Sol-gel as methodology to obtain bioactive materials

We employed the sol-gel methodology to obtain a silica matrix modified with calcium and phosphate ions. We prepared the matrix by hydrolysis and condensation of the precursors triethyl phosphate, calcium nitrate, and tetraethylorthosilicate, which were the sources of phosphate, calcium, and silicon, respectively. We dried and heat-treated the samples at 110 or 900°C and placed them in simulated body fluid (SBF) for three days. We conducted scanning electron microscopy, x-ray diffraction, and infrared spectroscopy analyses, which evidenced that the sample treated at 110°C contained calcium phosphate silicate and hydroxyapatite before and after contact with SBF, respectively. The sample treated at 900°C exhibited a hydroxyapatite phase before and after contact with SBF, but the crystalline phase was more evident after the contact. In conclusion, the sol-gel methodology provided bioactive samples for bone regeneration.


INTRODUCTION
Over the last 20 years, the term hybrid has been used to designate materials with organic-inorganic or bioinorganic characteristics.These materials present new properties and have a multifunctional nature, allowing for novel applications in various fields (Sanchez et al. 2011).In general, organic-inorganic hybrid materials are defined as materials with both organic and inorganic components; therefore, bioceramics cannot be classified as such.However, due to its bioactivity and the interaction established between its inorganic components and the organic constituents in the human body, bioceramics is also considered to be hybrid, which has led to its use in bone regeneration (Vallet-Regí et al. 2011).
Hydroxyapatite (HA) is bioceramic consisting of calcium and phosphate ions; it has the formula Ca 10 (PO 4 ) 6 (OH) 2 .The chemical and biological features of HA have enabled its application in bone and dental tissue restoration (Santos et al. 2005(Santos et al. , kawachi et al. 2000)).HA exhibits a bioactive characteristic; i.e., it can interact with surrounding tissue after implant (Liu et al. 2002).This material is useful in several applications, including its use as drug delivery system (Iafisco et al. 2012, Salimi et al. 2012, Chen et al. 2011, zamoume et al. 2011).Recent articles have drawn attention to the application of calcium phosphate as bone substituent, especially in the HA and tricalcium phosphate form (Bose and Tarafder 2012, Tofighi 2012, Alonso et al. 2012, Kim and Jeon 2012, Hong et al. 2011), mainly in the area of dentistry (Catel et al. 2012, Tirapelli et al. 2010

Various methodologies have been employed
to synthesize HA.Depending on the selected technique -mechanochemical method (Tabrizi et al. 2009), combustion (Ghosh et al. 2011), others (Rodriquez-Clemente et al. 1998, yoshmura et al. 1994, Lim et al. 1999) crystals of different morphology, size, and shape can arise.The sol-gel process is attractive because it uses mild conditions and easily available precursors.Countless literature works have reported the use of this route to prepare calcium-based compounds with applications in biomaterials (Radev et al. 2012, Wei et al. 2011, Heinemann et al. 2011, Jadalannagari et al. 2011, Bandeira et al. 2011, Chatzistavrou et al. 2011, Arcos and Vallet-Regí 2010).
In this study, we used the sol-gel methodology to obtain a silica matrix modified with calcium and phosphate ions.We dried the resulting sample at 50°C and treated it at either 110 or 900°C.To find out whether the synthesized materials were bioactive, we immersed them into simulated body fluid (SBF) for three days.We characterized the samples by thermal analysis (TG/DTA), x-ray diffraction (xRD), infrared spectroscopy (FTIR), and scanning electron microscopy (SEM).

SOL PREPARATION
The Si-Ca-P sol solution was prepared from the precursors triethylphosphate (TEP), calcium nitrate (Ca(NO 3 ) 2 ), and tetraethylortosilicate (TEOS).Briefly, 0.03 mol of TEP were dissolved in distilled water for 24 h, followed by addition of 0.05 mol of Ca(NO 3 ) 2 and stirring for an additional 24 h.Then, 0.05 mol of TEOS in ethanol solution was added, and the mixture was maintained under stirring until gel formation (~ 1 week), at room temperature.The samples treated at 110 and 900°C were placed in contact with simulated body fluid (SBF) for three days.After this period, the samples were centrifuged, washed, dried, and characterized by TG/DTA, xRD, FTIR, and SEM analyses.The solid sample was designated as HAS.
THERMAL ANALySIS (TG/DTA) The thermal analysis (TG/DTA) was carried out in a thermal analyzer TA -Instruments -SDT Q600 -Simultaneous DTA-TG, in nitrogen atmosphere, at a heating rate of 20°C.min - , from 25 to 1500°C.
x-RAy DIFFRACTION (xRD) The x-ray diffractograms were recorded at room temperature on a diffractometer Rigaku Geigerflex D/Max-c equipped with a CuKα radiation monochromator (λ = 1.5405Å).The diffractograms were registered with 2θ values ranging from 5 to 75°, at a resolution of 0.05°.

FOURIER-TRANSFORM INFRARED (FTIR) SPECTROSCOPy
The FTIR spectra were recorded on a Shimadzu model IR PRESTIGE 21 spectrophotometer with Fourier transform, using the kBr pellet technique and a sample/kBr ratio of 1:300.The spectra were measured over the 4000-400 cm −1 region with a 1 cm −1 resolution.Each spectrum was scanned over 1000 times, to increase the signal-to-noise ratio.

SCANNING ELECTRON MICROSCOPy (SEM) AND ENERGy DISPERSIVE x-RAy SPECTROSCOPy (EDS)
The samples were fixed on microscopic support and coated with a thin gold layer on SCD 050 SputterCoater, BAL Tec ® , using a sputtering method.SEM and EDS images were registered on a Carl zeiss Model EVO 50 Cambridge (Uk) microscope.SOL-GEL AS METHODOLOGY TO OBTAIN BIOACTIVE MATERIALS

RESULTS AND DISCUSSION
THERMAL ANALySIS (TG/DTA) Figure 1 depicts the TG, DTG, and DTA curves for the sample dried at 50°C, HAS, which displayed the following mass loss steps: between 26 and 128°C, ascribed to water molecules produced by hydrolysis of the precursors and the solvent (ethanol) employed during the synthesis; between 128 and 262°C, attributed to decomposition of the ethyl groups present in the precursors TEOS and TEP; from 262 to 465°C, due to residual organic product from the decomposition of the precursors; and from 445 to 604°C, assigned to the dehydroxylation of the silanol groups (Si-OH), which gives rise to the inorganic polymer (Si-O-Si) (Bandeira et al. 2011).
The HAS samples placed in contact with SBF and submitted to heat-treatment at 110 and 900°C displayed three mass loss stages.Table I lists the temperature ranges and the corresponding percent weight loss of these samples; Figures 2a and b illustrate their TG, DTG, and DTA curves.
Both heat-treated samples exhibited similar weight loss regions; the main difference lay on the percent weight loss.The first stage, with mass percent loss of approximately 7.5% for both samples, can be attributed to adsorbed water.The second step involved    29.7% and 7.4% mass loss for the samples treated at 110 and 900°C, respectively, and can be ascribed to the residual products of the synthesis and to the dehydroxylation of silanol groups.The last weight THIAGO J. RIBEIRO et al.
loss stage took place in the same region for both samples and can be assigned to HA decomposition, according to equation 1 (Trommer et al. 2007).
Ca 10 (PO 4 ) 6 (OH) 2 → 3 Ca(PO 4 ) 2 + CaO + H 2 O (equation 1) After contact with SBF, the differential thermal analysis (DTA) revealed endothermic peaks at 787 and 792°C for the samples treated at 110 and 900°C, respectively.This peak can be related to phase transition or HA decomposition.
x-RAy DIFFRACTION (xRD) Figures 3a, b, and c depict the xRD of the initial sample HAS, and of the material treated at 110 or 900°C, respectively.HAS, dried at 50°C (Figure 3a), displayed two halos centered at 2θ = 20 and 40°, which indicate the presence of amorphous materials due to the silica matrix.The xRD patterns of HAS treated at 110 and 900°C (Figures 3b and c) exhibited the characteristic amorphous silica halos as well as peaks corresponding to the onset of crystallization.HAS treated at 110°C presented main peaks at 2θ = 9, 22, 24, 25, 28, 33, and 40°, which refer to different phases containing the elements Ca, P, and Si and agree with card 1-471 for Ca(H 2 PO 4 ).xH 2 O, card 3-319 for SiP 2 O 7 , and card 3-335 for Ca(PO 3 ) 2 .xH 2 O. Hence, these phases and others can exist in this sample.HAS treated at 900°C contained peaks relative to HA (card 74-566), and peaks probably relative to other forms of calcium phosphate.The HA card is given in Figure 4.
Figures 5a and b represent the xRD for HAS treated at 110 and 900°C, respectively, after contact with SBF for three days.
The two samples had similar XRD profiles, with peak at 2θ = 25° as well as two groups of peaks, namely in the 2θ region between 30 and 35° and 45 and 55°.The cards 3-747 and 19-272, which correspond to HA (Ca 10 (PO 4 ) 6 (OH) 2 ) and carbonateapatite (Ca 10 (PO 4 ) 3 (CO 3 ) 3 (OH) 2 ), respectively, also present these groups of peaks.For the sample treated at 110°C, the wide peaks were probably due to the poor crystallinity of the material, but we did not detect these peaks before contact of this sample with SBF.The SOL-GEL AS METHODOLOGY TO OBTAIN BIOACTIVE MATERIALS sample treated at 900°C already presented HA before contact with SBF, but the xRD in Figure 5b evidenced increased crystallinity after contact.As previously discussed, the EDS analysis confirmed the presence of the Ca, P, O, and Si elements in both samples.The characteristic halo in the 2θ region from 15 to 35° in the xRD corresponded to amorphous silica matrix.

FOURIER-TRANSFORM INFRARED (FTIR) SPECTROSCOPy
Figures 6a and b show the FTIR spectra of HAS dried at 50°C and treated at 110 or 900°C, before and after contact with SBF.
In Figure 6a, the large band in the 3400 cm -1 region can be attributed to OH and H 2 O stretching.The intense band verified for the sample treated at 110°C suggests the presence of a major quantity of OH groups, which can be ascribed to incomplete condensation of the alkoxide precursors during the formation of the inorganic chain Si-O-Si.Dehydroxylation processes take place at temperatures above 400°C, as previously observed by thermal analysis.The water molecules existing in the sample appear mainly as crystallization water present in the calcium phosphate.Indeed, xRD identified phosphate phases, as discussed above.In the case of HAS treated at 900°C, the vibration modes at 3440 and 641 cm -1 can be assigned to OH groups present in hydroxyapapatite (with molecular formulae Ca 10 (PO 4 ) 6 (OH) 2 ) (Park et al. 1998).The bands at 2984, 2932, and 1430 cm -1 correspond to C-H bonds of organic groups present in the solvent and precursors for HAS treated at 110°C, and to N-O bond stretching of Ca(NO 3 ) 2 in the case of HAS treated at 900°C.The CO 3 2-occurring in the sample treated at 110°C can be identified by the bands at 1638 and 822 cm -1 (Ghosh et al. 2011).The sample treated at 110°C displayed vibration modes between 1200 and 1100 cm -1 , which refer to the precursor Si(OCH 2 CH 3 ) 4 .This indicates that part of the precursor did not react at this temperature.The phosphate groups exhibited vibration modes at 1348, 1226, 1062, and 960 cm -1 (Guan et al. 2005), corroborating the presence of phosphates previously evidenced by xRD.
As for Figure 6b, the free PO 4 3-ion presented tetrahedral symmetry in aqueous solution, and the corresponding vibration modes appeared at 938 cm -1 (ν 1 ), 420 cm -1 (ν 2 ), 1017 cm -1 (ν 3 ), and 567 cm -1 (ν 4 ).ν 1 relates to the non-degenerate stretching P-O, ν 2 refers to the doubly degenerate bending, and ν 3 and ν 4 correspond to the triply degenerate stretching (Park et al. 1998).When crystallized in the apatite form, the PO 4 3-ion can be distorted, and these bands are indeed displaced in the FTIR spectra of the samples after contact with SBF.In Figure 6, the non-degenerate band appeared as a shoulder at 959 cm -1 , and its low intensity can be attributed to a large and intense adjacent band.The wide band centered at 470 cm -1 can be ascribed to the doubly degenerate modes of asymmetric O-P-O.The stronger band between 1117 and 1038 cm -1 can be assigned to two components, namely asymmetric ν 3 and Si-O-Si stretching of the silica matrix.The bands at 603 and 562 cm -1 relate to the triply degenerate O-P-O modes.The stronger band at 3410 cm -1 refer to the stretching of water and/or OH from HA and the silanol groups present in the silica matrix (Park et al. 1998).The stretching at 3224 cm -1 can be ascribed to -C=O of the carbonate apatite (Mohamed et al. 2011).
SCANNING ELECTRON MICROSCOPy (SEM) AND ENERGy DISPERSIVE x-RAy SPECTROSCOPy (EDS) Figure 7 brings the SEM images obtained for HAS treated at 110°C.The sample displayed particles larger than 20 µm as well as undefined shape, and the particles presented cracks (Figure 7a).We used the region indicated in Figure 7a for the EDS analysis.We were able to detect the elements Ca, P, Si, O, and C all over the region, confirming the XRD and FTIR analyses.Figure 7b corresponds to enlargement of region 3 and shows different morphologies for the sample, suggesting the formation of two distinct phases.Region 3 (circle) in Figure 7b revealed aggregate particles, and the EDS analysis demonstrated the major presence of the elements Ca and P in this region, as compared to region 1 (square).This indicates that calcium phosphate and silica phases arose independently.
The sample treated at 900°C also displayed large particles and irregular shape, but the morphology attested to better homogeneity.Figures 8a and b correspond to the SEM micrographs for HAS treated at 900°C.In Figure 8a, regions 1 and 2 show where the EDS analysis was accomplished and where Ca, P, O, and Si elements were detected.The EDS evidenced different Ca/P molar for the sample treated at 900°C, suggesting loss of phosphate ions.More specifically, the Ca/P ratio was 5:1 and 3:1 in regions 1 and 2, respectively.The EDS analysis performed for the sample treated at 110°C furnished a Ca/P molar ratio of 1:8.The samples treated at 110 and 900°C were placed in contact with SBF for three days, centrifuged, washed, dried at 50°C for 24 h, and characterized by TG/DTA, xRD, FTIR, and SEM analyses.
The samples treated at 110 and 900°C and placed in contact with SBF for three days displayed different morphologies, as seen in Figure 9.
Figure 9 evidenced two kinds of particles for the materials, namely agglomerate and plates, as verified for the samples before contact with SBF (Figure 7).However, the agglomerate appeared in major quantity after contact with SBF, indicating formation of a new phase, probably HA, as revealed by the XRD and FTIR analyses.HAS treated at 900°C exhibited distinct morphologies before and after contact with SBF. Figure 9b

CONCLUSION
The sol-gel methodology is a chemical process that allows for the synthesis of biomaterials.The characterization studies accomplished here showed the presence of the inorganic components of the bone, carbonate and hydroxyapatite, in the synthesized samples after contact with SBF, indicating that the materials produced by this methodology act as osteoinductor.The low temperature required by the sol-gel method opens the perspective of obtaining biomaterials as bone substituent.Our findings are also important for drug incorporation into the samples, which can be used as drug delivery systems.

Figure 1 -
Figure 1 -Location of the study area on Areuá Island, Curuçá, State of Pará, Brazilian Amazon.

Figure 2 -
Figure 2 -TG, DTG and DTA curves for HAS treated at (a) 110°C or (b) 900°C after contact with SBF for three days.

Figure 3 -
Figure 3 -XRD of the sample HAS dried at 50°C (a), treated at 110°C (b) and treated at 900°C (c).

Figure 5 -
Figure 5 -XRD for the samples treated at (a) 110°C or (b) 900°C after contact with SBF for three days.

Figure 6 -
Figure 6 -FTIR of the samples HAS dried at 50°C and treated at (a) 110°C and (b) 900°C (A), and after contact with SBF for three days, (a) 110°C and (b) 900°C (B).

Figure 9 -
Figure 9 -SEM micrographs of the samples treated at (a) 110°C or (b) 900°C after contact with SBF for three days.
Figure9evidenced two kinds of particles for the materials, namely agglomerate and plates, as verified for the samples before contact with SBF (Figure7).However, the agglomerate appeared in major quantity after contact with SBF, indicating formation of a new phase, probably HA, as revealed by the XRD and FTIR analyses.HAS treated at 900°C exhibited distinct morphologies before and after contact with SBF.Figure9battested to the formation