β-tricalcium phosphate and octacalcium phosphate composite bioceramic material for bone tissue engineering

Introduction

With the development of transportation industry and the aggravation of population aging, bone graft treatment is becoming increasingly important due to the ever growing bone defects caused by trauma, tumor, and infection. ¹ Autogenous bone treatment has a preferable bone conductivity, superior osteoinductivity and reasonable mechanical strength.^2,3 However, there remains many disadvantages such as hemorrhage, trauma, donor infection, and limited choice of material (especially when bone defect is large). Allogeneic bone encounters limited donor source and safety problem.^4–6 In recent studies, metals and organics are also used as biomedical materials. However, metals tend to be corroded after being implanted into the human body which might produce toxic metal ions. ⁷ In addition, metal implantation may affect the surrounding biological tissues. As for organic materials, most of them exhibit relatively low strength and are insufficient to meet the required mechanical properties and durability. ⁸ Therefore, artificial synthesis of biomaterials and their implantation into the bone defects to repair the bone tissue are important. ⁹

Bone tissue engineering focuses on the use of bioactive materials to rebuild or repair diseased or defected bone tissue by in vitro culture or construction to strengthen its function. It is a typical inter disciplinary objective involving biology, material science, and engineering. ¹⁰ Scaffold material is a framework material supporting cells to grow into a complete tissue, and is the indispensable component in bone tissue engineering.^11–13 In recent years, with the development of life science, material science and medical clinic, such biomaterials have been deeply studied and widely applied. ¹⁴

TCP is a bioresorbable, bioactive and osteoconductive material.^15–17 The β-TCP phase attracts more attention due to its solubility, higher degradation rate and angiogenic activity, which are important for the neo-vascularization process of new tissues.^18–21 However, β-TCP exhibits insufficient osteoconductivity and poor osteoinductivity. OCP changes into HA spontaneously during new bone formation. ²² It has good biodegradability, osteoconductivity and osteoinductivity as well as biological activity. Many studies have shown that the implantation of OCP into animals can induce more new bone formation than other types of calcium phosphate ceramics.^23–26

In this study, OCP and β-TCP were prepared by co-precipitation method. A novel β-TCP/OCP biological composite material was fabricated using molding and cold isostatic pressing technique. We aim to integrate desired properties of β-TCP and OCP biomaterials in a composite scaffold to enhance the ability of bone regeneration.

Experimental procedure

(NH₄)₂HPO₄ (analytical reagent, A.R.), Ca(NO₃)₂4·H₂O (analytical reagent, A.R.), NaH₂PO₄·2H₂O (analytical reagent, A.R.), Ca(CH₃COO)₂·H₂O (analytical reagent, A.R.) were purchased from the company Aladdin. The β-TCP was prepared by co-precipitation method. ²⁷ The solution of Ca(NO₃)₂ (0.4 mol/L, 250 ml) was added to (NH₄)₂HPO₄ solution (0.6 mol/L, 250 ml) at 25°C for 0.5 h. After drying at 80°C for 4 h and at 120°C for 8 h, powders were sintered with a heating rate of 5°C/min to 800°C (kept for 3 h). OCP was prepared by the same method. The solution of Ca(CH₃COO)₂(0.04 mol/L, 250 ml) was added to NaH₂PO₄ solution (0.04 mol/L, 250 ml) at 70°C for 0.5 h. The above solution was washed with D.I. water for three to five times. Then the white floccus was freeze-dried. The initial temperature for pre-freezing was −60°C, and the pre-freezing time was 8 h. Then drying started at a rate of 2.5°C/h until 25°C. We put the two powders into a mortar and ground them together with polyvinyl alcohol as a binder. The β-TCP/OCP composite was molded at 80 MPa. Then, the composite was molded by cold isostatic pressing under 200 MPa.

XRD analysis was performed using X-ray diffraction (DMAX-2500PC, Rigaku, Japan) with CuKα radiation (λ = 0.1542 nm) at 45 kV and 150 mA. The 2θ range was from 10° to 90°at a step size of 10°/min for β-TCP. The 2θ range was from 3°to 60°at a step size of 10°/min for OCP. The chemical compositions of samples were investigated by the Fourier Transform infrared (FTIR) spectrometer (Vertex70, Bruker, Germany) with KBr pellet in a wavenumber range from 4000 to 400 cm⁻¹ at room temperature. The fracture surfaces were observed by scanning electronic microscopy (SEM) (Hitachi Model SU-70, Japan). In the test of compressive strength (CMT5105, Shenzhen new think metering technology company, China), samples were prepared in a rectangular shape with size of 5 mm × 5 mm × 10 mm. Universal testing machine crossbeam descending speed was set to 1 mm/min. The porosity was measured by the vacuum method. The specific operation steps are as follows: the sample was dried at 100°C, and weighed, marked as M1. Then, the sample was placed in a vacuum dryer with a pressure less than 0.0013 MPa for 10 min. Then the immersion liquid was injected until the sample was immersed. Pump down again until there were no air bubbles on the sample. Then weigh the sample in immersion liquid, marked as M2. Wipe off the liquid beads on the surface of the sample, weigh the sample immediately, marked as M3. The porosity was equal to (M3–M1)/(M3–M2) ×100%.

Results and discussion

The results of XRD patterns of β-TCP are shown in Figure 1. The position of the diffraction peak in the XRD diffraction pattern almost completely coincides with the diffraction peak on the JCPDS – ICDD standard card (#09–0169) of β-TCP, ²⁸ indicating the high purity of the obtained β-TCP.

OPEN IN VIEWER

Figure 1.

XRD patterns of β-TCP powder.

The results of XRD patterns of OCP are shown in Figure 2. According to the experimental data, the diffraction peaks at 4.7°, 26. 5°, 28.7°, 32.6°, and 33.6°are ascribed to the diffraction of (100), (421), (600), (031), and (700) crystal faces, respectively. Both the position and intensity of the diffraction peaks are consistent with the JCPDS – ICDD standard card (#79–0423) for triclinic OCP. ²⁹

OPEN IN VIEWER

Figure 2.

XRD patterns of OCP powder.

Figure 3 shows the FTIR spectrum of β-TCP and OCP. By comparing the infrared characteristic frequencies of various phosphates reported, the main infrared characteristic frequency of β-TCP is 550 cm⁻¹, 605 cm⁻¹, 948 cm⁻¹, 974 cm⁻¹, 1037 cm⁻¹, 1080 cm⁻¹, and 1120 cm⁻¹. The valleys at 3740 cm⁻¹ and 1635 cm⁻¹in the FTIR spectrum are attributed to the vibration absorption peaks of H₂O. While the 948 cm⁻¹–1120 cm⁻¹ is the stretching peak for PO₄³⁻, and 550 cm⁻¹–605 cm⁻¹ is the bending vibration peak for PO₄³⁻, indicating that the main component of the material is β-TCP. ³⁰

OPEN IN VIEWER

Figure 3.

FTIR spectra of β-TCP and OCP.

As for the FTIR spectrum of OCP (as shown in black line in Figure 3), the bands are classified as follows: the relatively wide peak with the wavenumber of 3448 cm⁻¹ is generated by the crystal water in OCP, and 1651 cm⁻¹ the hydroxyl group in the crystal water of OCP, which is the stretching oscillation absorption peak of O–H. The peak at 2364 cm⁻¹ should be attributed to the CO₂ in the air, namely C–O–C stretching oscillation absorption peak. ³¹ The spectral bands of 562 cm⁻¹, 601 cm⁻¹, 1031 cm⁻¹, and 1076 cm⁻¹ region are the characteristics of PO₄³⁻. Meanwhile, the bands at 526 cm⁻¹, 864 cm⁻¹, and 912 cm⁻¹ should be ascribed to the existence of HPO₄. The results of infrared spectroscopy further prove that the OCP was successfully synthesized, which are consistent with the XRD results.

The SEM image of β-TCP (Figure 4(a)) indicates that the particle size of the powder is uniformly distributed between 200 and 300 nm. The OCP powder exhibits a wide-plate-like or thin-blade-like crystal morphology (Figure 4(b)). The long strip is about 1–2 µm in length and 0.1–0.4 µm in width with a length-width ratio of more than 10. These strips are stacked on top of each other with varied size and length.

OPEN IN VIEWER

Figure 4.

SEM images of the β-TCP and OCP: (a) β-TCP; (b) OCP.

Figure 5 shows the porosity of the composites with different proportions. All materials possess a porosity of more than 33%. It is known that the porosity of materials implanted in living organisms must meet certain requirements. When the porosity exceeds 30%, the pores could communicate with each other, and new bone tissue could grow from the surface of the material into the interior. Generally, the porosity is controlled within the range from 40% to 60%, which could achieve the connectivity of the pores and also meet the requirements for material strength. As can be seen from Figure 5, as the proportion of OCP increases, the porosity of the material decreases first and then increases. This may be caused by the difference in the microscopic morphology of the two particles. The more different the mixing ratio between the two, the more uneven the mixing between the spherical β-TCP and the lath-like OCP. It creates more gaps and increases the porosity. The variation of porosity for all materials is very small and the difference is about 10% overall.

OPEN IN VIEWER

Figure 5.

Porosity and compressive strength of composites with different ratios. (1) β-TCP:OCP = 7:3; (2) β-TCP:OCP = 6:4; (3) β-TCP:OCP = 5:5; (4) β-TCP:OCP = 4:6; (5) β-TCP:OCP = 3:7.

The values of compressive strength of composites with different ratios are shown in Figure 5. The compressive strength of all materials is greater than 25 MPa, and the largest reached to 64 MPa. This basically meet the mechanical requirements for scaffold material. As the OCP content increases, the compressive strength of materials improves. The possible reason might be the lath-like OCP acts like a fiber-reinforced phase, preventing the crack from expanding, being able to withstand greater forces.

Figure 6 shows the SEM images of the fracture section of a composite material with different proportions. In Figure 6(a), the spherical β-TCP partially agglomerates, and the lamellar OCP partially laminates together. In Figure 6(b), the large piece of β-TCP and OCP is clustered together, and the two particles are hardly bounded together, which might be the reason for the lower strength at this ratio. In Figure 6(c), the two particles are more uniformly mixed together, and the void distribution is relatively uniform. In Figure 6(d) and (e), the microstructures of the two samples are similar. Spherical β-TCP is distributed in large holes, and the lamellar OCPs are stacked together, covering on the top. Some short rod-shaped OCPs are protruding from the gap. The combination is very close.

OPEN IN VIEWER

Figure 6.

SEM of the fracture section.(a) β-TCP:OCP = 7:3; (b) β-TCP:OCP = 6:4; (c) β-TCP:OCP = 5:5; (d) β-TCP:OCP = 4:6; (e) β-TCP:OCP = 3:7.

In order to verify the bioactivity of the prepared composites, scaffolds were immersed in SBF solution for 28 days. Figure 7 shows the surface morphology evolution after soaking in SBF. It could be seen that a sheet of material deposits on the surface of the material. This layer is a layer of hydroxylapatite. This indicated that the composite possesses certain biological activity. At the same time, with the increase of OCP, the compactness and uniformity of materials deposited on the surface are also enhanced, because OCP possess stronger biological activity than β-TCP, which could promote the deposition degree of hydroxylapatite.

OPEN IN VIEWER

Figure 7.

SEM of composites after 28 days of incubation in SBF. (a) β-TCP:OCP = 7:3; (b) β-TCP:OCP = 6:4; (c) β-TCP:OCP = 5:5; (d) β-TCP:OCP = 4:6; (e) β-TCP:OCP = 3:7.

Conclusions

In summary, we have successfully prepared β-TCP and OCP powder using the co-precipitation method. The results of XRD and FTIR show that the powder is pure with negligible impurities. Based on the results of compressive strength test and the porosity test, the composite exhibits excellent mechanical properties. In the meantime, the high porosity of more than 33% of the material is conducive to the attachment and growth of cells.