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.

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.

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.

Water-dependent micromechanical and rheological properties of silica colloidal crystals studied by nanoindentation.

Here we show the suitability of nanoindentation to study in detail the micromechanical response of silica colloidal crystals (CCs). The sensitivity to displacements smaller than the submicrometer spheres size, even resolving discrete events and superficial features, revealed particulate features with analogies to atomic crystals. Significant robustness, long-range structural deformation, and large energy dissipation were found. Easily implemented temperature/rate-dependent nanoindentation quantified the paramount role of adsorbed water endowing silica CCs with properties of wet granular materials like viscoplasticity. A novel "nongranular" CC was fabricated by substituting capillary bridges with silica necks to directly test water-independent mechanical response. Silica CCs, as specific (nanometric, ordered) wet granular assemblies with well-defined configuration, may be useful model systems for granular science and capillary cohesion at the nanoscale.

Bioactivity of wollastonite/aerogels composites obtained from a TEOS-MTES matrix.

Organic-inorganic hybrid materials were synthesized by controlled hydrolysis of tetraethoxysilane (TEOS), methyltrimethoxysilane (MTES), synthetic wollastonite powders and polydimethylsiloxane (PDMS) in an ethanol solution. Aerogels were prepared from acid hydrolysis of TEOS and MTES with different volume ratio in ethanol, followed by addition of wollastonite powder and PDMS in order to obtain aerogels with 20 wt% of PDMS and 5 wt% of CaO of the total silica. Finally, when the wet gels were obtained, they were supercritically dried at 260 degrees C and 90 bar, in ethanol. In order to obtain its bioactivity, one method for surface activation is based on a wet chemical alkaline treatment. The particular interest of this study is that we introduce hybrid aerogels, in a 1 M solution of NaOH, for 30 s at room temperature. We evaluate the bioactivity of TEOS-MTES aerogel when immersed in a static volume of simulated body fluid (SBF). An apatite layer of spherical-shaped particles of uniform size smaller than 5 microns is observed to form on the surface of the aerogels after 25 days soaking in SBF.

Structure of hybrid colloid-polymer xerogels.

Crack-free monolithic gels were prepared from different mixtures of colloidal silica with a sol solution containing tetraethoxysilane, under powerful ultrasonic agitation (sonosol). Recently, information on the structure of these gels, inferred from N2 adsorption and mercury intrusion porosimetry, was presented. In the present paper, these data were used to construct structural models of the gels using Monte Carlo calculations on the basis of random close packing (RPC) premises. In addition, the structure of gels under study was investigated by transmission and scanning electron microscopy. The material can be described as a composite in which the sonogel is the matrix and the colloid particles the reinforcing phase. For low colloid content, the colloid forms discrete clusters, and the main structural characteristic of sonogels, i.e., a network of uniformly sized particles of approximately 3-4-nm radius, remains unmodified. However, for high colloid silica content, a multimode distribution appears, the structure is discontinuous, and only colloid aggregates larger than 100 nm are observed. For medium colloid content, aggregates of approximately 50-100 nm can be seen, but the sonogel structure extends throughout the whole material. By the processing method and election of a suitable precursor concentration, it is possible to design the composite for specific purposes.