Enhanced mechanical properties, corrosion resistance, cytocompatibility, osteogenesis, and antibacterial performance of biodegradable Mg2Zn0.5Ca0.5SrZr alloys for boneimplant application. Magnesium (Mg) alloys have been widely used in bone fixation, bone repair, and cardiovascular stents as biodegradable boneimplant materials. However, their clinical application is limited due to their fast corrosion rate and poor mechanical stability. Here we report the development of Mg2Zn0.5Ca0.5Sr (MZCS) and Mg2Zn0.5Ca0.5Zr (MZCZ) alloys with improved mechanical properties, corrosion resistance, cytocompatibility, osteogenesis performance, and antibacterial capability for biodegradable boneimplant applications.
Enhanced mechanical properties, corrosion resistance, cytocompatibility, osteogenesis, and antibacterial performance of biodegradable Mg-2Zn-0.5Ca-0.5Sr/Zr alloys for bone-implant application Xian Tong, Yilong Dong, Runqi Zhou, Xinkun Shen, Yuncang Li, Yue Jiang, Huiyuan Wang, Jinguo Wang, Jixing Lin, Cuie Wen Dr X Tong, Dr J.X Lin Institute of Stomatology, School and Hospital of Stomatology Wenzhou Medical University Wenzhou 325027, China E-mail: jixing.lin@wmu.edu.cn Dr X Tong School of Materials Science and Engineering Xiangtan University Xiangtan 411105, China Y.L Dong, Dr X.K Shen Department of Orthopaedics This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record Please cite this article as doi: 10.1002/adhm.202303975 This article is protected by copyright All rights reserved The Third Affiliated Hospital of Wenzhou Medical University (Ruian People’s Hospital) Wenzhou 325016, China R.Q Zhou Chongqing Key Laboratory of Oral Disease and Biomedical Sciences and Chongqing Municipal Key Laboratory of Oral Biomedical Engineering, Higher Education and Stomatological Hospital Chongqing Medical University Chongqing 401174, China Prof Dr Y.C Li, Prof Dr C.E Wen School of Engineering RMIT University Melbourne Victoria 3001, Australia E-mail: cuie.wen@rmit.edu.au Dr Y Jiang Key Laboratory of Bionic Engineering of Ministry of Education, College of Biological and Agricultural Engineering Jilin University Changchun 130022, China This article is protected by copyright All rights reserved Prof Dr H.Y Wang, Prof Dr J.G Wang Key Laboratory of Automobile Materials of Ministry of Education, School of Materials Science and Engineering Jilin University Changchun 130025, China Abstract: Magnesium (Mg) alloys have been widely used in bone fixation, bone repair, and cardiovascular stents as biodegradable bone-implant materials However, their clinical application is limited due to their fast corrosion rate and poor mechanical stability Here we report the development of Mg-2Zn-0.5Ca-0.5Sr (MZCS) and Mg-2Zn-0.5Ca-0.5Zr (MZCZ) alloys with improved mechanical properties, corrosion resistance, cytocompatibility, osteogenesis performance, and antibacterial capability for biodegradable bone-implant applications The hot-extruded (HE) MZCZ sample exhibited the highest ultimate tensile strength of 255.8±2.4 MPa and the highest yield strength of 208.4±2.8 MPa among all alloy samples and an elongation of 15.7±0.5% due to the recrystallization and grain-refining effect of Zr The HE MZCS sample showed the highest corrosion resistance among all samples, with the lowest corrosion current density of 0.2±0.1 μA/cm2 and lowest corrosion rate of 4±2 μm/y obtained from electrochemical testing, and a degradation rate of 368 μm/y and hydrogen (H2) evolution rate of 0.83±0.03 mL/cm2/d obtained from immersion testing for 21 d in Hanks’ solution The MZCZ sample showed the highest cell viability in relation to MC3T3-E1 cells among all alloy extracts, indicating good cytocompatibility except at 25% This article is protected by copyright All rights reserved concentration Furthermore, the MZCZ alloy showed good antibacterial capability against S aureus Introduction Nowadays, biodegradable metal bone-implant materials are expected to be used as biomedical materials to promote bone tissue repair due to their good degradability, biocompatibility and no need for secondary surgery Among these metal implant materials, pure magnesium (Mg) has a density of ~1.79 g/cm3 and an elastic modulus of 45 GPa, similar to those of human cortical bone (1.75 g/cm3, 5–30 GPa), which can alleviate the stress-shielding effect caused by elastic modulus mismatch [1] Moreover, Mg is non-magnetic and has a high specific strength, good casting and cutting properties, and high thermal conductivity [2] Mg can be corroded and degraded in human body fluids containing chloride (Cl) ions and the degradation products can be absorbed or excreted from the body, which can effectively prevent the need for secondary surgical removal of implants after healing, thus reducing the pain and the economic pressure for patients [3] In addition, Mg is abundant in human bones and cells as an essential nutrient element and participates in the majority of body metabolic processes [4] Mg can induce the differentiation of bone-marrow mesenchymal cells into bone and cartilage tissue as a key regulator of osteogenesis and a low concentration of Mg2+s can promote bone healing without significantly changing local osmotic pressures [5] Furthermore, Mg is involved in protein synthesis as a metabolism-essential enzyme activator, regulating the activities of the neuromuscular and central nervous system, activating various enzymes in the body, ensuring normal contraction in the myocardium, regulating body temperature, and stabilizing DNA and RNA structures [6,7] Therefore, Mg alloys are favored by biomaterial researchers and medical professionals, This article is protected by copyright All rights reserved and this has become a hot spot in the research area of biodegradable metals At present, bone screws made of pure Mg and Mg alloys have been certified by Conformite Europeene and the Korea Food and Drug Administration [8] However, Mg has the lowest standard electrode potential compared to iron and zinc (Zn), indicating that Mg is chemically reactive and forms magnesia (MgO) products on its surface, which are soluble in water and thus ineffective in protecting its substrate [9,10] Therefore, the complete degradation cycle of Mg alloy implants is generally shorter than the time required for bone healing at the damaged site, which is normally more than 18 weeks, thus showing inadequate mechanical stability [11] In general, Mg alloy implants can only provide a stable mechanical environment at the initial stage of fracture healing and there is still no effective and sustained stress stimulation in the middle and late stages, resulting in local osteoporosis and refracturing [12] At the same time, the excessive degradation rate of Mg alloy implants leads to high concentrations of metal ions, production of a large amount of hydrogen (H2) gas, and alkalization of the surrounding site during the degradation process [13] This will cause bone cysts and osteolysis lesions, and affect the bone-healing and other physiological functions of the body, although low concentrations of Mg2+s and H2 can be absorbed, utilized, and metabolized through the kidneys [14] In addition, because implantation is associated with bacterial or microbial infection, bacteria can proliferate and form biofilms through the nutrients provided by the host in implant surgery and healing, which will cause a greater infection risk at the implantation site [15] Therefore, infection is one of the main factors leading to the failure of implantation therapy Currently, about 5% of implants need to be removed through a second operation due to aseptic loosening (~18% of failures) or implant infection (~20% of failures), resulting in implant failure [16] At the same time, due to the inevitable wear and shedding of corrosion products, a large number of fine particles will be generated around the Mg alloy implant, resulting in osteolysis and aseptic loosening [17] The abovementioned adverse factors seriously restrict the clinical application of Mg alloys Therefore, improving the mechanical properties, This article is protected by copyright All rights reserved corrosion resistance, biocompatibility, and antibacterial properties of degradable Mg alloys is a key issue to be urgently addressed The mechanical properties and corrosion resistance of Mg alloys are significantly affected by their chemical composition and microstructure [18] As a very effective alloying element of Mg alloys, Zn has the dual effects of solid-solution and grain-refinement strengthening, which significantly improve the mechanical properties of pure Mg [19] At the same time, the solid solution of Zn in Mg can increase the electrode potential of the α-Mg matrix due to its higher standard electrode potential than that of pure Mg, which enhances the corrosion resistance of Mg-Zn alloys [9] The addition of calcium (Ca) to Mg leads to the formation of an Mg2Ca intermetallic compound with a high melting point, which can refine the matrix phase size and reduce the precipitation and size of the second phase [20] Simultaneously adding Zn and Ca to Mg leads to the formation of a stable Ca2Mg6Zn3 intermetallic compound, which can significantly weaken the matrix texture and improve the mechanical properties [21] The combination of Mg and Ca promotes the deposition of inorganic substance in bone, which has a positive effect on the prevention of osteoporosis and the promotion of bone healing and osteoblast growth [22] Therefore, Mg-Zn-Ca alloys with low cost, good precipitation-hardening performance, and biocompatibility have broad prospects in biomedical applications Zareian et al [23] reported that an extruded Mg-2Zn-1Ca (ZX21) alloy showed an excellent combination of mechanical properties with an ultimate tensile strength (σuts) of 283 MPa and a failure elongation of 29% due to recrystallization and grain refinement by Ca Zhao et al [24] reported that a rolled and annealed Mg-2Zn-0.2Ca showed σuts of 285 MPa, tensile yield strength (σys) of 204 MPa, and fracture elongation (ε) of 24% due to an enhanced solid-solution strengthening effect Still, it is challenging for Mg-Zn-Ca alloys to meet the required mechanical properties (σuts of ≥300 MPa, σys of ≥200 MPa, and ε of ≥10% [25]) for bone implants This article is protected by copyright All rights reserved A low dose of strontium (Sr) salt can reduce bone absorption, maintain a high bone formation rate, and promote bone synthesis, development, and osteoid formation as a crucial component of human bone and teeth [26] Therefore, Sr-containing drugs such as strontium ranelate or alendronate are commonly used for osteoporosis treatment [27] Gu et al [28] reported that an Mg-2Sr alloy showed better corrosion resistance and mechanical properties than pure Mg, without cytotoxicity and with a good host response In addition, adding Sr to Mg alloys can refine α-Mg grains, improve the formability of Mg alloys, and increase their mechanical properties at room temperature (RT), along with high temperature and corrosion resistance [29] Therefore, as a biomaterial for internal orthopedic fixation, an appropriate amount of Sr can be added to Mg alloys to improve mechanical properties and corrosion resistance, promoting bone growth and healing Zirconium (Zr) was also reported to have good biocompatibility with no in vitro toxicity, mutagenicity, or carcinogenicity [30] Zr helps improve the strength and elongation of Mg alloy due to its good grain-refinement effect [31] Sun et al [32] reported that adding Zr refined the grain size of α-Mg and formed corrosion barriers, improving the corrosion resistance of an Mg-10Gd-3Y alloy According to the Mg-Zn, Mg-Ca, Mg-Sr, and Mg-Zr phase diagrams [33], the solid solubility of Zn, Ca, Sr, and Zr in Mg is 6.2 wt.% at 340 °C, 1.34 wt.% at 516.5 °C, 0.11 wt.% at 585 °C, and 0.443 wt.% at 653.6 °C Therefore, the Mg-2Zn-0.5Ca alloy was used as the base alloy in this study, and Sr and Zr were added as the alloying elements The mechanical properties, corrosion resistance, cytocompatibility, osteogenesis performance, and antibacterial properties of as-cast (AC) and hot-extruded (HE) Mg-2Zn-0.5Ca-0.5Sr/Zr alloys were comprehensively investigated for degradable bone-fixation implant applications Results and discussion 2.1 Microstructures of CP Mg, MZCS, and MZCZ samples Figure shows the microstructural characteristics of the AC and HE samples It can be seen This article is protected by copyright All rights reserved that the α-Mg matrix phase existed in all samples, while Mg7Zn3, Mg2Zn11, and Ca2Mg6Zn3 phases were found in both the MZCS and MZCZ samples (Figure 1a) In addition, an Sr2Mg17 phase was also found in the MZCS samples and there was no obvious diffraction peak of a Zr-containing second phase or pure Zr phase in the MZCZ samples Figure 1b–g show OM images of the AC and HE samples In the AC condition, CP Mg showed a coarse and irregular α-Mg grain with a grain size of 834.7±106.4 μm With the addition of Sr to MZCS and Zr to MZCZ, the α-Mg grains of the AC MZCS and MZCZ samples became polygonal and equiaxed, with more continuous and spot-like second phases uniformly distributed at grain boundaries (GBs) and inside grains Compared with CP Mg, the grain size was measured to be 163.2±12.9 μm for the MZCS and 89.4±7.8 μm for the MZCZ After hot extrusion, the grains showed an equiaxed structure with the grain size significantly decreased compared to their AC counterparts, indicating that recrystallization occurred during hot extrusion (Figure 1e–g) In addition to the equiaxed grains, the HE MZCS and MZCZ samples also showed black-striped deformation bands parallel to the extrusion direction This article is protected by copyright All rights reserved Figure Microstructural characteristics of AC and HE samples: (a) XRD patterns; (b) OM image of AC CP Mg; (c) OM image of AC MZCS; (d) OM image of AC MZCZ; (e) OM image of HE CP Mg; (f) OM image of HE MZCS; (g) OM image of HE MZCZ; (h) SEM image of AC MZCS; (i) SEM image of AC MZCZ; (j) SEM image of AC MZCS; (k) SEM image of HE MZCZ; (l) EDS mapping of HE MZCS; and (m) EDS mapping of MZCZ Figure 1h–k show SEM images of the AC and HE MZCS and MZCZ samples, and the corresponding EDS analysis results of the marked Spots 1–11 are shown in Table Spots and indicate the α-Mg matrix phases, which contain primarily Mg, Zn, and Ca, with small amounts of Sr in the AC MZCS and Zr in the AC MZCZ Spot shows a reticulated second phase on GBs in the AC MZCS that contains Mg, Zn, Ca, and Sr with atomic content of 64.1±2.7%, 20.4±1.8%, 13.6±1.0%, and 1.9±0.4%, respectively, and the Zn/Ca atomic ratio is ~3/2 Spot is a reticulated second phase in the AC MZCZ containing mainly Mg, Zn, and This article is protected by copyright All rights reserved Ca with atomic content of 67.6±3.1, 21.0±0.9%, and 11.4±2.2%, respectively, similar to the composition of Spot except for no Sr Spot is a fine granular second phase in the AC MZCZ containing a large amount of Zr with an atomic content of 40.0±1.8% and small amounts of Zn and Ca Spots and show the α-Mg matrices containing mainly Mg and Zn, with small amounts of Sr in the HE MZCS and Zr in the HE MZCZ, and the alloying element content is slightly higher than in Spots and Spot shows a fine second phase at the deformation band in the HE MZCS containing 78.5±0.7% Mg, 11.7±0.2% Zn, 7.7±0.7% Ca, and 2.1±0.5% Sr, with a Zn/Ca atomic ratio of ~3/2 Spot shows a coarse granular second phase with a phase size of 7.2±0.6 μm in the HE MZCS containing large amounts of Mg, Sr, and Zn and a small amount of Ca Spot 10 shows a fine granular second phase distributed in the deformation bond along the extrusion direction in the HE MZCZ containing 85.8±1.8% Mg, 9.9±0.9% Zn, 4.2±1.0% Ca, and 0.1±0.1% Zr Spot 11 shows a mixture of fine and coarse granular second phases distributed in the deformation bond along the extrusion direction in the HE MZCZ containing 72.2±2.8% Zr, 24.1±3.0% Mg, 2.7±0.9% Zn, and 1.0±1.1% Ca Table EDS analysis results for Spots 1–11 marked in Figure 1h–k of SEM images of AC and HE MZCS and MZCZ Spot no Mg Zn Ca Sr Zr Spot 99.2±0.1 0.5±0.1 0.2±0.1 0.1±0.1 – α-Mg Spot 64.1±2.7 20.4±1.8 13.6±1.0 1.9±0.4 – Ca2Mg6Zn3 Spot 99.0±0.3 0.7±0.1 0.2±0.1 – 0.1±0.1 α-Mg Spot 67.6±3.1 21.0±0.9 11.4±2.2 – Ca2Mg6Zn3 Phase (at.%) This article is protected by copyright All rights reserved 10