Introduction
β-Tricalcium phosphate (β-TCP) Ca3(PO4)2 is a promising biomaterial aiming at bone defect treatment [1]. Different from other bone substitutions, β-TCP is a kind of white powder ceramic material, with a good biodegradability, biocompatibility, and the ability to induce the formation of human bones [1, 2]. Additionally, when placed in human body this material can also be degraded into calcium and phosphorus which help the human body form new bones [1]. Due to these outstanding properties, β-TCP has been given close attention in the biomedical industry since 1991[3]. There is a wide range of application for this material like Scaffolds for bone orthopaedics, cartilage regeneration, etc. In recent years, successful clinical treatments have been operated, which illustrates that this bio-medical material has an excellent clinical performance. Detail information can be found by clicking the following URL link or scanning the QR code.
Figure 1: QR code for Beta-TCP in Material Library
https://hub.qmplus.qmul.ac.uk/view/view.php?homepage=beta-tcp&page=beta-tricalcium-phosphate
For obtaining information about the microstructures and chemical compositions of β-TCP powder, in this proposal, three characterization techniques have been used, which are X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). Table 1 indicates what characteristics can be obtained through the provided three techniques.
1. X-Ray Diffraction (XRD)
1.1 Principle
The basic principle of XRD is using regular atomic cells as X-ray diffraction gratings, because the distance between atoms is on the same order of magnitude as the wavelength of an X-ray, X-ray will have diffraction between different atom and finally get the different diffraction intensity value correspond to the different diffraction angle which is closely related to the crystal structure. Through XRD, the crystal structure can be characterized by using the equation
2dsinθ=nλ
(d: distance between crystal face; θ: diffraction angle; λ: the wave length of X-ray)
The X-ray wavelength after diffraction and the diffraction angle can be used to characterize the interplanar spacing and the value of diffraction intensity can be used to characterize the concentration of different interplanar.
1.2 Accuracy and precision
Due to the X-ray focusing issue, the XRD has ~25 micrometres accuracy, if the sample is in nanoscale, the XRD cannot characterize the sample accurately. During the sample rotation in the diffraction part, the angular accuracy of XRD is 0.01°. The XRD precision is related to the substance content, phase cannot be characterized by XRD when the content is lower than 5%[4].
1.3 Characterization result
In the characteristic of β-TCP, the sample is made into powder. During the rotation of the sample from 2θ=20 degree to 2θ=50 degree, the intensity of diffraction is shown in Figure 2. By analyzing the XRD diffraction pattern, the following information about crystal can be characterized:
- Phase and composition: By comparing the intensity with the standard cards, there are two phases in the sample which are β-TCP and β-Ca2P2O7, and from the number of the intensity peak, the concentration of each phase can be shown that the concentration of β-TCP is 71.2% and the rest, which was 28.8% of beta-calcium pyrophosphate.
- Crystal face concentration: From the analyze of intensity and diffraction angle, the highest peak corresponding to the crystallinity of beta phase was found at 2θ=31.5°, which corresponding to the (0 2 1 0) planes, so it means the most abundant crystal face is (0 2 1 0)
- Unit cell structure: The XRD also can reach the cell parameters of β-TCP, which are a=10.42497A ̊ and c=37.34332A ̊ and with the data of interplanar spacing, the structure of beta-TCP can be fully characterized [5].
Figure 2: The XRD diffraction patterns of β-TCP powders [5]
2. Transform Infrared Spectroscopy (FTIR)
2.1 Principle
Irradiate the specimen with a beam of infrared light which is the continuous wavelength. If the energy corresponding to a certain frequency is equal to the vibrational or rotational energy of a molecule, it would be absorbed by the molecule. So, molecules transition from low energy state to high energy state, resulting in energy level transition, then infrared molecular absorption spectra appear [6].
The infrared radiation from the infrared source is collimated by the collimator and then becomes a parallel infrared beam into the interferometer. After passing through the sample, the interferometric light becomes an interferometric light with sample information to be detected by a detector. The detector changes the interference optical signal into an electrical signal. Through the calculation of Fourier transform by computer, it is converted into the infrared spectrum [6,7]. FTRI has a high resolution, normally up to 0.1 ~0. 005 cm-1 [7].
2.2 Characterization result
2.2.1 Composition
Figure 3: FTIR diagram of β-TCP powders [5].
Based on the absorption frequency table, PO43− absorption peak is within 1120~940 cm−1 and P2O74- is 1220 – 960 cm−1, 950-850 cm−1 and 770-705 cm−1[8]. The main absorption bands found for the powders obtained coincide with the group (PO43−) around 1040 cm−1(shown in Figure 3). The V1 type vibration was presented at 960 cm−1, V2 at 460 cm−1, V3 was at 1040 cm−1 and in two different ranges. The first range was 1000–1100 cm−1, the second was 1020–1120 cm−1, which indicates the existence of both PO43− and P2O74-[5][8].
2.2.2 Processing information
By using the FTIR technique, a unique peak was found in Figure 3 around 720 cm−1, which refer to P2O74-. The presence of a residual quantity of P2O74- was the result of the transformation process from TCP to β phase, which would generate beta-calcium pyrophosphate phase (also confirmed by XRD results). Therefore, there is a large possibility that the processing method for β-TCP would be synthesized by sol-gel [9].
3. Scanning Electron Microscope (SEM)
3.1 Principle
SEM is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. In the SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using a secondary electron detector (Everhart-Thornley detector). The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography.[10] The Sigma 500 field emission scanning electron microscope launched by Zeiss adopts the mature GEMINI optical system design with a resolution of more than 0.8nm(at 30kV STEM ).[11]
SEM generally equips with the EDS, which uses the characteristic X-ray wavelength and frequency of the characteristic X-rays generated by the excitation of incident electrons and electrons in the inner shell of the sample to characterize the elemental composition, and uses the characteristic X-ray spectrum to quantitatively analyze the element content per unit volume in the micrometer scale. However, due to the principle of EDS, it cannot be used for the analysis of light atoms, and there are large errors in quantitative analysis (sometimes up to 5%), so it is considered to be a semi-quantitative analysis method [11].
3.2 Characterization Results
3.2.1 Micromorphology
Observing the microstructure of β-TCP power through SEM, the particle size was of 260–1000 nm and the size of the powder distribution presented an average of particle size 350nm (Figure 4(a)) The particles presented aspect ratios of 3 (3 um in large and 1 um in wide oval particles).
Figure 4: Results characterized by SEM: (a) Micromorphology; (b) Elemental composition [5].
3.2.2 Elemental composition
According to Figure 4(b), the β-TCP powders shown a Ca/P ratio of 1.79 by EDS. Since the main components in the material contain Ca3(PO4)2 and Ca2(P2O7) (based on XRD and FTIR analysis), the ratio of their amount of substances can be calculated as 11:3. This is inconsistent with the actual material ratio (7:3) because the material also contains cationic impurities such as Cu. The specific element composition and content are also shown in Figure 4(b).
Reference
[1] Liu Bin, Deng-Xing Lun. Current Application of β-tricalcium Phosphate Composites in Orthopaedics[J]. Aug. 2012.
[2] Donghyun Lee, et al. Injectable biodegradable gelatin-methacrylate/β-tricalcium phosphate composite for the repair of bone defects[J]. 2019.
[3] David. Xijing Hospital repairs long bone defect using Xi'an Particle Cloud's 3D printed artificial bone [OB]. April 20, 2018.
[4] Jeruzalmi D (2006). "First analysis of macromolecular crystals: biochemistry and x-ray diffraction". Macromolecular Crystallography Protocols, Volume 2. Methods in Molecular Biology. 364. pp. 43–62
[5] Criseida Ruiz-Aguilar (2018) “Characterization of β-tricalcium phosphate powders synthesized by sol-gel and chemosynthesis”
[6] Educational materials, Howard University, Physical Chemistry Laboratory, “Fourier Transform Infrared Spectroscopy” (2019).
[7] Huang Hongying, Yin Qihe (2011). “Principle and application of Fourier transform attenuated total reflection infrared spectroscopy (atr-ftir)”.
[8] L.Berzina-Cimdina, N.Borodajenko(2012), “Research of calcium phosphates using fourier transform infrared spectroscopy, Infrared Spectrosc”.
[9] Y. Wang, J. Gao, J. Hu, Solid reaction mechanism of CaHPO4 ·2H2 O + CaCO3 with and without yttria, Rare Met. 28 (2009) 77–81.
[10] Goldstein J.I., Newbury D.E., Michael J.R., Ritchie N.W.M., Scott J.H.J., Joy D.C. (2018) Electron Beam—Specimen Interactions: Interaction Volume. In: Scanning Electron Microscopy and X-Ray Microanalysis. Springer, New York, NY
[11] Carl Zeiss AG. (2020). ZEISS Sigma-Field Emission Scanning Electron Microscope. [Online] Available: https://www.zeiss.com/microscopy/int/products/scanning-electron-microscopes/sigma.html (June 7,2020)