Polyethylene Glycol (PEG) Modified Porous Ca 5 (PO 4 ) 2 SiO 4 Bioceramics: Structural, Morphologic and Bioactivity Analysis

: Bioceramics are class of biomaterials that are specially developed for application in tissue engineering and regenerative medicines. Sol-gel method used for producing bioactive and reactive bioceramic materials more than those synthesized by traditional methods. In the present research study, the effect of polyethylene glycol (PEG) on Ca 5 (PO 4 ) 2 SiO 4 (CPS) bioceramics was investigated. The addition of 5% and 10% PEG significantly affected the porosity and bioactivity of sol-gel derived Ca 5 (PO 4 ) 2 SiO 4 . The morphology and physicochemical properties of pure and modified materials were evaluated using scanning electron microscopy (SEM), X-ray powder diffraction (XRD), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR), respectively. The effect of PEG on the surface area and porosity of Ca 5 (PO 4 ) 2 SiO 4 was measured by Brunauer–Emmett–Teller (BET). The results obtained from XRD and FTIR studies confirmed the interactions between PEG and CPS. Due to the high concentration of PEG, the CPS-3 sample showed the largest-sized particle with an average of 200.53 µm. The porous structure of CPS-2 and CPS-3 revealed that they have a better ability to generate an appetite layer on the surface of the sample when immersed in simulated body fluid (SBF) for seven days. The generation of appetite layer showed the bioactive nature of CPS which makes it a suitable material for hard tissue engineering applications. The results have shown that the PEG-modified porous CPS could be a more effective material for drug delivery, implant coatings and other tissue engineering applications. The aim of this research work is to fabricate SBF treated and porous polyethylene glycol-modified Ca 5 (PO 4 ) 2 SiO 4 material. SBF treatment and porosity of material can provide a very useful target for bioactivity and drug delivery applications in the future.


Introduction
Ca5(PO4)2SiO4 bio-ceramic is a fully loaded compound consisting of Ca, P and Si elements [1]. The calcium-phosphate-silicate (CPS) ceramics are considered as biocompatible materials which is exclusively utilized as implantable materials for bone defects repairing [2]. Silicon-based bioceramics permit to form a functional silanol group with Si-O-H connectivity on the material surface [3]. The silanol group induces the formation of bone-like apatite by attracting Ca 2+ and PO4 3− through an ionexchange process in an artificial solution similar to blood plasma [4,5]. This apatite is broadly utilized biomaterial to repair and reconstruct hard tissue defects [6,7]. Thus, researches on silicon-containing bioceramics have received a considerable attention from the biomaterials scientists as such biomaterials are considered as bone substitute materials [8,9]. However, various drawbacks such as less mechanism properties, low chemical stability and reduced bioactivity, the clinical applications of CPS bioceramics are limited [10,11]. It was researched that for a reliable bone fixation, the porosity of implant material should be over 70% so that the body fluids can penetrate easily for the better bone growth [12,13]. In order to get a bioactive and porous material, sol-gel derived polyethylene glycolmodified CPS was prepared in which polyethylene glycol (PEG) was used to modify the phase of Ca5(PO4)2SiO4 via crosslinking of PEG diacrylate chain with PO4 3− and Ca 2+ probably by the OH¯ exchange reactions [14,15]. PEG has been widely accepted as a phase change material, which can be altered through its congruent melting behavior and low vapor pressure [16]. Low molecular weight polyethylene glycol (PEG) is a highly non-immunogenic, non-toxic, biocompatible and biodegradable polymer [17,18]. It possesses a straight poly-ether diol chain that has hydroxyl groups and also shows covalent binding with proteins, phospholipids, functional groups, fluorescent probes, etc [19]. PEG will be a promising agent that may enhance the biocompatibility of Ca5(PO4)2SiO4 by generating porosity in the material. The use of larger PEG chains resulted in more agglomerated hollow particles [20]. Sol-gel process is a flexible and favorable method due to its low-temperature, high purity, easy doping and cost effective approach to prepare various nanocomposites and other nanobiomaterials [21,22]. The reaction time may affect the particle size and stability in the sol-gel process [23]. During the reaction monomers converted into colloidal solution (sol) and then into gel or integrated network of discrete particles [24,25]. Due to bioactive properties, the silica-based glass networks are prepared in various shapes, sizes and hence applied for variety of biomedical applications [26]. Sol-gel synthesized bioceramics are highly biocompatible with controlled degradation rate and effortlessly metabolized in the body [27,28].
In the current research work, we synthesized a pure phase of Ca5(PO4)2SiO4 and PEG-modified porous Ca5(PO4)2SiO4 bioceramic materials through the sol-gel method and investigated various properties. The highly bioactive and porous PEG-modified CPS can be utilized for tissue engineering and drug delivery.

Synthesis of Porous Calcium Phosphate Silicate
Sol-gel synthesis was convenient because it permits direct fabrication of bioceramics with different configurations. The synthesis of PEG-modified Ca5(PO4)2SiO4 was carried out as follows: 12.17 mL tetraethyl orthosilicate (TEOS, ≥98% Sigma Aldrich, Saint Louis, MO, USA), 150 mL ethanol, 0.80 g P2O5 (≥99.9% Sigma Aldrich, Saint Louis, MO, USA) and 6.68 g Ca(NO3)2.4H2O (≥99.9% Sigma Aldrich, ) was mixed stepwise to get 680 mL homogenous mixture. To make composite, 5% and 10% w/v of PEG 400 were dispersed in 100 mL distilled water and mix with the above mentioned homogenous mixture. In acidic conditions, TEOS completely hydrolyzed and obtained Si(OH)4 which slow down the condensation rate [22]. Add ammonia (25%) solution to make pH 11 of the mixture which boosts the rate of the gelation process, ideal for the formation of smaller aggregates. TEOS works as a principal network forming agent during gelation. The change in synthesis conditions or parameters such as pH, temperature and additives affect the silica-based glass networks which produce various shapes, sizes and formats products [26]. The different steps used for the synthesis of PEG-modified Ca5(PO4)2SiO4 are shown through the schematic diagram, see Figure 1. Naming of the samples was done on the basis of concentration variation of additive, i.e., PEG in Ca5(PO4)2SiO4; for pure Ca5(PO4)2SiO4 without PEG was assigned to CPS-1. Similarly, for PEG 5% ND 10% by weight in Ca5(PO4)2SiO4 was assigned CPS-2 and CPS-3, respectively. Further, these three samples were used for different characterization.

Characterizations of Porous Calcium Phosphate Silicate
The phase identification of the synthesized materials was investigated using XRD (Rigaku Ultima IV, Tokyo, Japan). The measurement was taken at 45 kV voltage and 40 mA anodic current. XRD patterns were acquired at a diffraction angle from 15° to 60°. The compositional information of the prepared samples was investigated by FTIR (Perkin Elmer Frontier FTIR, MA, USA). First, the obtained powder was mixed with KBr in an appropriate ratio and then after applying pressure. The mixture was converted into pellets. For investigations of obtained spectra, the background spectra were calibrated with KBr. The morphology and elemental composition of the samples was examined using SEM (JEOL, JSM 6100, Akishima, Tokyo, Japan) and by energy dispersive spectroscopy (EDS) attached with same, respectively. On sputter was then to sputter coat the samples with a palladium layer. After 30 nm palladium coating, observations were done at an accelerating voltage of 20 kV and 10 Pa. The powerful size of the pore was figured as the mean distances across of sample pores. The nanoparticles synthesis confirmed through TEM (TECNAI 200 kV, Hillsboro, OR, USA) at SAIF in AIIMS, New Delhi. To provide contrast under magnification, nanoparticles were suspended in water (1 mg/mL), placed on copper grids of 0.037-mm size and then stained with a 2 g/100 mL uranyl acetate aqueous stain. Before viewing under 50,000 to 120,000 times magnification, surplus liquid on Mesh was wiped off with filter study and the grid was allowed to air dry. Observations were performed at 80 kV. Brunauer-Emmett-Teller (BET) (BELSORP mini II, Osaka, Japan) technique was used to measure the porosity and surface area of the prepared materials. Tris-HCl-buffered synthetic body fluid (SBF) was used to check bioactivity of the sample after 7 days at 37 °C in an incubator. The thermal stability of the prepared material was evaluated by the thermogravimetric analysis (TGA; Perkin Elmer STA 6000, MA, USA) operated under nitrogen flow in the temperature range from 50 to 800 °C at 10 °C/min. Using TGA, by inducing heat to the sample, the chemical reactions and physical changes due to dehydration, decomposition and oxidation can be evaluated.

Physiochemical Analysis of Calcium Phosphate Silicate Materials
The sol-gel derived white powdered samples of CPS and PEG-modified CPS were heated at 450 °C to get phase or structural transformation. In the XRD spectra, the distinct peaks of the pure phase of CPS-1 were identified and matched with the standard database card number 00-901-1950 and PDF 40-0393 [29].
The XRD patterns of the PEG-modified CPS bioceramics showed modification in peaks [30]. The addition of 5% and 10% PEG in CPS make some changes in the sample, generate and demolish several phases or peaks in CPS-2 and CPS-3 (Figure 2A). The heat treatment (450 °C) to CPS-2 (5% PEG) and CPS-3 (10% PEG) removed the precursor residues of PEG, limiting the densification of material [31]. During the heat treatment, PEG started to decompose within a temperature range of around 250-300 °C; this can completely remove PEG from the sample [32,33]. The removal/degradation of PEG may generate a porous structure due to structural rearrangement by various chemical reactions, based on several treatment temperature and duration. The sintered sample of CPS-2 (5% PEG) and CPS-3 (10% PEG) also confirmed the presence of wollastonite [PDF 50-0905].  [30,34]. The functional group SiO4 4− was also recognized by nearly at 612 and 878 cm −1 [30]. The absorption bands at 820 and 878 cm −1 represent Si-CH3 and SiO4 4− functional groups. The band between 1000-1200 cm −1 is associated with the Si-Ostretching, while the band at 930 cm −1 corresponds to Si-O-with one non-bridging oxygen [34]. The appearance of a medium stretching vibration at 1320 cm −1 was because of the C=O group. The pure PEG sample showed bands at 992, 1315, 1417 and 1560 cm −1 while PEG-modified sample showed bands at 1320 and 1365 cm -1 , denoting CH3 and C-O stretching vibrations.

Morphologic Characterizations of Calcium Phosphate Silicate Materials
The high-resolution 2 D TEM images revealed the particle size estimation of PEG-modified CPS-1 ( Figure 3A,B), CPS-2 ( Figure 3C,D) and CPS-3 ( Figure 3E,F) samples. Figure 3A shows a 506-nmsized crystalline particle of CPS-1. CPS-2 ( Figure 3C) revealed 265.4-nm-sized irregular particles while CPS-3 has not revealed any proper shape. Crystallite size is calculated using the Scherrer formula from XRD patterns for all synthesized samples. The calculated crystal size of CPS-1, CPS-2 and CPS-3 is 24, 18 and 15 nm, respectively. These calculated results are quite different from TEM results. Because the crystallite size determined using Scherrer formula from XRD patterns provided an average value of the bulk sample since the diffraction occurs from a considerable volume of the sample. Apart from that in TEM, we found the crystallite size from a very local area that may not be the representative size of the bulk sample. Removal of PEG at high temperatures during heat treatment may lead to a highly ordered porous structure, as observed in CPS-2 and CPS-3 (Figure 3). At the 200-nm-scale, all the samples showed significant structural differences; CPS-1 (0% PEG) looked like a dense material, while CPS-3 (10% PEG) displayed a better porous structure than the CPS-2 (5% PEG) sample.
The increment of PEG concentration (more than 10% w/v) led to the densification of materials, which reduced the porosity of the CPS. The SEM results revealed the irregular micro size crystalline particles of CPS-1 ( Figure 4A) and CPS-2 ( Figure 4B) revealed amorphous particles. The SEM image of CPS-3 showed a porous microstructure, observed after the removal of PEG after heat treatment, see Figure 4C. The morphology of this specimen significantly display that will be beneficial for future applications, for instance tissue engineering and drug loading. The optimized favorite sample CPS-3 was used investigated through EDS analysis ( Figure 4D) which showed the relative concentration of the Si, Ca, P and C elements in the synthesized sample. The results obtained from EDS analysis depend on several factors, to name a few, sample topography, beam parameters, field noises (electronic and external fields), acquisition settings, detector type and atomic number of the elements. From particle size distribution histogram ( Figure 4E), it is concluded that particle size of CPS-1 is to be under 10 µm, for CPS-2 it is estimated to be under 100 µm, while for CPS-3, it observed to be under 160 µm due to agglomeration of particles.

Porosity Measurements for Calcium Phosphate Silicate Materials
The Brunauer, Emmett and Teller (BET) method is a promising technique to determine the surface area through physical adsorption-desorption isotherm analysis [37]. The porosity of a material depends upon the concentration and type or nature of the template used during the fabrication process [38]. The reparative bone formation and inflammatory response are also influenced by the morphologies of the materials [39,40]. From BET analysis ( Figure 5A,B), CPS-3 revealed a surface area of 30.6672 m 2 ·g −1 , pore volume of 0.9722 cm 3 ·g −1 and pore diameter of 4.58 nm, while CPS-2 revealed a surface area of 27.9840 m 2 ·g −1 , pore volume of 0.6243 cm 3 ·g −1 and pore diameter of 2.84 nm. The addition of PEG affects the microstructure as well as the porosity of the CPS. The BET result revealed that the CPS-3 (10% PEG) has shown better porosity that will be a key factor in application part. At 450 °C, the decomposition of PEG generated the porous network. The bio factors include genes or cells, proteins and nutrients can easily exchange through the porous structure of materials. Porosity and pore size possess high impact on the application of the material [3,41].

Bioactivity Analysis
SBF provides the same environment as blood plasma, where surface dissolution starts mineralization through the slow release of ions in the solvent [33]. When materials were immersed in the SBF solution, a set of reactions such as ion exchange, precipitation and dissolution occurred for the apatite formation [42]. The SBF treatment generated two new groups, CO3 2− and PO4 3− , at 1420 and 1480 cm −1 , which are due to the absorbance of CO2 ( Figure  6D). The generation of new compounds are directed by both the immersion parameters (immersion time, temperature and pH, temperature) and surface characteristics of the materials [43]. The presence of CO3 2− and PO4 3− groups are associated with apatite formation on the surface of the sample after a seven-day immersion [30,44]. Further, CO3 2− and PO4 3− ions contribute to nucleation and subsequent surface mineralization that leads to actual apatite formation [45,46]. The in vitro apatite-forming ability of material often successfully predicts the actual bioactivity of biomaterials [47]. The bands at 1653 and 1470 cm −1 denoted CO3 2− , while peaks at 1314 and 2790 cm −1 were attributed to C-H group. The peaks appearing at 561 and 880 cm −1 are due to bending mode of O-P-O and variable symmetry of HPO4 3− , respectively [48]. The band at 1025 cm −1 was assigned to the presence of the PO4 3− group [49,50]. The surface of the material released Ca 2+ , HPO4 2− and PO4 3− ions and absorbed calcium and phosphate ions from SBF. The incorporation of other electrolytes, such as CO3 2− and Mg 2+ ions, started to generate the apatite layer [51]. The mineralization behavior of CPS-1, CPS-2 and CPS-3 was shown in Figure 6A-C and also confirmed through FTIR results, as shown in Figure 6D.

Thermal Gravimetric Analysis
The physical and morphologic transformations of the PEG-modified CPS samples analyzed through thermal gravimetric analysis. It is well known that the chemical structures are altered by heat treatment as it leads to the thermal decomposition of the materials. TGA results presented in Figure 7, weight loss in CPS-1 and CPS-2 divided into three main steps (S-1, S-2 and S-3): removal of -OH groups, polymeric phase (PEG) and burnout of the CPS mass (Ca, P and Si) while CPS-3 revealed weight loss in two stages. In all the samples, the initial weight loss was confirmed because of the release of absorbed moisture contents. CPS-2 and CPS-3 showed more weight loss than CPS-1 that was because of the PEG thermal degradation within a temperature range of around 250-300 °C. CPS-3 fabricated with 10% PEG but thermal decomposition of the organic groups, generated porous microstructure and microporous materials showed large specific surface that support more degradation at high temperature.

Conclusions
In summary, the effect of PEG on various properties of CPS was investigated. The use of PEG improves the morphology, physiology and bioactivity of CPS. Porosity and bioactivity of sol-gel-derived samples were greatly influenced by varying the concentration of PEG. The heat treatment at 450 °C plays an important role in the phase modification and porosity generation. This could be attributed, as the concentration of PEG increased, the densification and agglomeration in particles were observed. The formation of the apatite layer on the surface of SBF treated CPS exposed mineralization. PEG-modified Ca5(PO4)2SiO4 demonstrated better in vitro bioactivity than pure CPS, by tempting bone-like apatite in the artificial salt solution SBF. PEG-modified Ca5(PO4)2SiO4 bioceramic (CPS-3) is different from those in conventional material and may be a promising material for implant coatings, drug loading and bone regeneration applications. Future works should determine the optimum concentration for controlled porosity and their applications in soft as well as hard tissue engineering.