In recent years, biodegradable metal materials have attracted extensive attention due to their good combination of degradability and mechanical properties, especially in the field of orthopedics. Magnesium and its alloys are known as revolutionary metal biomaterials, which have been widely studied and clinically explored. Additive manufacturing technology has the ability to prepare complex geometric structures, which provides unprecedented opportunities for the preparation of biodegradable metal implants, especially for biodegradable magnesium alloys with appropriate mechanical properties and good biocompatibility. However, due to the inherent characteristics of magnesium alloys, such as low evaporation temperature, high vapor pressure and high oxidation tendency, there are a series of problems to be solved.

Ding Wenjiang, academician of Chinese Academy of engineering, wrote an article in the journal engineering of Chinese Academy of engineering, analyzed the challenges faced by additive manufacturing degradable magnesium based implants in the aspects of magnesium powder preparation, powder splashing and cracks, and put forward corresponding countermeasures. A new type of magnesium alloy powder with smooth surface and good roundness was successfully prepared, Then the additive manufacturing parameters were optimized. Based on the optimized parameters, three kinds of porous magnesium alloy scaffolds with different structures (bionic, diamond and minimal curved surface) were fabricated by selective laser melting technology, and their mechanical properties and degradation behavior were analyzed. Finally, the minimal curved scaffolds with the best performance were selected for calcium phosphate coating treatment, which greatly inhibited the degradation rate of scaffolds and improved their cytocompatibility. The article points out that the fabrication of magnesium alloy scaffolds with additive materials shows the prospect of clinical application as bone tissue engineering scaffolds.

一、

introduction

In recent years, biodegradable metal materials have attracted extensive attention due to their good combination of degradability and mechanical properties, especially in the field of orthopedics.

Among them, magnesium and its alloys are known as revolutionary metal biomaterials, which have received extensive research and clinical exploration.

Magnesium alloy has the following advantages: firstly, its mechanical properties are similar to natural bone, so it can avoid the stress shielding effect caused by the mismatch of elastic modulus; Secondly, magnesium is the fourth most abundant metal element in human body, which is very important for metabolism; In addition, studies have shown that magnesium ions can promote bone healing and new bone formation. However, the rapid degradation rate of pure magnesium and some magnesium alloys in physiological environment hinders its further clinical application.

Our team developed a new biodegradable mg nd Zn Zr magnesium alloy (jdbm), which has excellent corrosion resistance and antibacterial properties. At the same time, we also carried out long-term in vivo animal experiments of jdbm screw used in mandibular fracture and jdbm cardiovascular stent. The results further confirmed the application potential of the alloy in the field of medical implantation. However, these magnesium based implants are mainly prepared by traditional manufacturing process, and the traditional manufacturing process has limitations in the preparation of implants with complex geometry and ideal mechanical properties, so it is necessary to explore new manufacturing process.

Recently, additive manufacturing (AM) technology has attracted more and more attention, because its precise control of complex or porous structure is better than traditional manufacturing process, and it has gradually become one of the promising methods to prepare metal biomaterials. At present, traditional metal implants, such as titanium or stainless steel, have been widely used in the experimental research of additive manufacturing. However, am degradable magnesium alloy has made slow progress due to the difficulty in preparation of magnesium powder and its flammability. Recently, selective laser melting (SLM) technology has been used in the preparation and research of magnesium alloys, showing a certain medical application prospect; The porous scaffolds made of commercial magnesium alloy WE43 with diamond structure were prepared by SLM technology. The scaffolds meet the functional requirements of bone implants. However, the surface of the WE43 scaffold is not suitable for cell adhesion, and the structure of the porous magnesium scaffold also needs to be further improved.

This paper first analyzes the challenges faced by am degradable magnesium alloy, and puts forward strategies to deal with these challenges, including am parameter optimization and preparation of jdbm scaffold with better structure by SLM. At the same time, surface modification of AM scaffold is also studied.

2

Challenges of additive manufacturing degradable magnesium alloys

Due to the inherent characteristics of magnesium alloys, such as low evaporation temperature, high vapor pressure and high oxidation tendency, am degradable magnesium based implants are facing a series of challenges.

（1） Powder preparation is difficult

The preparation of magnesium powder is very demanding, and a little carelessness will cause an explosion accident. At present, only pure magnesium powder, AZ91D powder and WE43 powder are commonly used in the market. However, due to the biological toxicity of aluminum, AZ91D alloy contains 9% (mass fraction) aluminum, so only pure magnesium and WE43 powder are suitable for am degradable magnesium based implants.

The basic preparation methods of magnesium powder include mechanical crushing method, molten metal atomization method, evaporation condensation method and electrolysis method. The particle size of magnesium powder suitable for am degradable metal implants is 20 ~ 70 μ m. At present, most of these powders are prepared by gas atomization. However, the size range of magnesium powder prepared by inert gas atomization is from a few microns to 0.5 ~ 1.0 mm, which makes the utilization rate of powder for am research low.

（2） Powder spatter

Serious powder splashing occurs in the AM process of magnesium alloy, which is due to the low evaporation temperature and high vapor pressure of magnesium alloy. This phenomenon is very different from that of steel, titanium or aluminum alloy. Powder splashing will significantly reduce the stability of AM process of magnesium alloy, because some magnesium powder will be removed by steam along the scanning path, and defects are likely to occur here in the subsequent scanning passes. Therefore, the strategy of powder supplement must be adopted in the AM process of magnesium alloy. However, there is no research on the interaction between magnesium evaporation, gas flow and laser input. Reducing the tendency of magnesium evaporation is another possible solution. Zumdick et al. Successfully prepared WE43 block with very low energy input; In their method, the printing plane is slightly offset relative to the focal plane of the laser beam, resulting in about 125 μ M, which is about 90 μ The larger the spot diameter is, the energy input of WE43 is reduced by two times.

（3） Crack

Unfortunately, cracks occasionally appear in am magnesium alloy. The reason for the formation of cracks is not clear, which may be related to the above mentioned powder splashing, because the tendency of cracks decreases with the decrease of powder splashing at lower energy input. Figure 1 shows the typical crack formation of mg-15gd-1zn Zr magnesium alloy (gz151k) during SLM.

Figure 1 cracks on am gz151k block（ a) The crack is perpendicular to the printing direction（ b) Optical micrograph (OM) of crack on Y-Z plane in Fig. (a)

3

Coping strategies and research progress

Due to the complex physical mechanism of SLM process and the inherent characteristics of magnesium alloy, the two main variables affecting SLM process, namely process related parameters and raw material related parameters, must be carefully selected.

For the preparation of magnesium powder (i.e. raw material related parameters), even in ultra-high purity argon, the oxygen content must be kept as low as possible, which is due to the high oxidation tendency of magnesium powder; At the same time, the atomization technology used in the preparation of other metal powders should be further developed to obtain the appropriate powder size.

The main goal of SLM is to obtain high density and avoid possible defects. The most common way is to adjust the process parameters (laser power, scanning speed, scanning line width, etc.). The powder spatter can be reduced by increasing the laser scanning speed or reducing the laser power; In addition, the chemical composition of magnesium alloy should be selected according to its different crack sensitivity. At the same time, the initial temperature of the substrate should be increased to reduce the temperature gradient between the substrate and the metal powder, so as to further avoid the formation of hot cracks. By considering the above factors, we have successfully prepared am magnesium alloy. The related progress will be introduced in the following sections.

（1） Preparation of jdbm powder

In cooperation with Tangshan Weihao magnesium Power Co., Ltd., Shanghai Jiaotong University produced several magnesium powders for am by gas atomization, including gz151k and jdbm magnesium alloys. Figure 2 shows the scanning electron microscope (SEM) image of jdbm powder after size screening, and the particle size is 50 ~ 75 μ Most of the powders have smooth surface and good roundness. However, it can be found that some powder particles have smaller particles attached to their surfaces [shown by the white arrow in Fig. 2 (a)], while some powder particles have partial shells [shown by the black arrow in Fig. 2 (a)]. The corresponding enlarged SEM image of jdbm powder [Fig. 2 (b)] shows that the fine network of the second phase can also be observed on the surface of the powder.

Fig. 2 SEM image and corresponding enlarged image of gas atomized jdbm powder

（2） Optimization of manufacturing parameters of added materials

As mentioned above, powder splash and crack may occur in am process of magnesium alloy. In order to optimize the process parameters, the effects of laser power (P, w), scanning speed (V, mm · s-1) and scanning line width (HS, mm) on Microstructure of gz151k block are studied, and energy density is evaluated[ ψ = P / (HS · V · T)] and am bulk density, where t is the scanning layer thickness (t = 30 μ m) The test results are shown in Figure 3. It can be found that the bulk density increases with the increase of energy density, and tends to be stable at 2G · mm - 3; The higher the density of the sample, the less defects and the better the mechanical properties. According to this method, the AM optimization process parameters of jdbm alloy are listed in Table 1.

Table 1 am optimized process parameters

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Fig. 3 densification of am gz151k alloy（ ρ）- Energy density（ ψ） Where, ρ = – 0.44 exp (–17.54 ψ) + 2.02， ψ = P/(HS·V·t )， R2 = 0.9995

（3） Design and preparation of jdbm scaffold

Using rhinoceros ® 6.0 software designed three kinds of scaffolds, namely bionic structure (b), diamond structure (d) and minimal curved surface structure (g). B scaffold has a random structure similar to natural bone, D scaffold is a topological ordered rod structure, and G scaffold is a triple periodic minimal surface (TPMS) structure with zero mean curvature. The three scaffolds have the same porosity (75%) and average pore diameter (800%) μ m）。 Based on the optimization of process parameters and gas atomization of jdbm powder, scaffolds with diameter of 10 mm and height of 12 mm were prepared by SLM equipment under the condition of oxygen content less than 100 ppm. After preparation, all scaffolds were electropolished. The polishing electrolyte was composed of 10% (volume fraction) perchloric acid and 90% (volume fraction) anhydrous ethanol. The structure of the polished stent was analyzed by micro CT, and the scanning resolution was 17 μ m。 The micro CT reconstruction structure, the original CAD model and the macro image are shown in Fig. 4. According to the micro CT results, it can be found that the polished bracket has the same open hole structure as the design model.

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Fig. 4 CAD model of three kinds of scaffolds [bionic structure (b), diamond structure (d) and minimal curved surface structure (g)] and structural schematic diagram after polishing

Figure 5 shows the micro CT image cross section and SEM image of the polished scaffold. It can be found that the polished porous structure has smooth surface, no unmelted powder particles remain, and no cracks are observed. Therefore, am jdbm scaffold can meet the requirements of tissue engineering scaffold, that is, high porosity and fully connected porous structure.

Fig. 5 micro CT images [(a) ~ (c)], SEM images [(d) ~ (f)] and corresponding enlarged SEM images [(g) ~ (I)] of B [(a), (d), (g)], d [(b), (e), (H)] and G [(c), (f), (I)] stents

（4） Mechanical properties and in vitro degradation behavior of jdbm scaffold

The compression test was carried out on Zwick ag-100kn testing machine (Zwick Roell, Germany) at room temperature. The specimens were polished supports with a diameter of 10 mm and a height of 12 mm, and the compression speed was 1 mm · min – 1. The stress-strain curves were recorded in the form of engineering stress and Engineering strain, and each support was repeated for three times. The slope of the initial linear part of the stress-strain curve is taken as the young's modulus of the scaffold, the yield strength is calculated by the 0.2% offset method, and the strength at 30% of the strain is taken as the platform stress.

Figure 6 shows the compressive stress-strain curves of polished scaffolds. It can be found that although the three scaffolds have different structures, they all show similar compression characteristics of porous materials. The curves can be divided into three typical stages: elastic stage (I), stress plateau stage (II) and densification stage (III). At the stage of stress plateau, the stress-strain curves of D and G scaffolds fluctuated, and the peak stress almost did not increase until the densification stage; However, the stress-strain curve of B scaffold shows more strain hardening in the stress plateau stage, which indicates that the mechanical properties of AM scaffold largely depend on its porous structure.

Fig. 6 compressive stress-strain curves of supports B, D and G

Compared with topologically ordered structures (D and G supports) with uniform structural thickness, the mechanical strength of support B is relatively lower, which may be due to the larger failure tendency of some thin struts, as shown in Fig. 5 (a). It is found that the lamellar g stent exhibits tensile deformation behavior under compression load, so its mechanical strength is higher than that of the rod-shaped D stent, and the latter exhibits bending compression behavior. The fluctuation of stress-strain curve is the result of the joint action of scaffold structure and material. The fluctuation of stress-strain curve of D and G scaffold is more obvious than that of B scaffold, which may be due to the uniform distribution of structural elements along the compression direction of the first two kinds of scaffold and the layer by layer deformation during compression. However, because the number of structural units of D stent is larger than that of G stent, although the average pore diameter of the two kinds of stent is the same, they have different fluctuation times in the stress plateau stage. At the same time, the shear deformation mode at room temperature due to the inherent HCP structure of magnesium materials will also affect the fluctuation of the stress-strain curve.

Table 2 summarizes the corresponding mechanical properties of the three kinds of stents, and it can be found that the mechanical properties of the G stent are the best, and the platform stress is (32.34 0 ± 36) MPa, and Young's modulus was (0. 760 ±  0.020）GPa； The mechanical properties of B-type stent are the worst. The compressive strength and Young's modulus of cancellous bone were 0.2-80.0 MPa and 0.01-2.00 GPA, respectively; This indicates the potential of am jdbm scaffold in tissue engineering.

Table 2 mechanical properties of B, D and G stents

In order to characterize the degradation performance of am jdbm scaffold, porous disc samples with diameter of 10 mm and thickness of 3 mm were used. The samples were immersed in 3 ml cell culture medium (DMEM, GIBCO, USA) at 37 ℃ and 5% CO2. The cell culture medium contained 10% fetal bovine serum (FBS, GIBCO, USA) and 1% penicillin and streptomycin (GIBCO, USA), The cell culture medium was changed every 2 or 3 days. The test results of jdbm scaffold extract are shown in Fig. 7. It can be seen that the Mg2 + concentration of all scaffolds increases rapidly after soaking for 3 days, and the Mg2 + concentration of D scaffold is higher than that of the other two scaffolds; After 7 days, the Mg2 + concentration of D stent decreased to 1300 ppm, similar to that of B and G stent. However, the Ca2 + concentration of all samples decreased significantly after 6 h of immersion, and tended to be stable in the following days. The Ca2 + concentration of D stent was the lowest during the immersion period. In addition, the pH increment of all stents peaked on day 3 and decreased significantly on day 7; The osmotic pressure increment of all stents increased gradually during the 7-day immersion period, and reached a similar level on the 7th day.

Jia et al. Found that the sharp increase of Mg2 + concentration in the extraction solution of scaffolds was due to the large surface area of porous structure in contact with corrosive medium; The decrease of Ca2 + concentration in the extract was due to the deposition of calcium phosphate on the surface in alkaline environment due to the increase of pH. From the degradation test results, D stent showed more serious corrosion behavior than the other two kinds of stents three days before immersion, although the gap was narrowed on the seventh day, which indicated that B and G stents had better clinical application prospects.

Fig. 7 test results of jdbm scaffold extract（ a) Mg2 + concentration（ b) PH increment（ c) Ca2 + concentration（ d) Osmotic pressure increment

（5） Surface modification of g-stent

In order to improve the corrosion resistance and biocompatibility of am jdbm scaffold, the G scaffold with the best mechanical properties and degradation behavior was selected for surface modification, so that the surface of the scaffold was coated with DCPD. The detailed chemical composition of the mixed solution is shown in Table 3. Then, the porosity of bare scaffolds and coated scaffolds were measured by Archimedes method, and the degradation behavior and cell adhesion behavior of coated scaffolds in vitro were studied by immersion test and cell culture test.

Table 3 chemical composition of mixed solution for DCPD coating

Figure 8 (a) shows the macro morphology of G stent before and after DCPD coating, which is named g and g-dcpd respectively; Figure 8 (b) and figure 8 (c) show the SEM images of g-dcpd scaffolds and the corresponding enlarged images respectively. It can be found that the DCPD coating is uniformly coated on the surface of scaffolds and the coating has crystalline microstructure. Although the porosity of g-dcpd scaffold decreases with the increase of DCPD coating thickness, as shown in Fig. 8 (d), the g-dcpd scaffold still maintains a minimal curved surface structure.

Figure 8 g-dcpd bracket（ a) Macro image（ b) (c) SEM images（ d) Porosity

Figure 9 shows the changes of mg2+ and ca2+ concentration, pH and osmotic pressure of g-dcpd stent extract and g-stent extract. It can be seen that the mg2+ concentration of g-dcpd stent is significantly lower than that of G stent during soaking, and the osmotic pressure increment is also significantly lower than that of g-stent; Although the pH increment of g-dcpd stent was also lower in the first three days, the pH increment of g-dcpd stent was decreasing, so the values of the two were close in the later stage. In contrast, the concentration of ca2+ in g-dcpd stent was higher than that of g-stent. In conclusion, the degradation rate of g-dcpd stent in vitro was lower than that of g-stent. Niu has proved that CaHPO4 · 2H2O will deposit on the surface and be closely combined with jdbm matrix after DCPD treatment. The coating will play a role of barrier layer between magnesium matrix and corrosion medium, thus improving the corrosion resistance of coating support.

Fig. 9 test results of G and g-dcpd scaffold extracts（ a) Mg2 + concentration（ b) PH increment（ c) Ca2 + concentration（ d) Osmotic pressure increment

In the cell adhesion test, the samples were first placed in 12 well plates, and then MC3T3-E1 osteoblasts were cultured in 1 × The density of 105 cells / well was inoculated on the sample, and 3 ml of cell culture medium containing 10% fetal bovine serum (FBS, GIBCO, USA) and 1% penicillin and streptomycin (GIBCO, USA) was added（ α- MEM, GIBCO, USA) were cultured at 37 ℃ and 5% CO2 for 6 h, 1 D and 3 D, respectively. After culture, the scaffolds were gently washed with phosphate buffered saline (DPBs, hyclone, USA) and stained with calcein am and ethylene homeodimer-1 (Live / dead viability / cytotoxicity assay kit, Thermo Fisher Scientific Inc, USA) at 37 ℃ for 15 min. finally, the staining results were observed by fluorescence microscope (IX71, Olympus, Japan), The results are shown in Figure 10. It can be seen from Fig. 10 (a) and (d) that the number of cells adhering to g-dcpd scaffolds was much more than that on g-dcpd scaffolds after 6 h of culture; With the extension of culture time, the number of cells on the two groups of scaffolds increased gradually, but there was still a big gap in the number and activity of cells on g-dcpd scaffolds compared with g-dcpd scaffolds in the whole culture cycle; At the same time, FIG. 10 (f) showed that the cells adhered to the g-dcpd scaffold began to spread on the surface after 3 days of culture, indicating that DCPD coating can effectively improve the cytocompatibility of the scaffold.

图10 G支架[（a）~（c）]和G-DCPD支架[（d）~（f）]的细胞相容性，其中，（a）、（d）培养6 h，（b）、（e）培养1 d，（c）、（f）培养3 d

In addition, indirect cytotoxic tests were carried out to further characterize the effects of surface modification on cell activity and proliferation. First, the stent was placed in 3 ml under physiological conditions (5% CO2, 37 ℃) α- The extract was obtained by immersion for 3 days in MEM, and the extract was diluted to 15% and 30% respectively according to the recommendations of the cytotoxicity test of the degradable magnesium alloy in vitro. Then, MC3T3-E1 osteoblasts were inoculated in 96 pore plates with 2000 cell / pore density. After 24 hours of culture, the culture medium was replaced with dilution extract, and then cultured for 6 hours, 1 day and 3 days respectively. Add 10 to each hole after culture μ The L cell count kit-8 (CCK8, beyo time, China) solution was cultured in the cell culture chamber for 2 hours, and then the absorbance of each hole was measured at 450 nm wavelength. The test results are shown in Figure 11. For 15% diluted extract, both groups showed the effect of promoting cell growth, and there was no significant difference between g-stent and g-dcpd stent; However, in 30% diluted extract, the cell activity of g-dcpd scaffold was higher than that of g-stent, and g-dcpd stent had a significant effect on osteoblast proliferation, which indicated that g-dcpd stent had certain osteogenic performance.

Compared with coated scaffold, one of the main causes of poor cell compatibility of bare scaffold may be its excessive corrosion rate; It is also reported that the surface wettability also has an effect on cell adhesion, and DCPD coating has been proved to be suitable for cell proliferation. In order to further verify the potential clinical application of am jdbm stent after surface modification of DCPD, related in vivo research is also in progress.

Fig. 11 cell viability of MC3T3 osteoblasts cultured in the diluted extract of G and g-dcpd scaffolds for 6 h, 1 D and 3 D, respectively

4

conclusion

In this paper, the challenges and corresponding strategies of AM magnesium based implants were discussed. Three kinds of magnesium scaffolds with the same porosity and average pore size were designed and fabricated by SLM process. It was found that am-jdbm scaffolds showed completely connected structure, suitable compression performance and moderate degradation behavior, which met the basic requirements of tissue engineering scaffolds; At the same time, combined with cell culture test, am degradable magnesium scaffold with complex structure shows the prospect of clinical application.

In addition, DCPD modified scaffolds can significantly promote cell proliferation, which is due to the better corrosion resistance and cell compatibility compared with bare scaffolds, indicating the necessity of subsequent surface modification of AM degradable magnesium based implants. In conclusion, the combination of degradable and am technology makes degradable magnesium alloy a promising candidate for the next generation of orthopedic implants with complex structure.

Note: the content of this article is slightly adjusted. Please refer to the original if necessary.

Adapted from the original text:

Yinchuan Wang, Penghuai Fu, Nanqing Wang, Liming Peng, Bin Kang, Hui Zeng, Guangyin Yuan, Wenjiang Ding.Challenges and Solutions for the Additive Manufacturing of Biodegradable Magnesium Implants[J].Engineering,2020,6(11):1267-1275.

This paper is selected from the 11th issue of engineering, Chinese Academy of engineering, 2020

Authors: Wang Yinchuan, Fu Penghuai, Wang Nanqing, Peng liming, Kang bin, Zeng Hui, yuan Guangyin, Ding Wenjiang

Source: challenges and solutions for the additional manufacturing of biodegradable magnetium implants [J]. Engineering, 2020,6 (11): 1267-1275

Introduction to the author

Ding Wenjiang, light alloy research expert, academician of Chinese Academy of engineering.

He has been engaged in the research of advanced magnesium alloy materials and its precision forming for a long time, combining magnesium with rare earth to carry out systematic research and form Chinese characteristics.