility of tailoring membrane surfaces with osteoconductive potential, in particular in biodegradable devices, to create modified biomaterials that stimulate osteoblast response should make them more suitable for clinical use, hopefully enhancing bone regeneration. Bioactive inorganic materials, such as silica, have been suggested to improve the bioactivity of synthetic biopolymers. An in vitro study on HOB human osteoblasts was performed to assess biocompatibility and bioactivity of SiO2 functionalized poly(lactide-co-glycolide) (PLGA) membranes, prior to clinical use. A 15 nm SiO2 layer was deposited by plasma enhanced chemical vapour deposition (PECVD), onto a resorbable PLGA membrane. Samples were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy, and infrared spectroscopy (FT-IR). HOB cells were seeded on sterilized test surfaces where cell morphology, spreading, actin cytoskeletal organization, and focal adhesion expression were assessed. As proved by the FT-IR analysis of samples, the deposition by PECVD of the SiO2 onto the PLGA membrane did not alter the composition and other characteristics of the organic membrane. A temporal and spatial reorganization of cytoskeleton and focal adhesions and morphological changes in response to SiO2 nanolayer were identified in our model. The novedous SiO2 deposition method is compatible with the standard sterilization protocols and reveals as a valuable tool to increase bioactivity of resorbable PLGA membranes.

Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction.

New biomaterials for Guided Bone Regeneration (GBR), both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB®) HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide) (PLGA) membrane foil functionalized by a very thin film (around 15 nm) of TiO2 (i.e., TiO2/PLGA membranes), designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors.

Light induced hydrophilicity and osteoblast adhesion promotion on amorphous TiO2.

We have studied the effect of the UV induced superhydrophilic wetting of TiO(2) thin films on the osteoblasts cell adhesion and cytoskeletal organization on its surface. To assess any effect of the photo-catalytic removal of adventitious carbon as a factor for the enhancement of the osteoblast development, 100 nm amorphous TiO(2) thin layers were deposited on polyethylene terephthalate (PET), a substrate well known for its poor adhesion and limited wettability and biocompatibility. The TiO(2) /PET materials were characterized by X-ray photoelectron spectroscopy, and atomic force microscopy and their wetting behavior under light illumination studied by the sessile drop method. The amorphous TiO(2) thin films showed a very poor photo-catalytic activity even if becoming superhydrophilic after illumination. The illuminated samples recovered partially its initial hydrophobic state only after their storage in the dark for more than 20 days. Osteoblasts (HOB) were seeded both on bare PET and on TiO(2) /PET samples immediately after illumination and also after four weeks storage in darkness. Cell attachment was much more efficient on the immediately illuminated TiO(2)/PET samples, with development of focal adhesions and cell traction forces. Although we cannot completely discard some photo-catalytic carbon removal as a factor contributing to this cell enhanced attachment, our photodegradation experiments on amorphous TiO(2) are conclusive to dismiss this effect as the major cause for this behavior.

Mitochondrial bioenergetics and distribution in living human osteoblasts grown on implant surfaces.

Osseointegration of implants is crucial for the long-term success of oral implants. The periimplant bone formation by osteoblasts is strongly dependent on the local mechanical environment in the interface zone. Robust demands for energy are placed on osteoblasts during the adhesion process to solid surfaces, and mitochondria are capital organelles in the production of most of the ATP needed for the process. We have assessed the relationship between osteoblast differentiation and mitochondrial bioenergetics in living cells grown on two different titanium surfaces, in order to provide valuable information for the design of material surfaces required for the development of the most appropriate osteogenic surface for osteoblastic anchorage. Combined backscattered and fluorescence confocal microscopy showed that in flat cells grown on a machined surface, highly energized mitochondria were distributed along the cell body. In contrast, cells grown on the rough surface emitted long protrusions in search of surface roughness, with actin stress fibers clearly polarized and highly energized mitochondria clustered at focal adhesion sites. This report using normal human osteoblastic cells indicates that these cells are especially sensitive to surface cues through energy production that enhances the necessary adhesion required for a successful osseointegration.

Internalization of the receptor for advanced glycation end products (RAGE) is required to mediate intracellular responses.

To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE-EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE-EGFP protein compatible with an internalization of the AGEs-RAGE complex. Furthermore, while N2a cells expressing the RAGE-EGFP showed an increase in ERK1/2 phosphorylation and NF-kappaB DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.

Neuropeptides bombesin and calcitonin inhibit apoptosis-related elemental changes in prostate carcinoma cell lines.

BACKGROUND: Etoposide-induced apoptosis in prostate carcinoma cells is associated with changes in the elemental content of the cells. The authors previously reported that calcitonin and bombesin inhibited etoposide-induced apoptosis in these cells. In the current study, the authors investigated whether these neuropeptides block the etoposide-induced changes in elemental content. METHODS: Cells from the PC-3 and Du 145 prostate carcinoma cell lines were grown either on solid substrates or on thin plastic films on titanium electron microscopy grids, and they were exposed to etoposide for 48 hours in the absence or presence of calcitonin and bombesin. After the exposure, the cells were frozen and freeze dried, and their elemental content was analyzed by energy-dispersive X-ray microanalysis in both in the scanning electron microscope and the scanning transmission electron microscope. RESULTS: Etoposide treatment consistently induced an increase in the cellular Na concentration and a decrease in the cellular K concentration, resulting in a marked increase of the Na/K ratio and also an increase in the phosphorus:sulphur (P/S) ratio. Both bombesin and calcitonin inhibited the etoposide-induced changes in the cellular Na/K ratio, and calcitonin, but not bombesin, inhibited the changes in the P/S ratio. No significant elemental changes were found with bombesin or calcitonin alone. CONCLUSIONS: The neuropeptides bombesin and calcitonin, which inhibited etoposide-induced apoptosis, also inhibited the etoposide-induced elemental changes in prostate carcinoma cells. This important fact strengthens the link between apoptosis and changes in the intracellular elemental content. This correlation provides an objective basis for the study of neuropeptide target points and may be helpful for alternative therapeutic protocols using neuropeptide inhibitors in the treatment of patients with advanced prostatic carcinoma.