Document Type : Original Article
Authors
1 Department of Materials Engineering, Tarbiat Modares University, Tehran, Iran
2 Department of Clinical Medicine, Aarhus University, Denmark
3 Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract
Graphical Abstract
Highlights
Keywords
Recent research on bone tissue engineering has been carried out in various branches of this science [1-6]. Bone defects caused by skeletal diseases, traumatic events, congenital anomalies, and malignancies annually bring millions of patients to hospitals for bone remodeling and transplantation [7, 8]. Bone marrow transplantation by replacing damaged bones with the use of its own patient (autograft) or donors (allograft) has been done since two centuries ago, and autograft is a better option because of high biological acceptance [7, 9]. However, issues such as access restrictions, poisoning of donor sites and increased procedural costs have led research to be found on methods and bio-materials [9].
Among the range of proposed synthetic materials, calcium phosphates (CP) have attracted a lot of attention due to their similar bone composition and excellent properties including biocompatibility, bioactivity, osteoconductivity, and non-immunogenicity, non-toxicity [10-12]. The CP family contains HA, TeCP, α-TCP, β-TCP, DCPD, DCPA, and OCP. One of the applications of this material is coating on metal implants that increase corrosion resistance and biological properties [13], because biological compatibility and biological function of the surface are considered an important issue for biomaterials [14]. Another use of these materials is bone cement [13].
Since the initial formulation in the 1980s, CP cements have been increasingly used as bone substitutes, and many have been commercialized with various compounds. CP cements are produced by a chemical reaction between two phases (solid and liquid) which, when mixed, become doughy and gradually solidifies. The solid phase contains one or more CP compounds. CP is injectable and hardening is achieved in situ through the needle shaped or plate crystal entanglement [7, 8, 15].
These properties make CP cement widely used as fillers in dentistry and orthopedics. It is easily formed and can be easily injected into the cavities with a syringe without the need for an open portal through the tissue [16].
Formulation of composite materials of CPs and resins is difficult as the ceramic usually act as stress concentration centers in resin base composites and cause crack growth [17]. Due to the poor effect of CP particles on the resin matrix, it is important to keep its contents as low as possible without sacrificing the potential for regeneration [18].
Currently, despite the numerous combinations of cement of CP cements, there are only two final products, brushite and apatite, such as HA or CDHA (Calcium Deficient HA), and these two products are further obtained by two types of hydrolysis and acid-reactive chemical reactions. The major difference between these two products is that bromide solubility is 1-2 times higher than apatite solution in physiological pH. However, the bruchite is a metastable phase and may become apatite under physiological conditions [7]. With special conversion processes, Brushite can be converted to monetite with superior Osteoconductive and Osteoinductive properties in vivo. Unfortunately, this cement has shown a decrease in weight, porosity and weakening of mechanical properties in vitro [19].
Brushite, Dicalciumphosphate dehydrates, can be made as hydrolyzed cement with a wide range of applications. In addition to the ability to regenerate bone, it can be replaced in vivo faster than most CPs with reconstructed tissue [20]. It has been used as bioactive filler, with a relatively high solubility with a calcium-phosphorus ratio of 1 and the presence of structural water molecules. One of its interesting features as a filler in repairing composites is that the refractive index is 1.54-1.55, which is better than hydroxyapatite (1.63-1.67) and suitable for photopolymerization [20]. DCPD is low cost and can easily be placed on metal surfaces [21]. CPs must have the same mechanical properties as bone tissue to be used as a substitute for repair agents [22]. One of the ways to improve mechanical properties is to use reinforcements.
Studies on the use of carbon nanomaterials in orthopedic medical applications, especially graphene, have steadily increased during the past 5 years [23- 25]. Graphene has excellent mechanical properties (for example, the Young or E modulus) due to the sp2 carbon bonding network. The single-layer graphene theoretically shows the Young's modulus (E) 1.02 TPa (ν = 0.149) which is approved for a defect-free
Table 1. The primary chemicals used in the powder synthesis phase. |
||||
No. |
Chem. |
Co. |
Purity |
Formulation |
1 |
Graphene oxide |
Abalonyx |
25% |
GxOy |
2 |
Calcium nitrate tetrahydrate |
Merck |
> 99% |
Ca(NO3)2.4H2O |
3 |
Diammonium hydrogenphosphate |
Merck |
> 99% |
(NH4)2HPO4 |
graphene sheet with a failure strength of 42 N.m-1 [26]. Graphene oxide (GO) is a combination of carbon, oxygen, and hydrogen in variable proportions with a single atomic layer, which is produced by graphite exfoliation with strong oxidants. The bulk product is a solid brown / yellow substance that maintains the structure of the graphite layer, but has a larger, more irregular spacing. The basal plate is decorated with oxygen-containing groups such as hydroxyl, carboxyl and epoxy. GO is hydrophilic and it can be suspended in deionized water, N-Methylpyrrolidinone (NMP), Dimethylformamide (DMF), Tetrahydrofuran (THF) and other solvents that have the same behavior with water as these groups have a higher relative tendency to molecules of water. Graphene oxide can recover most of the properties of pure graphene through its light, heat, or chemical reduction [27- 30].
The chemical depositions of CPs at the graphene nanosheets (GNS) are generally accomplished by dispersing GNS in a chemical bath that CPs are deposited. GNS coated with CPs ensures that CPs and GNS are distributed uniformly. GNS is suspended in calcium carbonate / hydroxide / calcium nitrate in a bath and then mixed together. Then, hydrogen phosphate diammonium / phosphoric acid are added to a highly stirred bath to form a drop in order to create a CP precipitation on a suspended GNS surface. Deposition parameters, such as temperature and pH, should be optimized to ensure that the desirable phase is formed HA, CaHPO42H2O or Ca3(PO4)2 phases. GNS is uniformly distributed in CPs precipitation to powder form via chemical deposition [31- 35].
Recently, graphene and its derivatives have been studied to increase the biological and mechanical properties of CP structures and their results have been satisfactory [36- 39]. In this study, the main objective is to investigate the physical properties of GO/DCPD powders, which has been synthesized via a simple precipitation method and has not been addressed so far. According to the above, these powders will have a lot of potential for modifying the properties of bone cement.
The primary chemicals used in the powder synthesis phase with the specifications are listed in Table 1.
2.1. Powders synthesis
For synthesis of powders, solutions containing calcium and phosphorus were prepared with calcium to phosphorus ratio of >1. For this purpose, 2.5 g of (Ca(NO3)2.4H2O) in 110 cm3 distilled water and 1 g of ((NH4)2HPO4) in 75 cm3 distilled water were prepared as solutions. First, the solution containing calcium was added dropwise to 20 mL of GO colloid (5 mg/mL) and was stirred for an hour. Then, the solution containing phosphorus was added dropwise to the above solution. The process was assessed by analyzing the precipitated powders after drying.
2.2. Characterization
Phase constituents of the samples were identified by X-ray diffraction (XRD, X’ Pert Pro, Panalytical Co.) with a detector using Cu Kα radiation (λ=1.5406 Å) generated at 40 kV and 40 mA and a 2θ scanning range from 5° up to 80° in steps of 0.02°. The
Williamson-Hall method was used to calculate the crystallite size (Equation 1):
β.Cos θ= + 2η.Sin θ (1)
Where d is grain size, θ is Bragg diffraction angle, λ is wave length of used X-ray (Cu), β is full width at half height (FWHM), and η is crystalline lattice strain.
Fourier transform infrared spectroscopy (FTIR, VERTEX 70, Brucker Co.) was carried out to identify the functional groups of the composites with a resolution of 4 cm-1 and a scan number of 8, with a spectral region from 400 to 4000 cm-1 using 2 cm-1 steps. The samples were prepared and mixed with potassium bromide (KBr) in a concentration of 1 mg powdered samples and 300 mg KBr. The mixture was pressed into discs having 1 mm thickness by applying 200 MPa pressures. The spectra were collected at room temperature (25 °C) and 60% relative humidity.
Micro-Raman spectra were carried out using a Reinshaw invia spectrometer in the range of 250-1200 cm−1 (recording 5 times for 10 seconds of each accumulation) with a wavelength of 532 nm (green laser line in a backscattering configuration using a microscope with a 100× objective, 100% power) and an acquisition time of 10 s, which had been excited from an argon ion laser. An optical microscope was used with the Raman spectrometer. In order to remove the fluorescence background, which is a serious challenge in the Raman analysis of apatite, the samples were subjected on Al foil.
The morphology of the powders was observed by a Field Emission Scanning Electron Microscope (FESEM, Hitachi S4700 equipped with energy dispersive X-ray spectroscopy) and a Portable Scanning Electron Microscope (SEM, TM-1000). The samples were mounted in an adhesive carbon film and Au coated by sputtering for its observation.
HRTEM images were obtained on a TALOS F200A with a twin lens system, X-FEG electron source, Ceta 16M camera and a super-X EDS detector. Spatially resolved elemental analysis, with a spatial resolution higher than 2 nm, was obtained using the same TALOS microscope in STEM mode.
The other instruments used to characterize the samples include: inductively coupled plasma (ICP) (DV7300, Optima Co.), TEM (CM120, Philips), FTIR (VERTEX 70, Brucker Co.), and X-ray photoelectron spectroscopy (XPS).
Figure 1. The XRD and TEM images of the hybrid powders after precipitation process.
The XRD pattern and TEM images of the hybrid powders after precipitation process is shown in Figure 1. The structure of powders, with its x-ray pattern shown in Figure1a, is monoclinic (JCPDS 72-0713). The crystallite size according to this pattern, Williamson Hal method is about 138 nm (Figure 1b). The characteristic peak of GO (2θ=10) is covered with the peak of the (020) diffraction surface [41, 42]. In addition, there are no signs of graphite peaks due to the presence of DCPD peaks in their vicinity and their small content GO, while their presence is confirmed by TEM, which indicates that GO composition has no effect on stability of the DCPD and the absence of GO peaks is likely to be related to the reduced graphene oxide layer structure partially. TEM images show the GO which is composed of DCPD deposit on its surfaces. These images also confirm the directional growth of DCPD that the particle size is between 100 and 200 nm.
Figure 2 shows the HRTEM, FFT and IFFT analysis of hybrid powders after precipitation process. HRTEM analysis images confirm the presence of GO layers in final powders. The distance between the carbon atoms is 0.14 nm. Changing the mode in two FFT images is due to the GO surface folding and wrinkling.
Figure 3 shows the schematic diagram for DCPD/GO interaction in hybrid powders. According to XRD-analysis, the d-spacing of (020) planes in DCPD are 0.76 nm. The distance between the GO sheets is 0.83 nm in cross section. So, mismatch for this state is much less than the incoherent limitation (0.25). Therefore, the interface between the two phases is coherent.
Figure 2.HRTEM, FFT, and IFFT analysis of GO sheets.
Figure 3. Schematic diagram for DCPD/GO interaction in hybrid powders.
Figure 4 shows the FTIR spectrum of GO, starting materials, and final samples along with XPS spectra of the GO/DCPD powders. In FTIR analysis, two conditions have been investigated, the first one is when GO is doped with Ca ions and then the phosphorus ions are added to it. In the second one, phosphorous ions are first added to GO. In both cases, the DCPD eventually precipitates (Figure 4a). But, when calcium ions are first mixed with GO, there will probably be more bonds between Ca ions and GO because calcium ions have a negative charge and are more absorbed by GO. However, phosphorous ions are also partially absorbed by GO [47]. Figure 4b shows the FTIR spectrum of powders after precipitation. The wide absorption peak between 2500 cm-1 and 3500 cm-1 is related to the stretching vibrations of the O-H band. Absorption peak in 1650 cm-1 is due to bending of H-O-H and the one at 1065 cm-1,1130 cm-1 and 1215 cm-1 are related to stretching vibrations of the O=P band. Absorption peaks at 980 cm-1,870 cm-1 and 790 cm-1 are related to the P-O-P asymmetric stretching vibrations while the peaks in 525cm-1 and 580 cm-1are due to (H-O-) P=O for acid phosphates [43, 48, 49]. Due to the presence of GO in the initial solution, the peaks associated with this material and the peaks associated with the DCPD are likely to be overlapping.
Figures 4c, d show the XPS spectra of the GO/DCPD powders. The GO spectrum should contain carbon (284- 289 eV) and oxygen. The emergence of other couriers including Ca 2p (350) are signs of DCPD formation (Figure 4c) [45]. The high resolution XPS spectrum of O 1s of the GO- DCPD powders is shown in Figure 4d, in which the peak centered at 531 eV is attributed to the O in DCPD and OH groups, whereas those at 533 and 532 eV correspond to the O in C–O and H-C=O groups [46]. The XPS results further reveal the presence of DCPD and GO, which agree with the Raman analysis results.
Figure 4. (a, b) The FTIR spectrum of GO, starting materials, and final samples, (c, d) XPS spectra of the GO/DCPD powders.
Figure 5 shows FESEM images for the final hybrid powders. Figure 5a shows plate-shaped DCPD particles, which have grown preferentially and in certain directions. Figures 5b and 5c show DCPD particles at higher magnifications, which are evidence of their directional growth. Figure 5d shows FESEM image of GO layers which are composed of a DCPD deposit on its surface. In a study published by other researchers, the directional growth of DCPD is mentioned [40].
Figure 6 shows the Raman spectrum of the hybrid powders after precipitation process. The main bonds in Figure 6b spectrum are the phosphate bonds. DCPD Raman vibrations are associated with four different modes of four internal tetragonal states. ν1 correspond to a totally symmetric stretching mode of the tetrahedral PO4 3- group (P-O bond). ν2 is a doubly degenerate bending mode of the phosphate group (P-O-P bond). ν3 is a triply degenerate asymmetric stretching mode of the tetrahedral PO43- group (P-O bond), and ν4 is a triply degenerate bending mode of the PO4 group (O-P-O) [43, 46]. All three shifts obtained in G, 2D and D (Figure 6a, c) are related to carbon compounds such as GO. In fact, these shifts along with previous microscopic images are the best evidence of the presence of GO compounds in the final synthesized powders.
Figure 5. FESEM images of the final hybrid powders.
Figure 6. Raman spectrum of the hybrid powders after precipitation process
Figure 7 shows the energy dispersive X-ray spectroscopy analysis, and map images for the final hybrid powders. The EDS analysis confirms the presence of trace elements in the synthesized composite which is accordance with the ICP analysis. The following reaction occurs in this mechanism, in which the final solution, S, contains some residual calcium and phosphorus ions:
Ca(NO3)2.4H2O+(NH4)2HPO4+H2O→CaHPO4.2H2O+S (1)
The first analysis was inductively coupled plasma mass spectrometry (ICP-MS) of the final solution(S) which indicated that almost the ratio of calcium to phosphate should be 1.
In the sample, the powders were easily separated by centrifugation. Based on the findings of the characterization, a large number of primary ions have not reacted at this stage and the ratio of calcium to phosphorous in these powders was approximately 1. Also, the map of the elements shows that the trace elements are dispersed homogeneously in the powders.
Figure 7. Energy dispersive X-ray spectroscopy analysis, and map images for the final hybrid powders.
Figure 8 shows the mechanism of nanostructured powders formation by precipitation. GO basal planes are more covered with epoxy and hydroxyl groups, while carboxyl groups (-COOH or COO) are located at the edges. The charge of the GO surface is negative in terms of functional groups. The structure of GO affects the formation of DCPD and induces epitaxial and directional growth. Also, as anchoring sites, oxygen-containing groups induce the formation and bonding of particles to the surface of GO. In the chemical precipitation, when adding calcium nitrate to a solution containing GO, Ca2+ cations are absorbed and bound to GO by electrostatic interactions with hydroxyl groups (C-O-C, OH) or ion exchange with carboxyl groups (H+) and play the role of the primary sites for nucleation and growth of DCPD particles. Calcium ions can react in situ with phosphate ions and form calcium phosphate nanoparticles through electrovalence reactions. In this process, the solution is strongly stirred and the GO layers are homogeneously dispersed, then the same nucleation and the controlled growth occur.
Figure 8. mechanism of nanostructured powders formation by precipitation method. a) GO. b) GO+Ca2+. c) GO+DCPD
Dicalcium phosphate dihydrate was precipitated in the presence of graphene oxide. Raman Spectroscopy and XRD results showed that dicalcium phosphate dehydrate (DCPD) formed the hybrid powders along with graphene oxide. DCPD crystallite size was estimated at 138 nm. Microscopic images confirmed the preferred directional growth of DCPD particles in plate-shaped. FTIR and XPS results confirmed the emerging bands. ICP and EDS results confirmed the presence of trace elements in the synthesized powders. The result of this study is likely to improve the biological and mechanical properties of bone cements.
Conflict of interest
The authors declare that they have no conflict of interests.
Acknowledgments
None declared.