Influence of Synthesis Parameters on the Structure , Pore Morphology and Bioactivity of a New Mesoporous Glass

The main objective of the present work was to investigate the effect of surfactant type and synthesis temperature on the structure, porosity and the bioactivity of 92S6 (92% SiO2, 6% CaO, and 2% P2O5 mol %) mesoporous sol-gel glasses. The aim was to provide a basis for controlling the bioactive behavior of the different 92S6 samples used for tissue regeneration and for biomedical engineering in order to obtain sufficient performances by controlling the porosity of the glass. In this paper, a series of mesoporous bioactive glasses were synthesized using three different surfactants (C10H20BrN, C19H42BrN, C22H48BrN) at different aging temperatures (20 ̊C, 40 ̊C and 60 ̊C). The surfactant was removed by calcination, which was carried out by increasing the temperature to 650 ̊C for 6 h. A comparison among these synthesized glasses was conducted and the research emphasis was placed on the synthesis temperature and the surfactant type dependence on the textural properties and particularly porosity that were ultimately responsible for glass bioactivity.


Introduction
Various kinds of bioactive materials like hydroxyapatite, bioglasses, bioglass-ceramics and calcium phosphate ceramics have attracted a strong interest of research over the last decades and have shown significant potential in biomedical research field.These glasses could facilitate bone integration of the implant [1], they are intended to replace bone through a bone defect area in the human skeleton [2] [3].The bioactive behavior of those glasses, which is identified as capability of bone bonding, is attributed to the formation of an apatite-like layer, whose composition and structure are equivalent to the mineral phase in bone [4].
In the last couple of decades, there has been some interest in porous glasses in applications where bone ingrowth is needed or as coatings in applications such as implants [5] [6], where a good bone-implant interface is required [7] [8].Porous biomaterials have been produced by several techniques such as the use of polymeric sponge, foaming processes and techniques using organic additives such as n-hexadecyltrimethylammonium bromide CTAB [7].
The porous structure is induced by the condensation of silica around supramolecular surfactant aggregates acting as a structure-directing agent, and the pores are formed upon removal of the surfactant from the matrix by calcination [9].The resulting amorphous material is biocompatible and degradable in aqueous solution as well as in simulated body fluid (SBF) [9].It's well known that a bioactive material is one that elicits a specific biological response at the interface of the material and allows the attachment to the tissue, such as soft tissue and bone [2].When bioactive materials are implanted in human body, their interface can be transformed to calcium phosphate phase (apatite phase) through of series of surface reactions including dissolution-precipitation and ion exchange denoted as "bioactive process" [3].For bioactive glasses, variables such as chemical composition, structure, surface morphology, and synthesis conditions, such as temperature, pH, and reactant ratios, are influent parameters on the bioactivity [6] [10]- [15].All these factors must be taken into account when applying this synthesis route to biomaterials, as the bioactive process is a surface process and the response of the living tissue will be ruled by the surface characteristics of the implant [16] [17].
The objective of this paper is to report the preparation of mesoporous glasses in the system SiO 2 -CaO-P 2 O 5 through sol-gel route.In order to analyze changes in 92S6 textural properties, synthesized at different aging temperatures and using different length of CTAB chain, and in order to estimate the values of parameters describing the properties of pore structure, nitrogen adsorption isotherms were used.In vitro bioactivity of glasses was investigated to analyze its feasibility as biomaterial.

Preparation of Mesoporous Bioactive Glass Samples (92S6)
Tetraethyl orthosilicate (TEOS), Triethyl phosphate (TEP) and calcium carbonate CaCO 3 were used, respectively, as a silica source, source of phosphorous, and a source of calcium.The surfactant n-hexadecyltrimethylammonium bromide CTAB was used as a structure-directing agent.
Samples were prepared by the solgel process.The synthesis was carried out according to the modified Stöber's method described for synthesis of monodisperse silica spheres MCM-41 [18].In the first step, the solution was prepared as follows: 2.5 g of commercial surfactant (C 10 H 20 BrN, C 19 H 42 BrN, C 22 H 48 BrN) was dissolved in 50 mL of deionized water and mixed with 75 mL of absolute ethanol, at room temperature.3.6 g of TEOS is further added to this solution and kept under strong stirring for one hour.Under stirring, 0.16 g of TEP and an acidic CaCO 3 solution were added.In the second step, the mixture was stirred for 24 hours at 20˚C.Finally 18.53 mL of aqueous ammonia solution (25% wt, Fluka, France) was added to this clear solution and stirred for 15 min.At the end of the addition of aqueous ammonia solution, the gel formation immediately began.The white precipitate was isolated by filtration and washed several times with water and ethanol until neutral pH was reached.In order to study the effect of the temperature, we used the sample prepared with C 19 H 42 BrN and we chose to vary aging temperature (20˚C, 40˚C and 60˚C).Samples were dried at 90˚C overnight.The surfactant was removed by calcination, treating the dried gel at 650˚C for 6 h.The resulting bioactive glass was crushed and sieved to select grain size less than 63 µm.Samples were then characterized.

Samples Characterization
Scanning electron microscopy (SEM) was performed on a JEOL-JSM-6301F instrument.Specific surface area and pore size distribution of the materials were determined by nitrogen N 2 adsorption-desorption using a Micromeritics ASAP-2010.The pore size distribution was determined using the BJH method (desorption) [19].

In Vitro Bioactivity Test
Samples were immersed in SBF solution at 37˚C ± 0.5˚C, for 7, 15, 21 and 30 days.This solution has ion concentrations and pH nearly equal to those of human blood plasma [20].The glass specimen before soaking in SBF is termed as zero days' specimen.A solid to liquid ratio of 1 mg/2 ml was maintained for all immersions, and the SBF solutions were not renewed during the experiments.The samples were removed from the incubator, rinsed with deionized water and left to dry at ambient temperature over night.

Effect of CTAB Carbon Chain Length
Nitrogen isotherms of calcined 92S6-C10, 92S6-C19 and 92S6-C22 are shown in Figure 1.In all cases, the isotherms were of type IV and exhibited hysteresis loops of H3 type for 92S6-C10 and 92S6-C19 glasses, and H4 type for 92S6-C22, according the IUPAC classification, which is associated with the presence of mesopores.Table 1 summarizes the different pore sizes, specific areas and pore volumes in samples.Synthesized glasses show specific surface area (S BET ) in the range of 88 to 248 m 2 •g −1 .It shows that the use of different surfactants has a significant influence on glass specific area.On the other hand, a decrease of pore volume (Vp) is observed, the value is declined from 0.28 cm 3 /g to 0.13 cm 3 /g with increasing the number of surfactant carbons.The N 2 adsorption experiments revealed that these materials have an average pore size varying from 2.6 nm to 8.24 nm.It seems that the length of carbon chains plays an important role in the formation of the mesoporous structure.The large pore diameter is related to relatively long chain of carbon of surfactant.These results are associated to the mechanism of formation of these glasses.CATB surfactant is formed by a hydrophilic head facing the outside of the micelle and by the hydrophobic carbon chains facing the center.The increase of the number of carbons results in an increase of pore diameter in glasses prepared with different surfactants [21].

Effect of Aging-Temperature
To investigate the effect of the aging-temperature on the changes of glasses pore structure, textural properties were studied by the analysis of nitrogen adsorption-desorption isotherms.The nitrogen adsorption-desorption of the glasses synthesized at various temperatures are depicted in Figure 2. The curves are identifiable as Type IV with H3 hysteresis loops for 92S6-T20 glass, H4 hysteresis loops for 92S6-T40 glass and H2 hysteresis loops for 92S6-T60 glass.Type IV isotherm is typical of mesoporous materials.The results of the glasses textural properties are shown in Table 2.All synthesized glasses show high specific surface area (S BET ) in the range of 224 to 559 m 2 •g −1 .The increase of specific surface area can be related to stronger micelle-silica interactions,    which favors the incorporation of the surfactants, increasing the number of the mesopores, and consequently, increasing S BET [22].Some differences were observed in the adsorption isotherms as a function of the aging temperature.The positions of the adsorption branches for 92S6 glasses shifted toward higher pressures as the synthesis temperature increased, indicating no presence of microporosity.
With the increase of the synthesis temperature, the capillary condensation step is shifted to higher relative pressures (Figure 2), corresponding to the formation of larger cavities, which can explain the increase of the pore volume.

In Vitro Bioactivity of the 92S6 in SBF
The in vitro physico-chemical analyses are useful to explain the observations on ex vivo specimens [23].We presented here ICP-OES results obtained for glasses synthesized using three different surfactants, and SEM results obtained for glasses synthesized at different aging temperature.

Chemical Reactivity Investigation Using ICP-OES
Figure 3 shows the evolutions of calcium and phosphorous ionic concentrations with the soaking time in SBF for 92S6-C10, 92S6-C19 and 92S6-C22.As it can be observed, variation of Ca and P concentration in SBF followed the same tendency for the three glasses.However, these evolutions were different for each glass.For 92S6-C22, Ca and P concentrations reached a maximum after 15 days of soaking in SBF and gradually decreased up to 30 days, while for 92S6-C19 and 92S6-C10 Ca and P concentrations, show a an increase the first 7 days of soaking, followed by a decrease in concentration.This is due to the small surface area of 92S6-C22 compared to the surface area of the two other glasses, as reported before.This leads to faster formation of hydroxyapatite layer at the surface of 92S6-C19 and 92S6-C10 than at the 92S6-C22 surface.
These observations highlight ionic exchanges between the surface material and the medium (ionic release from the glass followed by ionic uptake by the glass surface), which lead to the formation of the apatite layer.According to Hench, these changes in ionic concentrations demonstrated the dissolution/precipitation process: [24] a hydrated silica layer formed at the surface of bioactive glass prior to the deposition of HA, and these silanol groups (Si-OH) could be specific sites for apatite nucleation.The variations of Ca concentration can be explained by Ca 2+ ions release from the glass network, and the consecutive apatite layer formation that causes the Ca concentration decrease in SBF.92S6-C10 and 92S6-C19 glasses have been shown to nucleate HAP more rapidly than 92S6-C22 glass.This was mainly attributed to the large surface area afforded by the glasses synthesized with C 10 TAB and C 19 TAB surfactants.

Scanning Electron Microscopy
Figure 4(a), 4(c), 4(e) show the micrographs of the 92S6-T20, 92S6-T40 and 92S6-T60 samples, respectively, before soaking in SBF.The morphological structure results in the formation of spherical mesoporous particles exhibiting a dense and smooth appearance (high coalescence degree) whatever the synthesis temperature; but the size of the individual particles increases with the synthesis temperature.At 20˚C, the size of the individual particles tends to be smaller with a size of 30 -50 nm.The particles of the material synthesized at 40˚C are made of agglomerates of spheroidal nanoparticles with size of 50 -150 nm.When increasing the synthesis temperature to 60˚C, the size of these spheroidal particles is larger (100 -300 nm) than those obtained for 92S6-T20 and 92S6-T40.This is related to the increase of the nucleation rate (higher supersaturation and polycondensation rate).The formation of hydroxyapatite deposition on the glasses surfaces after immersion in SBF was also observed by SEM.

Conclusions
The solid surface reaction is of particular importance in the field of biomaterials, since they will be in contact with an aqueous medium and in presence of cells and proteins.
Factors such as the length of carbons chain of the surfactant and the temperature of the reaction mixture determine, as it has been shown here, the particle morphology and the porous characteristics of the final mesoporous glasses.

Figure 4 (
b), 4(d), 4(e) show the surface morphology of glasses specimen incubated in SBF for 2 weeks.The surface morphology changed as the synthesis temperature was increased.92S6-T20 surface (Figure 4(b)) shows the formation of submicron apatite grains.With increasing aging temperature to 40˚C, apatite grains (Figure 4(d)) grows like spherical balls with an average diameter of ~1 µm size.The density of the micro spherical particles of 92S6-T60 glass was approximately 3 times higher than those of 92S6-T40 glass (Figure 4(e)).Furthermore, the surface shows clusters with cauliflower like morphology covering the entire surface.

Table 1 .
Textural properties of sol-gel glasses synthesized with different length of the carbon chain template.

Table 2 .
Textural properties of sol-gel glasses synthesized at various temperatures.