FIBROUS SILICA-HYDROXYAPATITE COMPOSITE BY ELECTROSPINNING

New nanocomposite membrane was fabricated by electrospinning. The nanocomposite combines a glass and hydroxyapatite (HA). This research proposed the incorporation of glass to counteract the brittleness of HA in a composite formed by coaxial fibers which will be used for bone replacement. Calcium phosphate ceramics are used widely for dental and orthopedic reasons, because they can join tightly through chemical bonds and promote bone regeneration. Precursors HA and SiO2 were synthetized through the sol-gel method and then incorporated into a polymeric PVP matrix; later they were processed by coaxial electrospinning to obtain fibers with an average diameter of 538 nm which were characterized with techniques such as Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy, Differential Thermal Analysis and Scanning Electron Microscopy. Chemical and physical characterization of the membranes evidenced fibers in a coaxial disposition with a glass core and hydroxyapatite cover. The microporous fibers have many potential uses in the repair and treatment of bone defects, drug delivery and tissue engineering. Through ATR-FTIR and SEM-EDX analysis the presence of characteristic chemical groups, the fiber composition and microstructure were determined.


Introduction
Until very recently the repair of the skeletal system was performed with the use of bone grafts, however, the difficulty of obtaining such materials from natural sources and donors allowed the introduction of non-biological materials which can be easily obtained through synthetic processes while they are also less likely to produce antigenic reactions (Dinarvand et Roveri et al., 2003). Significant efforts have been made to the field of biomaterials and bone tissue engineering. Glasses and ceramics can be bioactive they have the capacity to form an interphase with living tissue through physical and chemical interaction (Hench and Kokubo, 1998). Bioactive ceramics are used primarily for the reconstruction or substitution of parts of the skeletal system (Boccaccini, 2005). Calcium phosphate ceramics and silica bio glasses are used widely for dental and orthopedic reasons, due to their bioactive properties, while promoting bone regeneration in the area (Leeuwenburgh et al., 2008).
Hydroxyapatite (Ca10(PO4)6(OH)2) is the primary mineral component of human bones and teeth, it is a bioceramic, and can be obtained through a variety of chemical reactions such as hydrolysis or sol-gel method. Although this compound has been researched for the replacement of hard tissue, there are several characteristics that restrict its use in biomedical applications: absence of antimicrobial properties, brittleness and limited contact with the host tissue (Basu and Balani, 2011 Through the electrospinning technique membranes of nanofibers of different compositions can be produced, to be used as scaffolds for tissue reconstruction purposes. There is no doubt that the electrospinning has gained popularity in recent years for bio-dental applications mainly for tissue engineering scaffolds. The progress of tissue engineering is promising for the regeneration of oral and dental tissues. Various polymer and ceramic composite materials have been electro spun to fabricate scaffolds for regeneration of dental tissues (Zafar et al., 2016). The objective of this contribution was to prepare core/shell fibers composed of silica glass and hydroxyapatite. The fibers were analyzed regarding their morphology, chemical composition, thermal properties and crystalline phases.

2.2.Synthesis of Silica Sol
The silica sol-gel was prepared dissolving tetraorthosilicate in ethanol-water solution; the molar proportions for tetraorthosilicate, ethanol, deionized water and concentrated HCl were 1:2:2:0.1. First the TEOS was dissolved in ethanol, followed by the mixing of water and HCl used as catalyst under constant stirring for 30 min at room temperature.

2.3.Synthesis of Calcium Phosphate Sol
This synthesis was carried out using the method described by Lee

2.4.Composite Electrospinning
For the electrospinning process, a solution of PVP in ethanol with concentration of 10 w/v% was made. This solution was mixed with the silica sol at 10 w/v% and calcium phosphate sol at 15 and 20 w/v% for PVP weight. A hygroscopic white solid gel was obtained from the sol. The solutions were charged in syringes of 30 mL connected to a coaxial nozzle with double feeding. For the electrospinning process, a Nabond NEU-Pro device was used. The flow of the solutions was set in a range of 0.4 to 2.0 mL/h in intervals of 0.2 mL/h. The voltage used was in a rage from 10 to 15 kV and the distance between the nozzle and the collector in a range between 10 and 20 cm in intervals of 5 cm. For the obtainment of the ceramic fibers, the green fibers were dried at 50°C for 24 h in a stove (Thermoscientific® mod. OSG60) and later heat treated in an muffle (Thermoscientific® mod.FB1410M) at 800 and 900ºC for 1 h with a temperature ramp of 0.5 ºC/min. Figure 1a shows the fibers as spun with SiO2-HA flow of 1.2 mL/h, the fibers were cylindric, with homogeneous surface and random orientation. The average diameter of fibers was 560 nm ±82 nm. Figure 1b shows fibers obtained with SiO2 flow of 8.0 mL/h which were well shaped with an average diameter of 545 nm ±104 nm. There were some regions in the membrane with accumulations of phosphate and a contrast zone throughout the inner part the fibers. A further reduction in the inner flow to 0.4 mL/h to produce fibers with cylindric morphology and homogeneous surface and an average diameter of 510 nm ±90 nm is shown in Figure 1c. The same pattern of contrast is observed throughout the fibers (the contrast zone is segmented). All membranes obtained had HA particle agglomerations on the surface due to the instability of the jet during electrospinning causing the expulsion of droplets ( Http://www.granthaalayah.com ©International Journal of Research -GRANTHAALAYAH [42] arranged longitudinally of what is presumed to be SiO2 encapsulated in hydroxyapatite ( Figure  1d). TGA and DSC analysis were performed on the green fibers before the heat treatment. In Figure  2, thermogram shows an initial weight loss (4%) around 90°C due to evaporation of ethanol and water. At 350°C there is a second weight loss of approximately 29% corresponding to the removal of residual and polymer. Around 440°C the weight of the sample was reduced by 62% due to the densification process in the ceramic material. Increasing the temperature beyond 600°C does not seem to produce any additional weight loss. Hence, the fiber at 600 °C is likely to produce a non-volatile residue containing predominantly hydroxyapatite and silica. In the DSC of the green fibers it can be seen an endothermic peak corresponding to the loss of solvents at 100°C. An endothermic peak is located at 474°C due to the loss of organic material from the PVP and the metalorganic precursors. An exothermic peak is shown at 697°C because of the release of CO2 from the crystals of CaCO3 formed by the substitution of PO4-3 by (CO3-2) during the sol-gel synthesis. The material presents an endothermic peak around 1160°C corresponding to the final loss of OH-and densification of the ceramic network (Meejoo et al., 2006). The IR spectrum of the fibers before the heat treatment is shown in Figure 3. At 25ºC the spectra show absorption bands related to functional groups in the PVP, silica and the calcium phosphate (see Table I). Bands at 450, 560 and 960 cm-1 correspond to vibrations of PO4-3 groups. A characteristic vibration of the C=O group appears at 650 cm-1 and at 1291 and 1424 cm-1 belong to nitrogen of tertiary amine in PVP. At 1652 and 2955 cm-1 bands correspond to bending vibrations of H2O molecules and C-H group in the polymer. At 800ºC the IR spectrum changed and the bands corresponding to groups in the PVP disappeared (see Table II). At 450, 560 and 960 cm-1 bands belonging to PO4-3 groups in HA appear. The bands at 800 and 1066 cm-1 are related to deforming vibrations of Si-O-Si groups and at 1405 cm-1 there is a slight bump in a band that corresponds to α-TCP (tricalcium phosphate).   After the heat treatment, the fiber diameter was reduced and in all the membranes. The fibers exhibited spindle-thread morphology and they were interconnected as seen in Figure 4, which shows fibers prepared with SiO2 flow of 1.8 mL/h (a), 0.8 mL/h (b) and 0.4 mL/h (c). The average diameter of the fibers in each membrane was 330 nm ±85 nm, 320 nm ±95 nm and de 300 nm ±85 nm respectively. There were zones in the membranes with accumulation of HA, specifically in Figure 4c and 4d. The membranes were dense, and the fibers had an overall smooth surface. The morphology of the fibers after the heat treatment shows patterns of thread and spindle, or throttling of the fiber which is attributed to the presence of SiO2 on the inside the hydroxyapatite cover which prevents the contraction thereof during sintering (Tian et al, 2015;Sun et al., 2006).

Conclusions & Recommendations
Glass-Hydroxyapatite fibrous networks with average fiber diameters between 300 nm to 400 nm were produced after calcination of electrospun PVP/HA/TEOS/sol mixtures. X-ray diffraction and infrared analysis indicated hydroxyapatite and silica to be the dominant inorganic phases remaining after firing. A continuous fibrous network could be obtained with a uniform dispersion of hydroxyapatite. For dental tissue engineering the present Glass-Hydroxyapatite fibrous network is a suitable microenvironment for dental regeneration, while it is expected that the combination of the two ceramics in a nanofiber membrane improved the mechanical properties and functionalities for biological aspects in dental applications.