Introduction

At present, due to the difficulty in reconstructing large bone segments, large load-bearing bone defects after trauma, infection or tumor resection are still the main problems faced by surgeons [1,2,3]. The challenge in the field of porous scaffolds is to develop a product that matches the biomechanical properties of bone and also has sufficient biological activity to stimulate new bone regeneration [4,5,6]. Due to the need for effective and comprehensive repairing of bone defects, bioactive ceramics and their composites are moving toward high mechanical strength, good bioactivity and controlled biodegradation [7, 8].

β-tricalcium phosphate (β-TCP) has been considered a valuable implant material for many years [9,10,11]. In fact, β-TCP has a good potential application in bone defects due to its good human compatibility and chemical composition similar to the organic composition of bone [12]. As a bio-absorbable material, β-TCP bio-ceramics are considered to be an ideal temporary scaffold in bone tissue engineering. It will promote the initial formation of new bone tissue and eventually be replaced by new bone [13]. In order to further improve the application characteristics of β-TCP in tissue engineering, people are now paying attention to the improved synthetic calcium phosphate, which changes its physical and chemical properties and biological properties by ion doping [14,15,16,17,18,19]. Mg is the abundant cation in the human body and ranks fourth. It is present in calcified living tissues (about 0.5% in bone and enamel, and more than 1% in dentin). The lack of Mg in bones has been considered to be one of the causes of osteoporosis [20]. When part of Ca2+ in β-TCP is replaced by Mg2+, the rate of degradation of the porous scaffolds in vivo can be regulated [21]. There are many methods for preparing composite calcium phosphate bio-ceramics, such as solid-phase reaction, sol–gel technique, microwave synthesis method and liquid-phase precipitation method [22,23,24,25,26]. The liquid-phase precipitation method is simple in process and low reaction temperature. Therefore, it is one of the most commonly used methods. In this study, MgxCa3−x(PO4)2 composite ceramics are expected to be used in preparing specific porous scaffolds for enhanced large-area segmental bone defect regeneration and in situ repair.

There are many methods for preparing porous bio-ceramic scaffolds, such as solvent casting and particle leaching, combination of direct foaming and sacrificial template methods and 3D printing [27,28,29]. Although the traditional method can also prepare porous bone repair materials, there are some defects: for example, the pores in the material are not coherent, the pore diameter cannot be accurately controlled, and the shape of the pores is single. Therefore, researchers began to study the application of 3D printing technology in the preparation of porous scaffolds. The combination of 3D printing technology and bone tissue engineering scaffolds can improve the osteogenic performance of the graft, reduce the amount of autologous bone and effectively solve the problem of insufficient bone graft in clinical practice. The cancellous bone of the human body is a porous structure, which is arranged by intertwined bone trabeculae and is distributed inside the bone. 3DGP is a rapid prototyping technology based on the gel-molding process. It is a new type of rapid prototyping technology that can be “tailored” and can also print the simulated trabecular micropores to promote bone growth and healing [30, 31]. The slurry is extruded from the nozzle of the printer by gas pressure, and the layer material is scanned and processed in two dimensions under the computer control using the geometric information determined by the CAD model of the part. From the point to the line, from the line to the surface and from the surface to the body, the layers are stacked to obtain a three-dimensional stent blank, and then the body is dried and sintered to obtain the desired stent [32].

In the paper, the effect of Mg2+ on MgxCa3−x(PO4)2 composite scaffolds and preparation process and powder characteristics of MgxCa3−x(PO4)2 composite powders were studied. The influence of different Mg2+ doping contents (0, 5 mol%, 10 mol% and 30 mol%) on degradability and mechanical properties of β-TCP scaffolds was analyzed. The research results show that the proper doping of Mg2+ is helpful for the preparation of MgxCa3−x(PO4)2 scaffold with good mechanical properties and degradability for the repair of cancellous bone.

Experimental

Firstly, MgxCa3−x(PO4)2 powders with different magnesium contents were synthesized by a simple liquid-phase precipitation method, and then the MgxCa3−x(PO4)2 slurry for printing is prepared. Next, the MgxCa3−x(PO4)2 porous scaffolds were printed by 3DGP, and then the scaffolds were degreased and sintered. Finally, the MgxCa3−x(PO4)2 scaffolds were tested.

Materials

The materials used in this study are as follows: diammonium phosphate (AR, Sinopharm Chemical Reagent Co., Ltd), calcium chloride (AR, Sinopharm Chemical Reagent Co., Ltd), magnesium chloride hexahydrate(AR, Sinopharm Chemical Reagent Co., Ltd), ammonia (AR, Sinopharm Chemical Reagent Co., Ltd), acrylamide monomer (AR, Tianjin GuangFu Fine Chemicals), N,N′-methylenebisacrylamide (AR, Sinopharm Chemical Reagent Co., Ltd),ammonium citrate (AR, Beijing Yili Fine Chemicals), PVA (AR, Jiangxi Alpha Gaoke Pharmaceutical Co., Ltd), oleic acid (AR, Shandong West Asia Chemical Co., Ltd), citric acid (AR, Sinopharm Chemical Reagent Co., Ltd) and deionized water (University of Science and Technology Beijing). The characteristics of the materials used in this study are shown in Table 1.

Table 1 Characteristics of materials used in this study
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Preparation of MgxCa3−x(PO4)2 powders

Figure 1 shows the preparation process of MgxCa3−x(PO4)2 powders for 3DGP. Different Mg2+ doping contents (0, 5 mol%, 10 mol% and 30 mol%) of MgxCa3−x(PO4)2 precursor powders were synthesized by liquid-phase precipitation. Anhydrous calcium chloride (CaCl2), diammonium hydrogen phosphate ((NH4)2HPO4) and magnesium chloride hexahydrate (MgCl2·6H2O) were used as starting chemical precursors for magnesium calcium phosphate (MgxCa3−x(PO4)2). When Ca/P ratio is 3.5,the β-TCP scaffolds were obtained by sintering at 1100 °C for 3 h. In order to replace some of calcium ions (Ca2+) in β-TCP with Mg2+, an appropriate amount of MgCl2·6H2O was dissolved slowly in CaCl2 and (NH4)2HPO4 mixed solution to achieve a predetermined (Ca + Mg)/P molar ratio of 3.5 [22], and Mg2+/(Ca2++Mg2+) molar ratio was 0, 5, 10 and 30, respectively. The pH value of mixed solution was adjusted to be about 5.0–6.0 by aqueous ammonia. After the addition was completed, the mixture was stirred at a constant rate and reacted at 40 °C for 2 h. The reacted solution was allowed to settle for 24 h, and the precipitate was separated by filtration and dried at 80 °C. The dried precipitate was studied and printed for scaffolds.

Figure 1
figure 1

The preparation process of MgxCa3−x(PO4)2 powders for 3DGP [22]

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Slurry preparation and characterization

The representative slurry formulations of MgxCa3−x(PO4)2 scaffolds are shown in Table 2. When the molar ratio of (Ca + Mg)/P is 3.5, and the molar ratio of Mg2+/(Ca2 + Mg2+) in the four groups of samples is 0, 5, 10 and 30, respectively, the corresponding X in MgxCa3−x(PO4)2 is 0, 0.15, 0.3 and 0.9. The last two columns are the amount of MgxCa3−x(PO4)2 precursor powder that can be uniformly dispersed in the 3 g premixed solution. MgxCa3−x(PO4)2 composite powders were mixed with premixed solution which was stirred by an appropriate amount of acrylamide, N,N′-methylenebisacrylamide, ammonium citrate and PVA. An appropriate amount of oleic acid can be added to make the slurry more evenly dispersed [22].

Table 2 Chemical composition of MgxCa3−x(PO4)2 ceramic slurry
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Preparation of MgxCa3−x(PO4)2 scaffolds

Table 3 shows the printing parameters of 3DGP for porous MgxCa3−x(PO4)2 scaffolds. The size of the experimental design bracket is 15 mm × 15 mm × 8 mm. The large hole size of the bracket is 1.5 mm × 1.5 mm × 1.5 mm, and the diameter of the needle used for printing is 0.2 mm. From this calculation, it can be known that the porosity of the bracket is about 61%. The UG software is used to design a cube and save it as an STL file, and 2D and 3D printing diagram of the scaffolds is shown in Fig. 2. The file is imported into the matching printing layering software, and the inside of the cube is uniformly distributed with a rectangular body structure. After the G-code is generated, the stand is printed with 3D gel-printing device. And remove it after the stand is sufficiently dry. The temperature was raised to 1100 °C at 2 °C/min in a high-temperature pipe furnace (BFG-12A), kept for 3 h, and cooled to room temperature.

Table 3 Printing parameters of 3DGP for porous MgxCa3−x(PO4)2 scaffolds
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Figure 2
figure 2

Print path generated by the UG software

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Performance Testing

The phase composition of the scaffolds sintered at 1100 °C was verified by X-ray diffraction (XRD; SmartLab) at 40 kV/40 mA. Scanning electron microscopy (SEM; Zeiss EVO18) was used to observe the surface morphology of the C-coated powder and scaffold at an accelerating voltage of 10 kV. The scaffolds porosity was measured by the drainage method [33, 34]:

$$ P = (W_{2} - W_{1} )/(W_{2} - W_{3} ) \times 100\% $$
(1)

where P is the porosity of the stent, W1 is the dry weight of the stent, W2 is the weight of the stent saturated with water and W3 is the weight of the stent suspended in the water. Five samples were tested to calculate the average porosity. Mechanical testing was performed using a static mechanical tester (SUST-CMT5605) and a 10 kN load cell. And the samples were tested at a constant crosshead displacement rate of 0.5 mm/min.

To evaluate the biodegradability of the scaffolds, the PBS buffer having an initial pH value of 7.4 was used with an S/L ratio of 1.0 g/200 ml. At the predetermined time period, the scaffold was rinsed with ethanol, dried at 80 °C for 12 h and weighed (Wt) to determine the mass change of the scaffold and pH change of the solution.

$$ W = W_{0} - W_{\text{t}} /W_{0} \times 100\% $$
(2)

where W0 is the initial weight of the scaffold and Wt is the weight of the scaffold for different soaking times [35].

Results and discussion

The morphology and particle size distribution of MgxCa3−x(PO4)2 powders were important because they directly affect the performance of the printing slurry and also directly affect the performance of the printing scaffolds. In this part, the morphology and particle size distribution of MgxCa3−x(PO4)2 powder, the rheological properties of the slurry, mechanical properties and degradation performance of the printing scaffolds were discussed.

Characteristics of MgxCa3−x(PO4)2 powders

The MgxCa3−x(PO4)2 powders are characterized by SEM, EDX and XRD. Figure 3 shows that SEM images and particle size distribution of MgxCa3−x(PO4)2 precursor powders prepared in this study. When Mg2+ doping content is 0, 5 mol%, 10 mol% and 30 mol%, the particle size of the precursors are about 44.51 μm, 41.91 μm, 38.38 μm and 31.93 μm, respectively. The particle size of the powder is suitable for 3D printing. As shown in Fig. 3, the powders have an irregular shape and a certain agglomeration phenomenon. The agglomeration of ceramic powders during the drying process is due to the existence of the solid–liquid interface and the surface tension of the liquid. The collapse of gel pores and the accumulation of particles can easily occur. After the addition of Mg2+, the agglomeration of the powder is improved that attributes to the stability of β-TCP structure. Mg2+ can be present as a stabilizer in MgxCa3−x(PO4)2.

Figure 3
figure 3

SEM images and particle size distribution of precursor powders for 3DGP: a TCP-0 Mg, b TCP-5 Mg, c TCP-10 Mg and d TCP-30 Mg

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The purpose of EDX spectrum analysis is to verify whether Mg2+ participate in the reaction and form a composite phase. As shown in Fig. 4,EDX spectrum analysis revealed the presence of oxygen (O), calcium (Ca), phosphorus (P) and a small amount of Mg. Mg element was detected in MgxCa3−x(PO4)2 precursor powders prepared in this study. Mg2+ was successfully doped into β-TCP powders by liquid-phase precipitation. The magnesium-doped composite powder has the same elemental composition, but the content of each element is different. As Mg2+ content increases, the peak of Mg element is also more pronounced. Figure 5 shows XRD pattern of different Mg2+ content powders after sintering. When Mg2+ content is different, XRD diffraction peaks of the powders are consistent with the standard peak of β-TCP (PDF#09-0169). XRD diffraction pattern of the powders and EDX analysis confirmed that when Mg2+ replaced a part of Ca2+ in β-TCP, a crystallized MgxCa3−x(PO4)2 phase was formed.

Figure 4
figure 4

EDX component analysis of MgxCa3−x(PO4)2 precursor powders for 3DGP: a TCP-0 Mg, b TCP-5 Mg, c TCP-10 Mg and d TCP-30 Mg

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Figure 5
figure 5

XRD pattern of MgxCa3−x(PO4)2 powders after sintering: a TCP-0 Mg [22], b TCP-5 Mg, c TCP-10 Mg and d TCP-30 Mg

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Rheological properties of slurry

Figure 6 shows the apparent viscosity of slurry with different Mg2+ contents. At a solid content of 50 vol% [22], the slurry viscosity decreases with increasing shear rate, exhibiting the characteristics of a pseudo-plastic fluid. Since the colloidal particles in the slurry belong to a chain polymer, when the shear rate is low, the long-chain molecules are entangled with each other, and the viscosity of the slurry is large. As the shear rate increases, the long-chain polymer shrinks, and the viscosity decreases with the shear stress. When the Mg2+ content is different, the viscosity of the slurry changes, and the viscosity gradually decreases as the shear rate increases. When the shear rate is less than 160 s−1, the viscosity of TCP-30 Mg slurry is higher than TCP-10 Mg at the same shear rate. When the shear rate is greater than 160 s−1, the viscosity of the two is close, and the viscosity of TCP-30 Mg is slightly lower.

Figure 6
figure 6

The apparent viscosity of MgxCa3−x(PO4)2 slurry with different Mg2+ contents at different shear rates

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Characteristics of MgxCa3−x(PO4)2 scaffolds

The scaffolds for tissue engineering applications are expected to have porous structures with pore interconnect. The porous structure enables cells to attach better to the scaffold, facilitating cell proliferation and internal growth of new tissues and transport of nutrients to developing bones. The macro view of the MgxCa3−x(PO4)2 scaffolds is shown in Fig. 7. The addition of Mg2+ has no obvious effect on the macroscopic morphology of the green body stent, and the stent shows good formability. As the Mg2+ content increases, the contraction of the stent also gradually increases after sintering. From Fig. 7(d1–d4), it can be seen that when the magnesium content is 30 mol%, the stent has obvious contraction. And the stent has a slight phenomenon of warping around the periphery.

Figure 7
figure 7

The macro view of MgxCa3−x(PO4)2 porous scaffolds printed by 3DGP

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Figure 8 shows the shape integrity, and surface morphology of MgxCa3−x(PO4)2 porous scaffolds printed by 3DGP were investigated by SEM. As shown in Fig. 8a1–d1, the surface of the printed silk is smooth, and the macroscopic pores of the stent exhibit a regular shape. The size of the pores is about 1.5 mm × 1.5 mm. As shown in Fig. 8a2–d2, at the junction, the connection between the two printing wires is good, and there is no delamination or smashing. As shown in Fig. 8a3–d3, in addition to the pre-designed macroporous structure, the scaffolds also have an open-cell structure formed by the removal of the binder, and the micropores are distributed on the walls of the large pores. Since the structure and size of the micropores produced by the binder removal are irregularly distributed, the scaffolds exhibit a broad interconnected distribution in the range of 10-750 um. As shown in Fig. 8a4–d4, the micropores number on the printed silk decreases as Mg2+ content increases, indicating that the sintering of the powder is improved as the Mg2+ content in the sample increases, which is due to Mg2+ replaces part of Ca2+ in β-TCP, the particle size of the powder becomes smaller, the degree of activation is improved and the sintering condition of the powder is improved.

Figure 8
figure 8

SEM images of MgxCa3−x(PO4)2 porous scaffolds printed by 3DGP

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Shrinkage and porosity of sintered MgxCa3−x(PO4)2 scaffolds

Figure 9 shows the shrinkage and porosity of MgxCa3−x(PO4)2 porous scaffolds with different Mg2+ contents. The shrinkage and porosity of the scaffolds are affected by Mg2+ content. As Mg2+ content increases, the shrinkage of the scaffolds gradually increases. At the same time, the porosity of the scaffolds gradually decreases. When Mg2+ content was increased to 30 mol%, the shrinkage ratio of the scaffolds in the three directions of length, width and height was 35.12%, 32.69% and 33.08%, respectively. The shrinkage of TCP-30 Mg scaffolds increased compared to pure β-TCP stent, which is caused by an increase in the electrostatic binding force between Mg–O and Ca–O in the structure and a closer distance between Mg–O and Ca–O in the MgxCa3−x(PO4)2 structure. When the Mg2+ content is 0, 5 mol%, 10 mol% and 30 mol.%, the porosity of the sintered stent is 72.11%, 70.26%, 69.83% and 65.15%. Both are higher than the theoretical porosity of 61%. The reason is that the removal of organic matter after sintering makes the pores on the printing yarn increase. With the increase in Mg2+ content, the contraction rate of the stent gradually increased. When the Mg2+ content increased from 5 to 10 mol%, the shrinkage of the stent increased to a small extent, while the porosity decreased. When the Mg2+ content increased to 30 mol%, the shrinkage of the stent significantly decreased, and the porosity also appeared to a large extent decline. In order to further study the effect of Mg2+ content on the porosity and shrinkage of the stent, we will conduct a more in-depth study of the role of Mg2+ in the next study.

Figure 9
figure 9

Shrinkage and porosity of MgxCa3−x(PO4)2 scaffolds

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In vitro degradation of MgxCa3−x(PO4)2 scaffolds

The biodegradable scaffolds provide initial structure and stability for tissue formation. The scaffolds degrade as the tissue is formed, providing space for matrix deposition and tissue growth. The degradation of MgxCa3−x(PO4)2 scaffolds in SBF solution is shown in Fig. 10. Compared to pure β-TCP scaffolds, the degradation of the Mg-doped stent scaffolds is more significant. As Mg2+ content increases, the weight loss rate curve of MgxCa3−x(PO4)2 scaffolds for the soaking time is shown in Fig. 11a. There was no significant change in the solubility of pure β-TCP scaffolds throughout the impregnation. At the first week, the quality of Mg-doped stent increased significantly. The dissolution of the porous scaffolds and the precipitation of the apatite phase during the impregnation are simultaneously performed. When the precipitation rate of the apatite phase is faster than the degradation rate of the stent, the mass of the stent will increase. In the second and third weeks, the quality of the stent gradually began to decline. After 3 weeks, the degradation rate of the stent tended to be flat. As the immersion time is extended, the solubility of MgxCa3−x(PO4)2 scaffolds as a whole decreases as Mg2+ content increases, which may be caused by a white block phase on the MgxCa3−x(PO4)2 surface.

Figure 10
figure 10

Degradation of MgxCa3−x(PO4)2 scaffolds in SBF

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Figure 11
figure 11

Weight loss rate of MgxCa3−x(PO4)2scaffolds and pH change of SBF

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Figure 11b shows pH change curves of solution in the buffer during the impregnation stage. The pH value decreases with the prolongation of the soaking time. At the beginning of the soaking, the pH value of SBF solution drops sharply. Due to the decomposition of MgxCa3−x(PO4)2 and the precipitation of apatite phase, a large amount of Ca2+, Mg2+, PO43− and CO32− exists in the solution. Ca2+ and Mg2+ are deposited on the surface of the sample, so a large amount of PO43− and CO32− remains in the SBF solution, causing a sharp drop in the solution pH value. The degradation rate of the material and the deposition rate of the apatite phase change from an unbalanced wave state to an equilibrium state. After soaking for 8 weeks, the solution pH value was about 5.5.

Compressive strength of MgxCa3−x(PO4)2 scaffolds

The compressive strength curves of MgxCa3−x(PO4)2 scaffolds with different Mg2+ contents are shown in Fig. 12. The maximum force in the original chart report generated by the mechanical performance tester is greater than the true maximum force the material can withstand. Due to the presence of large bore passages in the stent, when the powder structure is compressed, the large pores close, creating a more robust structure that provides greater resistance to the applied force. Therefore, the first peak in the resulting graph is considered to be the maximum compressive strength that the stent can withstand. When Mg2+ doping content is 0, 5 mol%, 10 mol% and 30 mol%, the compressive strength of the stent is 1.04 ± 0.01 MPa, 0.73 ± 0.17 MPa, 0.23 ± 0.01 MPa and 1.89 ± 0.21 MPa, respectively. Mg2+ can inhibit the growth of tricalcium phosphate crystals. The decrease in crystallinity may be the reason for its compressive strength reduction [36]. Previous studies have shown that when magnesium-substituted hydroxyapatite is used to prepare apatite, the incorporation of magnesium reduces the compressive strength of the apatite [37]. As the magnesium ion content increases, the stent shrinks sharply and the porosity decreases, so the stent strength increases. When Mg2+ doping amount is 30 mol%, the scaffold has the largest shrinkage rate and the highest strength. The mechanical strength of the scaffolds makes them unsuitable for load-bearing applications. However, they can be used in other applications such as sponge bone, cancellous bone or bone fillers [38,39,40].

Figure 12
figure 12

The compressive strength curve of MgxCa3−x(PO4)2 porous scaffolds printed by 3DGP: a TCP-0 Mg [22], b TCP-5 Mg, c TCP-10 Mg and d TCP-30 Mg

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Conclusions

MgxCa3−x(PO4)2 composite powders were prepared successfully by liquid precipitation method. And the porous MgxCa3−x(PO4)2 composite scaffolds were printed by 3DGP technology. When Mg2+ doping content is 30 mol%, the degree of the precursor agglomeration is the smallest, and the particle size of the powder is about 31.93 μm. After TCP-30 Mg scaffolds were sintered at 1100 °C for 3 h, the strength reached a maximum of 1.89 ± 0.21 MPa, and the porosity was 65.15%. In addition, the addition of Mg2+ plays a role in regulating the degradation of the scaffolds. Compared to pure β-TCP scaffolds, the degradation of the Mg-doped scaffolds is more significant. And the weight loss rate of the scaffolds decreases with increasing Mg2+ content. This approach provides an idea for developing biodegradable, high-strength bio-ceramic scaffolds, which is a huge application prospect in bone defects repairing.

In summary, it was found in the study that Mg2+ can replace the Ca2+ in β-TCP during the liquid-phase reaction and form a new calcium–magnesium phosphate phase. The addition of Mg2+ has a certain regulatory effect on the compressive strength and degradability of the β-TCP scaffold, but no obvious law was found. And a clearer understanding of the specific mechanism of action of Mg2+ is lacking. Therefore, the specific mechanism of action of Mg2+ will be further explored in the next research work.