Alginate/Bioactive Glass Beads: Synthesis, Morphological and Compositional Changes Caused by SBF Immersion Method

Composites of biodegradable polymers/bioactive glasses (BG) are of particular interest in bone repair, where ceramic phase can improve osteogenic features. This work reports the development of organic–inorganic beads based on alginate prepared by incorporating at polymer suspension different contents (25, 50 and 75 wt% with respect to alginate) of BG present at precursor solution, whose system (SiO2–CaO–P2O5) was obtained by hydrolysis of both tetraethoxysilane (TEOS) and triethylphosphate (TEP) alkoxides, molar ratio TEOS:TEP of 1:0.1317, and which calcium source came from the same cold CaCl2 solution used to gelling the hybrids. Beads were synthesized by extruding mixtures, drop by drop, in CaCl2 solution. Were also investigated the behavior resulting upon soaking of hybrids into SBF. Samples were characterized by SEM, ATR-FTIR, TGA and XRD analysis. Results indicated decrease in spheres size with increase of glass and presence even pos-immersion, of Si, Ca and P associated to BG phase.


Introduction
Bioactive glass particles, when in contact with body fluids, are able to bond to living bone through the formation of a surface layer of hydroxyapatite (HAp), as a result of their high reactivity in aqueous media. Moreover, the products of the ionic dissolution of bioactive glasses (Si and Ca) can lead to favorable intracellular and extracellular responses, actively stimulating bone formation [1][2][3][4][5][6] . Another advantage of bioactive glasses includes their antibacterial properties against a wide range of pathogenic bacteria 7,8 .
Hench identified the compositional limits for bone and soft tissue bonding in the SiO 2 -Na 2 O-CaO-P 2 O 5 glass system, showing the best composition as 45%SiO 2 -24.5%Na 2 O-24.5%CaO-6%P 2 O 5 wt% 1,9 . Over the course of the years, several other types of bioactive glasses were synthesized with silicon varying from 21 to 75wt%, for materials synthesized by conventional meltquenching 10 . As stable bioactive gel-glass could be made by sol-gel processing, bioactivity in SBF was demonstrated for gel-glass compositions up to 90% SiO 2 9,11 . The bioactivity of bioactive glass is not an absolute concept determined only by the composition, but is also affected by the size and shape of the material 12 .
When compared with microparticles, nanoparticles have a significantly higher surface area and this drastically changes the material characteristics, such as surface energy, wettability, surface topography, and surface chemistry. Furthermore, studies have shown that nanoscale materials have higher biocompatibility 13 .
A problem that can be observed with the use of very fine-grained materials to aid in the formation of bone tissue, is associated with its degradation, where it can found that the use of sol gel derived silica nanoparticles show faster surface dissolution, associate with high area and reactivity 14 .
Some actual bone substitutes can be found in different forms, as example in block or granular form, which they are then used to fill the bone defects. However, each form is associated with problems. The block form tends to have a low filling factor 15 and therefore requires curettage or other related measures at the filling site 16 and cells exist only in the area near the surface of scaffolds 17 . With the granular form, variations tend to arise in filling density, and pore maintenance is difficult 16 . Nevertheless, recent studies pointed out the favorable properties with use of granules 15,17,18 , with some systems been obtained by adding powders at micrometer scale on polymeric materials 6 . As result scaffolds can be fabricated by accumulating spheres to create a porous network and act as osteoconductive material 15 .
Another advantage of bioactive beads used as filler have been demonstrated by acquire a macropore architecture, where the convex arc of beads was markedly beneficial for bone formation in comparison of pore architecture with concave arc shape. The suggested mechanism for scaffolds osteogenic activity was attributed to which convex surfaces were more conductive to liquid flow and cell growth 19 . In addition, adjusting the packing of the beads can promote porosity and pore size compatible with cell proliferation and blood vessel migration 20 .
If osteoblast-like cells can adhere and grow at the surface of polymers based microparticles where these substrates could be used for: (i) nonload bearing applications or as part of a 3D-construct; (ii) cultivating anchorage-dependent cells in a dynamic bioreactor and (iii) encapsulating bioactive molecules in the microparticles and simultaneously growing cells at the surface of the microparticles that would release encapsulated growth factors to stimulate proliferation and differentiation of adherent cells 21 .
Thus, produce polymer in hydrogels form associate with a bioactive glass phase, it can bring additional functionalities 22 , including crosslinking which was shown to affect the elasticity, Young's modulus, and the cell adhesion 23 .
As example for polymer that is able to produce hydrogels we have alginate 24,25 , which can stablish crosslink from its G blocks 26 . Due to its biocompatibility, biodegradability, ability to chelate calcium ions and to absorb large amounts of water, alginate has been widely used in a variety of biomedical applications including tissue engineering and drug delivery [27][28][29] .
In regard with hybrid hydrogels, one of the approaches to induce crosslinking of anionic polysaccharide including the so-called internal gelation, employing such inorganic fillers as Ca 2+ ions which crosslink the polymer suspension leading to the formation of a homogeneous hydrogel network 27,28 . Another inorganic compound, that can be used as a source of Ca 2+ ions for internal gelation, as well as for source of silicon groups are the bioactive glasses 8,30 .
In this context, this work presents the production and characterization of hybrid microparticles based on alginate and bioactive glasses it can be able to promote polymeric structure blended with a dispersed inorganic phase of bioactive glass at nanometric scale (of Si-Ca-P sol-gel system), where the chemical composition and morphology can promote a significant effect on the bioactive properties of material. In addition, we hypothesize that production of spheres by controlling its diameter can be able to control the pore size of beads accumulated as 3D scaffolds in bone defect sites, and fulfill requirements for osteoconduction and vessel migration. Moreover, they can regulate the release of glass degradation products in order to increase bioactivity, compatible with the time required in the process to repair the calcified tissues. Finally, samples were kept in SBF following by morphological and chemical characterization.

Preparation of hybrid samples
The processing route used for synthesizing the organicinorganic hybrids beads was based on sodium alginate and sol-gel-derived bioactive glass (BG) as illustrated in Figure 1.
To obtain the composite was used alginic acid sodium salt from brown algae, medium viscosity, supplied by Sigma Aldrich and selected considering aspects such as the molecular weight (Mw = 100,000 g.mol -1 ) and mannuronate/guluronate ratio (M/G = 1.56). Deionized water (DI-water, Millipore Simplicity) with resistivity of 18MΩ•cm was used in glass solution preparations and distilled water in dissolution of simulated body solution (SBF).

Preparation of the sodium alginate suspension (Sol 1)
Aqueous suspension of alginate was prepared with concentration 1 wt. (%) by dissolving the alginic acid powder (1 g) in 100 mL of distilled water, under constant stirring, for 24 h. The pH of the suspension was adjusted to 5.3±0.1 by acetic acid (AcOH) solution 1N.

Preparation of the starting bioactive glass solution (Sol 2)
The starting sol solution (Sol 2) was synthesized by mixing tetraethoxysilane (TEOS, Sigma), D.I. water, triethylphosphate (TEP, Sigma), in presence of hydrochloric acid solution 2N. In this context the solution was obtained using reagents with sufficient amounts to reach a system of 86.5%SiO 2 -13.5%P 2 O 5 wt%, which has the same weight ratio of SiO 2 /P 2 O 5 found at 58%SiO 2 -33%CaO-9%P 2 O 5 wt% system of our previous works 31,32 . However, in the present study, specifically regarding calcium content, the addition was achieved by crosslinker solution, with internal gelation taking place due slowly diffusion of calcium after the mixture of Sol 1 with Sol 2 to be dropped on cooled CaCl 2 solution, as describe the item 2.1.3.
To obtain that glass solution, deionized water acidified with hydrochloric acid (H 2 O/H + ) had adjusted to pH = 2. Then solution was prepared in a separate beaker with 2.1 mL H 2 O/H + and 2.2 mL tetraethylorthosilicate (TEOS) mixed in the given order and then allowed to react for 45 minutes at ambient temperature, when 0.2 mL triethylphosphate (TEP) was added and reacted for a further 30 minutes. The mole ratio of (H 2 O/H + ):TEOS:TEP was 12:1:0.1317 and HCl was added to catalyze TEOS and TEP hydrolysis.

Preparation of the Alginate-Bioactive glass hybrid beads
The compositions of hybrids for all prepared samples are shown in Table 1. The beads were obtained using a procedure similar to the one described in our previous work 33 . In short, an appropriate amount of starting glass solution was added to alginate suspensions and was stirred for approximately five minutes at the temperature of 25±5 °C. Sol 1/Sol 2 in the final solution were made up of volume ratios 1:0.33, 1:1 and 1:3, resulting in final volumes of about 101.5, 104.5 and 113.5mL, respectively. After that 10 mL of each mixture was introduced dropwise from a 15mL syringe with a 27.5 gauge needle into 100 mL of an aqueous calcium chloride solution 1M, whose container where then sealed, and cooled down to the temperature of 7±2 °C for 7 days, as diagram shown in Figure 1. These hybrids were prepared in triplicates (n=3) and referred as indicated in Table 1. After synthesis all samples were submitted to dry process in a desiccator (silica gel, under vacuum) for 7 days at the temperature of 25±5 °C.

Structural and Chemical Characterization of Hybrids
The particle size determination of wet beads obtained was carried out using a high-resolution digital camera with a ruler placed into the image, which was used as reference scale. The average sizes and standard deviation were determined for each formulation using images acquired by thirty beads randomly selected and evaluated by image analysis software ImageJ. Data were analyzed by one-way analysis of variance (ANOVA).

Morphological analysis of particles by scanning electron microscopy (SEM)
The SEM analysis was utilized to provide an initial qualitative evaluation of the changes on the surface structure and morphological aspects of the composite hydrogels when bioactive glass was added. The samples were dried in a desiccator (silica gel, under vacuum) at the temperature of 25±5 °C. After that the samples were covered with gold and analyzed using an SSX-550 Superscan (Shimadzu) microscope operated at 15 kV and equipped with an energy dispersive X-ray spectroscopy (EDS) unit (Shimadzu).
Before examination, the samples were coated with a thin gold film by sputtering at a low deposition rate using a SC701 Quick Coater device (Sanyu Electron) at the current 1.5mA for 3 minutes. The maximum distance between target and sample was used to avoid sample damage.

Chemical characterization by FTIR spectroscopy
Fourier transform infrared spectroscopy (FTIR) was used to characterize the presence of specific chemical groups in the Alginate/Bioactive glass hybrid samples. FTIR spectra were obtained using attenuated total reflectance (ATR, 4000-400 cm −1 using 68 scans and a 4cm −1 resolution -Shimadzu Corporation, model IRPrestige-21), equipped with ATR accessory DuraSampl IR II, using a ZnSe crystal.

Crystallinity and phase characterization by
X-ray diffraction X-ray diffraction (XRD) patterns were obtained from hybrid Alginate/Biactive Glass using CuKα radiation with λ=1.54056 Å (XDR 7000, Shimadzu). XRD analyses were conducted in the 2θ range from 5.0 -80.0 o with steps of 0.02 o . The observed peaks were identified using the "Software XPowder".

Thermal analysis
Simultaneous TG-DTA curves were obtained by using the thermal analysis equipment SII EXSTAR, model TG/DTA 7200, using N 2 as purge gas with flow rate of 90 mL.min -1 . Heating rates of 10 ºC (N 2 ) was adopted using alumina crucibles between 25 and 900ºC for the samples with masses around 5.0 mg

Degradation upon immersion in SBF -In Vitro Assay
Bioactivity and dissolution behavior of the hybrids were studied by immersion in Simulated Body Fluid (SBF) prepared following the Kokubo protocol 34 at the ion concentrations (Na + 142.0; K + 5.0; Mg 2+ 1.5; Ca 2+ 2.5; Cl -147.8; HCO 2 -4.2; HPO 4 2-1.0; SO 4 2-0.5 mM) similar to that of blood plasma. Samples of particles alginate/bioactive glass were soaked in SBF at an approved rate of 0.01g.mL -1 in sealed polyethylene containers. The flasks were place inside an incubator at controlled temperature of 37 o C during 28 days. The samples were analyzed for periods of 0, 7, 14, 21 and 28 days, with the solution being refreshed every 7 days. Once was complete immersion period, the samples were rinsed gently in deionized water, and dried at 25 o C for 48 hours in a desiccator for further characterization.

Characterization of synthesized samples
Biomaterials at form of beads have been used in various biomedicine-related fields, as vehicle for drug delivery system 35 , carriers for cell culture in vitro 36 and bead assembled system of 3D implant 20 which spheres accumulated scaffolds can act as macropore platform. It is generally accepted that macropore size for osteogenesis should be > 100 μm, and for vascularization should be 300 μm 37,38 .
Therefore, some studies showed beads of radius R packed presenting macropore size ranging from 0.73R to 2R, formed by arrange of 3 and 5 spheres, respectively 19 . At this case it can be found that macropore size is > 600 μm for beads with radius about 900 μm. Moreover, if spheres are arranged in hexagonal close packed, hypothetical pore radius that could fit into the tetrahedral arrange will be 0.225R and for octahedral arrange will be 0.414R 39 .
In this study the production of beads composed of alginate/bioactive glass with desired size and spherical shape were obtained for different concentration of the inorganic phase added on the polymer suspension. We note a decrease in the medium diameter size of wet beads, from 1,892μm in samples composed exclusively of alginate, to about 1,686μm for beads with larger concentrations of bioactive glass (75 wt.%), as showed in the Table 2.
According to ANOVA analysis there was no significant differences (F-ratio = 3.89; p-value = 0.0002) between samples of 100AS-0BG and 75AS-25BG. In addition, ANOVA indicated significant variation in the size of all beads with more than 50 wt% of bioactive glass in the composition (p<0.05). However, all samples showed size of beads able to produce macropores associate to osteoconduction.

Scanning Electron Microscopy (SEM)
Several reports show hydrogel beads prepared by using an extrusion method 20,40 , that approaches have been studied incorporating bioactive inorganic material into alginate, mainly using phosphate or silicate 6,41 based bioactive inorganics. In general, the beads are obtained incorporating a small amount of inorganic powders at micrometer scale into alginate suspension 6,42 . On the other hand a silica-alginate hybrid was obtained by the in-situ synthesis of silica networks via sol-gel process from (TEOS) and (APTES) precursors in an alginate matrix 43 . In our proposal was used a sol-gel route introducing a solution of both silicon and phosphorus hydrolyzed alcoxides into polymer suspension prior to the gelation process aiming to produce hybrids specially in the form of beads.
The morphology of dried particles in all formulations studied could vary depending upon preparation compositions 40 . For samples 100AS-0BG were verified shape like pear, with one region round and narrowing towards the opposite side, but without a distinct tail, as shown in the Figure 2a.
In general, increasing the glass leads to decreasing on the polymer concentration of feed solution, which tended to increase the corrugated degree of particle surface to a more irregular shape that it departs considerably from a circle, as the raisin-like particles presented in the Figures 2b, 2c and 2d.
The observed results for such morphological changes suggest the role of alginate as a matrix in the composite, where the structure does not remain stable during the drying process in low polymer concentrations.
Scanning electron micrographs of the microparticles are presented with magnifications in Figure 2. Alginate microparticles without glass (detail of Figure 2a) exhibited surface of smoother morphology. This finding is consistent with the data of Laia 44 who demonstrated that alginate blends had a smooth and regular surface without irregular cavities.
The microparticle composites of 75AS-25BG, 50AS-50BG and 25AS-75BG samples (Details of Figure 2b, 2c and 2d) were more grooved and rough compared with alginate beads. The addition of (25 wt.%) of bioactive glass led to surface microrelief more corrugated with striation in random orientation. The composition with (50 wt.%) of bioactive glass had the similar morphology of (25 wt.%), however the striation had a narrower size. A concentration of (75 wt.%) bioactive glass showed thin and aligned grooves in a single direction.
Crosslinking the hybrids by adopting the low temperature allowed to control the gelation and permitted to extend the chelation through internal structure of beads. That it may arises due slowly diffusion of calcium to inner regions, associate with decrease in the degree of gelation at the interface of hybrids, by cooling the CaCl 2 solution 28 .
These findings associating an increase in surface smoothness of the microparticles with an increase in degree of crosslinking may be due to the high calcium-binding capacity to alginate and bioactive glass. Where showed that the polymer nature and degree of crosslinking had a significant effect on the morphology of alginate-bioactive glass beads.

Fourier transform infrared spectroscopy (FT-IR)
Representative IR spectra of pure alginic acid powder and the hybrid particles alginate/bioactive glass (AS/BG)  are shown in Figure 3. The broad band observed from 3100 to 3600 cm -1 in all spectra may be assigned to O-H stretching due the strong hydrogen bond of intramolecular and intermolecular type 45,46 . The band at 2850-2950 cm -1 was attributed to alkyl stretching mode (νCH). The absorption bands at 1600 cm -1 and 1410 cm -1 decreases due to the asymmetrical and symmetrical stretching vibrations of COO − in alginate.
Other typical peaks which can be associated with alginate are located at 1290cm -1 and 1030cm -1 both assigned to C-O-C group 46,47 . In the FTIR spectrum of the hybrids with bioactive glass, presented in Figure 3, the bands related to Si-O-Si asymmetric and symmetric stretching modes are observed at approximately 1070 cm -1 and 450 cm -1 , respectively [48][49][50] . Also, the vibrational band at 950 cm -1 has been credited to the presence of silanol groups (Si-OH) 31 usually found in silica synthesized via sol-gel method. FTIR spectrum of hybrid AS/BG, presented in Figures 3c, 3d and 3e, have also presented broad bands in the frequency ranging from 3000 to 3650 cm -1 attributed to both contributions of hydroxyls (AS) and silanols (BG). In the range 1500-900 cm -1 there is a superposition of the bands derived from the bioactive glass and the alginate components. Therefore, bands 1600cm -1 and 1410cm -1 are associated with the asymmetric and symmetrical stretches, respectively, of the carboxylic group (COO -) [45][46][47]51 , which was intense in samples prepared with (50 wt.%) bioactive glass and had a significant reduction for samples prepared with addition of (75 wt.%) glass.
The bands presented in 1978, 2023 and 2160cm -1 can be associated with carbon based gases from surrounding atmosphere on pores of samples, originated in the matrix structure during the drying process. It is also observed the presence of bands in 953, 600 and 560cm -1 associated with the phosphate groups formed in the bioactive glass 4,52 .

In vitro study in SBF solution of alginatebioactive glass hybrid beads 3.2.1. Hydrogels surface analysis
The results of SEM analyses for beads samples, after immersion in SBF for 28 days, are presented in Figure 4. As a general trend the morphological aspects of hydrogels showed as corrugated for all compositions. Since the particles were produced from alginate suspensions (1.0% m/v), and that the sol of glass added may resulting in mixtures with water rate above 95%, it can be expected to the obtain spheres with high degree of hydration. Therefore, a high contraction for the samples should be plausible through drying process 33,53 . Furthermore, must also be considered that soaking time in SBF is a favorable occasion when can occur reactions between solution and materials promoting degradation of its structure [54][55][56] .
Moreover, immersion in SBF permits to evaluate the ability of ions to precipitate on the surface of particles 34 , which was verified for all samples. Even alginate samples showed regions with precipitations, although less prominent,

Thermal analysis by TGA/DTA
As presented in Figure 5, the mass loss profile was considerably similar for all hydrogel compositions, where few differences were observed in their thermal decomposition behavior.
Thermal analysis of samples pre immersion in SBF showed mass losses in the temperature range of up to 200 o C between 20 and 25%, compatible with the dehydration of the samples 57,58 , as shown in Figure 5. The range between 200 o C and 530 o C indicated mass reduction around 47% for alginate beads and approximately between 31% and 43% for hybrid samples.
Although a significant reduction in mass loss would be expected for gradual reduction of polymer, according to Fiume et al. 59 most of the mass reduction of glass can be  attributed to residual water in the gel and decomposition of organic compounds of sol precursor. This can indicate that the reduction in polymer would be compensated to the same extent with higher levels of water associated on glass, that must justify the loss of mass with values very close for such temperature range.
Analogously, the results shown a similar trend associated with residues levels below of glass added. However, if the water content associated with glass on hydrogels is not considered in the calculation, it can found values agreed with the nominal composition.
From the DTA curves were verified an evident thermal event for samples 75AS-25BG, in temperature ranging from 550 and 660 o C, which corresponds to the sintering zone of bioactive glasses 60 . Both results can be associated with the presence of glass network around the polymer matrix, without complex formation between silicon and alginate. The segregation can be confirmed by the surface texture on SEM images for samples with (25 wt.%) glass.
The TGA curves revealed at 7th and 28th days posimmersion that the water content of samples had a gradual decrease until 200 o C, for an increase of immersion time. Moreover, the residual mass accounted had highest values associated with longer soaking time. Both results are associated to the manner for calculate the mass loss, where a number used as denominator (initial mass values of wet beads) varies from a higher value at time zero, to a smaller value with the increment in the soaking time (lower degree of hydration). Where hybrids, before immersion on SBF, may have a high degree of water coupling by the gel of glass incorporated on the composites 59 .
Mass reductions were also observed for TG curves at range of 800 o C for all studied samples, which can be associated to conversion of the remaining materials to carbon residues and calcium carbonate 61 , which may suggest the significant presence of calcium based compounds after 28 days in SBF.
Regarding the analysis by DTA, similar behaviors were verified for all curves, with emphasis for samples with (25 wt.%) and (75 wt.%) of glass, immersed for up to 7 days, whose indicated exothermic peak at 540 o C associated with the glass content in the material composition, that the literature 62 identified to appear upon heat treatment of bioactive glasses at about 600°C. For samples with (50 wt.%) of glass, such peak is not observed, likely caused by several chemical interactions involving glass and alginate structure at the hybrid particles.

Characterization by energy dispersion X-ray spectroscopy (EDS)
A chemical analysis of EDS was carried out and the results are shown in the Table 3 and the graph inserted in Figure 6. The chemical elements Si, P, and Ca were identified and corresponding to the composition of bioactive glass.
At the 28th day it is possible to identify a peak related to sodium chloride (ICDD 9-432), that may have precipitated from the SBF solution, once time this solution has a high concentration of Na + and Clions.
Percentage concentrations calculated for phosphorus, calcium and silicon, by dividing the concentration of each element by the total concentration of these three elements and multiplying by 100%, demonstrated phosphorus and silicon were higher in all hybrids pos-immersion, while Figure 6. The change in the surface concentration of silicon, phosphorus, and calcium over time after hydrogels immersion in SBF. Table 3. EDS compositional analysis performed on particles.

Chemical characterization of hydrogels by FTIR spectroscopy
The FTIR analysis comparing samples before and after immersion in SBF are shown in Figure 7. For FTIR spectra of 100AS-0BG samples, it was verified that the band at 3500cm -1 associated with OH groups, has its intensity increased after one week of immersion, followed by decrease with 28 days. These changes may indicate a high initial ratio of alginate degradation in the initial 7 days, especially for regions with lower degree of cross-linking by the calcium ions. Therefore, residual structure of samples upon SBF soaking for 28 days must correspond to the portion of alginate with a high degree of gelation 63,64 , which limits the polymer chains ability to bind water molecules 65 , as they have fewer carboxylate groups. These observations also can be associated with EDS results, which shows that amount of Ca decrease at 7 days of immersion and increased after 28 days. Furthermore, it can be verified a reduction followed by an increase in the bands at 1600 and 1400cm -1 of COOgroups and by the same oscillation for the rate of volatiles observed in thermogravimetric analysis, which can be associates to the fewer loss mass of organic phase.
Regarding intensity of bands 1030 and 1070cm -1 , a progressive increase was observed for the periods of 7 and 28 days. This result may be associated with phosphate precipitation, which is also confirmed by an increase in phosphorus observed in EDS. A speculation for this event may be associated with large amount of calcium available from alginate gelation, where it can increase the precipitation of phosphates for 7 days, following the same behavior for 28 days. The high precipitation of salts may explain the reduction at OH, COOand COOH bands, as well as the maintenance in the intensity of the band in 1070cm -1 , and the appearance of the band centered at 780 and 600cm -1 .
At the opposite extreme, samples with (75 wt.%) BG showed high intensity for the 3500 bands; 1600; 1400; 1080; 1030; 950; 820; 600 and 560cm -1 meanwhile there were a reduction in all these bands after 7 days. For 28 days immersion a decrease of 3500cm -1 , stability of 1600cm -1 and an increase for bands 1400, 1030 and 950cm -1 were observed, which may suggest the presence of bioactive glass combined with the polymer. In addition to these findings, a tendency for the presence of phosphate was also supported by EDS analysis.
For samples with (75 wt.%) AS the FTIR spectra posimmersion in SBF at times of 0 and 7 days were practically the same, with exception for a less intense band at 3500cm -1 for the period of 7 days. Compared to samples of 100% AS, all bands for (75 wt.%) AS samples were less intense. Increase in the bands of these samples was observed at 28 days, and the vibrations 1020, 820 and 600cm -1 showed increases significantly higher than those of the other vibrations, which may indicate the presence of the glass stabilizing the structure of material 4,52 .
Similar characteristics regarded for the samples with (50 wt.%) AS were showed in FTIR at 0 and 7 days, including their intensities. After 28 days of immersion in SBF all bands increased slighty, including the 3500 cm -1 . Hence, the suggestion is that the glass may have physically blocked the polymer, results that can be supported by SEM images, with the presence of aligned polymer structures, as shown in Figure 7b and represented by scheme of Figure 8.
Considering FTIR characterization and the SEM images of Figure 7a, it is found particles of 100% alginate of flat and smooth surface covered with precipitates, samples of (25 wt.%) of AS resulting in a glass structure with polymeric incrustations and samples with (50 wt.%) of AS revealed grooves of polymers associated with fine particulate, suggesting also the interaction between the materials, as shown in Figure 7c.
Silica-alginate hybrid hydrogels prepared by a solgel were also performed by Hernández-González et al. 43 . The authors observed that silica nuclei grown through the surface and inner face of the alginate matrix. In addition, the catalyst absence favored an interpenetrated silica/alginate network in which the alginate structure was preserved, and the polycondensation of Si-OH groups took place within this structure resulting in a better distribution in the hybrid structure.
Thus, the samples with (50 wt.%) AS obtained in our work, which represents intermediate levels of silica should limit the growth of glasses in the alginate matrix. In addition, the alginate content of these hybrids may be sufficient to restrict the growth of the glass nuclei. These results may indicate a composition of matrix capable to act as of bioactive glass reservoir, modulating its degradation and function as support for cells growth.

Crystallinity and phase characterization by
X-ray diffraction Figure 9-II present the XRD patterns of alginate spheres cross linked with calcium at 0 and 28 days of immersion in SBF solution media. The XRD patterns of the hydrogels before immersion revealed a broad diffraction bands, which is typically associated with guluronic acid blocks at 15° and manuronic acid blocks at 22° [66][67][68] . It can be observed that diffractograms of hybrids based on alginate with bioactive glass start to present a reduction on peak intensity mainly for adding of 25% glass (Figure 9-III). In addition, a slight reduction in both peaks at 15 and 22° were observed for the other glass concentrations, whose diffractograms were not inserted, but which showed a similar aspect between them, indicating the contributions from alginate with semi-crystalline structure 69 and amorphous phase of bioactive glass, as found by Fiume et al. 59 for glasses obtained by the sol-gel process. This result suggests that glass phase was well dispersed in the alginate matrix 43 .
Conversely, the results of XRD diffraction of all hydrogels (Figure 9) after 28 days of SBF assay presented peaks at 27 o , 32 o , 45°, 57°, 66°and 75°, which evidenced the formation of new crystalline phases and values matched well with the standard ICDD reference pattern 9-432. According to the . SEM analysis of hybrids (at magnification 1000x) presented in details showed in rectangle of (a) thin striation; arrows of (b) grooves of aligned polymer structures and circles of (c) polymeric incrustations. literature, the appearance of these peaks can be associated with sodium chloride 70 resulting from the precipitation of SBF salts during the drying of samples.
Although several peaks associated with sodium chloride can be overlapping with calcium phosphate compounds, the inserts of Figure 9-III show details of the peaks localized

Conclusion
Alginate/bioactive glass particles in desired size and spherical shape were successfully obtained with varying structural properties for different concentration of glass added.
The processing route used for synthesizing the inorganicorganic hybrids controlling gelling temperature was able to have incorporated the elements Si, Ca and P that play a special role in bioactivity.
Samples were analyzed by studying the surface of the beads with SEM, EDS, FTIR and XRD after they have been immersed in the SBF for some time. Even upon soaking into SBF for 28 days, the structure of beads remains with your surface available to interact with chemical compounds of medium that can significantly favored apatite deposition with increasing immersion time in SBF. As shown on the surfaces with some precipitate, which are formed by small crystalline aggregates.
Between the compositions of beads, we can propose as better the sample with (50 wt.%) AS. Although these samples have intermediate levels of glass in their composition, such ratio of bioactive glass based gel compared to alginate demonstrated ability to maintain the sphere shape. In addition, (50 wt.%) AS beads seems do not inhibits the activity of glass, where could be observed gradual reduction in silicon levels and progressive increase in phosphate and mainly the calcium levels during SBF tests. The results were also confirmed by phosphate groups at FTIR and by precipitates showed in SEM images.
Thereby, the modifications in the hybrids features from surface erosion include the partially degradation due to polymer disentanglements forming some products which may diffuse outwards. Moreover, the inorganic components may also be degraded which can provide controlled release of ionic specimens to act as potential use in bone repair, particularly to low-load or nonload bearing applications.