Recent Progress on Nanocrystalline Metallic Materials for Biomedical Applications

Nanocrystalline (NC) metallic materials have better mechanical properties, corrosion behavior and biocompatibility compared with their coarse-grained (CG) counterparts. Recently, nanocrystalline metallic materials are receiving increasing attention for biomedical applications. In this review, we have summarized the mechanical properties, corrosion behavior, biocompatibility, and clinical applications of different types of NC metallic materials. Nanocrystalline materials, such as Ti and Ti alloys, shape memory alloys (SMAs), stainless steels (SS), and biodegradable Fe and Mg alloys prepared by high-pressure torsion, equiangular extrusion techniques, etc., have better mechanical properties, superior corrosion resistance and biocompatibility properties due to their special nanostructures. Moreover, future research directions of NC metallic materials are elaborated. This review can provide guidance and reference for future research on nanocrystalline metallic materials for biomedical applications.


Introduction
Biomedical metallic materials have good mechanical properties, biocompatibility, etc. However, the properties of conventional coarse-grained (CG) metallic materials do not fully meet clinical requirements. Nanocrystalline (NC) metallic materials have substantially improved mechanical properties, corrosion behavior, and biocompatibility than conventional CG metallic materials [1,2], thus it is obvious that the properties of nanocrystalline metallic materials can better meet clinical needs compared with conventional coarse-grained metallic materials. Therefore, NC metallic biomaterials are receiving increasing attention in recent years [3]. NC metallic biomaterials can be divided into two categories: bioinert NC metallic materials and biodegradable NC metallic materials. Currently, interest in bioinert NC metallic materials include NC pure titanium (Ti) and its alloys [4][5][6], NC SMAs [7], NC SS [8], and NC biodegradable metallic alloys including Fe-based alloys and Mg-based alloys [9]. The nanostructured metallic alloys open new avenues and concepts for medical implants, providing benefits in all areas of medical device technology.
Data from several studies suggest that after NC processing, NC metallic biomaterials have desirable characteristics, such as good manufacturability, and superior physical and mechanical properties [10,11]. This review summarizes the mechanical properties, corrosion behavior, biocompatibility, and clinical applications of bioinert NC metallic materials and biodegradable NC metallic materials in recent years and provides future directions for the development of biomedical NC metallic materials.

Biomedical NC Pure Ti and Its Alloys
The excellent biocompatibility of medical devices manufactured from conventional CG pure Ti and its alloys for clinical applications has been demonstrated in several clinical investigations [12][13][14]. However, the mechanical strength and hardness of commercial CG Ti and its alloys need to be further improved. Nano-structuring of pure Ti and its alloys have recently been demonstrated to be a new horizon and promising alternative method for increasing the mechanical characteristics of conventional CG Ti alloys.
Researchers find that the mechanical performance of NC pure Ti is superior to that of conventional commercial CG Ti [15]. The strength of the NC Ti is nearly twice than that of conventional CG Ti: the σ YS and σ UTS of CG pure Ti were tested to be about 530 MPa and 700 MPa, respectively; after equal-channel angular pressing (ECAP), the CG Ti transformed to NC Ti and showed much higher σ YS of 1267 MPa and σ UTS of 1330 MPa [16]. In another study, the NC Ti-15Mo alloy showed significantly enhanced elastic modulus, microhardness, and tribological properties compared to its CG counterpart because of its higher relative density, sealed porosity, and grain size refinement. The binary NC Ti-15Mo alloy exhibited a microhardness of 315 HV 0.02 and a modulus of elasticity of 95 GPa at 1373 K [17]. NC Ti-20Nb-13Zr has an average grain size of 70-140 nm with a duplex microstructure of the α-Ti (hcp) region surrounding the β-Ti (bcc) matrix, leading to a hardness of 660 HV, and the NC alloy also showed stimulation of new bone formation [18]. The σ YS and σ UTS of the NC Ti-5Ta-1.8Nb were reported to be 800 MPa and 750 MPa, respectively, and the fracture surface of the NC alloy exhibited shear bands and more ductile dimples compared to its CG counterpart which showed dimples and microvoids [19].
Not only have the strength and hardness of the NC Ti alloys increased, but their elongation has also been improved considerably compared to their CG counterparts. Shahmir et al. [20] obtained a good combination of strength and elongation in high-pressure torsion (HPT)-processed NC Ti at elevated temperatures ranging from 573 to 773 K. The strength, microhardness, and elongation of the NC pure Ti (Grade 2) with a grain size of 70 nm were measured as 945 MPa, 300 HV, and 9% at room temperature and approximately 350 MPa, 230 HV, and 130% at 673 K, respectively. Filho et al. [15] fabricated NC pure Ti (grade 2) with ECAP followed by cold rolling and found that this method gives the best strength-ductility combination. González-Masís et al. found that the nanocrystalline Ti-Nb-Zr-Ta-Hf had a combination of high hardness of 564 HV and moderate elastic of 79 GPa [21]. The mechanical properties of NC Ti better meet the requirements of bone replacement and repair. Two main factors can be identified to determine the strengthening mechanisms of NC metallic materials: grain refinement and increased dislocation density induced by the processing of the NC materials [22].
Elias et al. [23] compared the compressive and fatigue strength of dental implants made from the NC Ti (grade 4) processed by ECAP and the microcrystalline Ti (grade 4) and found that the NC Ti implants exhibited both higher compressive and fatigue strength for 5 × 10 6 cycles than the microcrystalline Ti implants, and the ECAP-processed NC Ti exhibited transgranular fracture with no striation at the fatigue crack initiation and propagation regions. The increased compression and fatigue strengths of the NC Ti make it a very good material for dental implant applications. Javadhesari et al. produced Ti-50 at%Cu alloy which showed excellent mechanical properties: ultra-high microhardness of 10 GPa and acceptable toughness of 8.14 MPa·m 1/2 [24]. All the abovementioned studies have demonstrated that NC Ti and its alloys have better mechanical properties for implant applications.
In addition to the enhanced mechanical properties, an enhanced biological response can also be anticipated from NC metallic materials. For instance, the fibroblast mice cells L929 covered 53% commercially pure Ti (CP-Ti) surface and 87.2% nanostructured Ti surface after 72 h of cell culturing, indicating superior cytocompatibility of the NC Ti compared to its CG counterpart [4]. In another study, NC Ti showed an improvement in in vitro biosafety and long-term cellular functionalization in cytobiology and in vivo biostability [25]. Figure 1 shows the histotomy of bone contact of NC Ti at four weeks implantation as compared to CG Ti, indicating a higher osseointegration ability with freshly generated bone development direction and kinetics following implantation of NC Ti. Cell adhesion and proliferation test for nanocrystalline Ti25Nb16Hf showed lower adhesion and higher proliferation when compared to Ti grade 2 [26].
compared to its CG counterpart [4]. In another study, NC Ti showed an improvement in in vitro biosafety and long-term cellular functionalization in cytobiology and in vivo biostability [25]. Figure 1 shows the histotomy of bone contact of NC Ti at four weeks implantation as compared to CG Ti, indicating a higher osseointegration ability with freshly generated bone development direction and kinetics following implantation of NC Ti. Cell adhesion and proliferation test for nanocrystalline Ti25Nb16Hf showed lower adhesion and higher proliferation when compared to Ti grade 2 [26]. NC Ti-29Nb-13Ta-4.6Zr (the so-called TNTZ alloy) showed greater hardness than its CG counterpart, which is up to 310 HV [27]. Lin et al. [28] reported that the β Ti-35Nb-3Zr-2Ta alloy exhibited ultrafine equiaxed grains of approximately 300 nm after ECAP for 4 passes at 500 °C; the ECAP-processed alloy showed a longitudinal microhardness of 224 HV, a σYS of 390 MPa, and a σUTS of 765 MPa, while maintaining a good level of elongation of 16.5% and elastic modulus of 59 GPa. In another study, the microstructure of the HPT-processed TNTZ exhibited a single phase of β grains with diameters of a few hundred nanometers and high-angle boundaries, and due to the severe plastic deformation of the HPT process, the grains exhibited non-uniform subgrains with high dislocation density. The tensile strength of the nanocrystalline TNTZ alloy increased significantly [29]. In summary, the microhardness of NC TNTZ alloy is consistently much higher than its CG counterpart [30].
Xie et al. [31] reported that HPT-processed NC β-Ti alloy (Ti-36Nb-2.2Ta-3.7Zr-0.3O, at.%) showed markedly improved mechanical and biocompatibility properties; the hardness and elastic modulus of the NC Ti alloy were, respectively, 23% higher and 34% lower than those of its CG counterpart. The decrease in the elastic modulus of a metallic implant biomaterial is of critical significance because it helps prevent stress shielding that occurs when the implant is stiffer than its surrounding host bone. The β-type Ti-24Nb-4Zr-8Sn alloy processed by warm swaging and warm rolling with a uniform microstructure comprising a β phase with a size less than 200 nm and the precipitation of nanosized α phase, exhibited high ultimate tensile strength of 1150 MPa, low elastic modulus of 56 GPa, and good ductility with an elongation of 8%, along with large-scale nonlinear deformation behavior with a recoverable strain of up to 3.4% [32]. Kent et al. [33] reported the mechanical properties of the Ti-25Nb-3Zr-3Mo-2Sn alloy processed by a modified accumulative roll bonding (ARB) technique, and found that after 4 cycles of rolling, the ARB-processed sample exhibited significantly refined β grains heavily elongated in the rolling direction and NC α precipitates distributed on the β grain boundaries, with an ultimate tensile strength of 1220 MPa, a 0.5% proof stress of 946 MPa, which were, respectively, ~70% higher and almost double those of the CG solution treated NC Ti-29Nb-13Ta-4.6Zr (the so-called TNTZ alloy) showed greater hardness than its CG counterpart, which is up to 310 HV [27]. Lin et al. [28] reported that the β Ti-35Nb-3Zr-2Ta alloy exhibited ultrafine equiaxed grains of approximately 300 nm after ECAP for 4 passes at 500 • C; the ECAP-processed alloy showed a longitudinal microhardness of 224 HV, a σ YS of 390 MPa, and a σ UTS of 765 MPa, while maintaining a good level of elongation of 16.5% and elastic modulus of 59 GPa. In another study, the microstructure of the HPT-processed TNTZ exhibited a single phase of β grains with diameters of a few hundred nanometers and high-angle boundaries, and due to the severe plastic deformation of the HPT process, the grains exhibited non-uniform subgrains with high dislocation density. The tensile strength of the nanocrystalline TNTZ alloy increased significantly [29]. In summary, the microhardness of NC TNTZ alloy is consistently much higher than its CG counterpart [30].
Xie et al. [31] reported that HPT-processed NC β-Ti alloy (Ti-36Nb-2.2Ta-3.7Zr-0.3O, at.%) showed markedly improved mechanical and biocompatibility properties; the hardness and elastic modulus of the NC Ti alloy were, respectively, 23% higher and 34% lower than those of its CG counterpart. The decrease in the elastic modulus of a metallic implant biomaterial is of critical significance because it helps prevent stress shielding that occurs when the implant is stiffer than its surrounding host bone. The β-type Ti-24Nb-4Zr-8Sn alloy processed by warm swaging and warm rolling with a uniform microstructure comprising a β phase with a size less than 200 nm and the precipitation of nanosized α phase, exhibited high ultimate tensile strength of 1150 MPa, low elastic modulus of 56 GPa, and good ductility with an elongation of 8%, along with large-scale nonlinear deformation behavior with a recoverable strain of up to 3.4% [32]. Kent et al. [33] reported the mechanical properties of the Ti-25Nb-3Zr-3Mo-2Sn alloy processed by a modified accumulative roll bonding (ARB) technique, and found that after 4 cycles of rolling, the ARB-processed sample exhibited significantly refined β grains heavily elongated in the rolling direction and NC α precipitates distributed on the β grain boundaries, with an ultimate tensile strength of 1220 MPa, a 0.5% proof stress of 946 MPa, which were, respectively,~70% higher and almost double those of the CG solution treated counterpart. He et al. [34] investigated the mechanical properties of the Ti 60 Cu 14 Ni 12 Sn 4 M 10 (M = Nb, Ta, Mo) alloys prepared using arc melting and copper mold casting. The alloys exhibited a composite microstructure containing a micrometer-sized dendritic β-Ti(M) phase dispersed in an NC matrix with a compressive elastic modulus in the range of 59-103 GPa, a compressive yield strength in the range of 1037-1755 MPa, and a compressive plastic strain in the range of 1.68-21.34% [35].
Some studies have also shown that NC Ti alloys have superior electrochemical corrosion resistance. Yilmazer et al. [36] evaluated the corrosion behavior of the HPT-processed Ti-29Nb-13Ta-4.6Zr alloy in simulated body fluid (SBF) using electrochemical impedance spectroscopy (EIS) and found that the NC alloy exhibited improved corrosion performance than its CG counterpart due to a passivated surface layer contained a titania (TiO 2 ) matrix dispersed with zirconia (ZrO 2 ), niobia (Nb 2 O 5 ), and tantala (Ta 2 O 5 ) oxides. Furthermore, an NC Ti alloy exhibits better hydrophilic property due to the NC structure [37]. This will lead to improved protein adsorption properties because of the increased contact points between the protein and the NC surface. The adsorption of bone morphogenetic proteins on a material surface affects the cell adhesion, spreading, and proliferation on the material. M. A. Hussein showed that nanocrystalline Ti20Nb20Zr alloy had a hydrophilic nature compared with a CP Ti [38]. Xie et al. [31] showed that HPT-processed NC β-type Ti-36Nb-2.2Ta-3.7Zr-0.3O (at.%) showed enhanced cell attachment and proliferation of human gingival fibroblasts (HGF) after 30 min cell seeding compared to its CG counterpart.

Biomedical NC SMAs
Nitinol refers to a family of SMAs composed of nickel (Ni) and Ti with a unique combination of properties including superelasticity and shape memory properties. The word nitinol is originated from its composition (Ni-Ti) and its place of discovery of Naval Ordnance Laboratory (USA) by William J. Buehler and Frederick Wang in 1963 [39]. Nitinol are the most widely used biomedical SMAs due to their large recoverable strains (~8%) in polycrystalline forms [40]. The phase transformation temperature of medical Ni-Ti SMAs is close to that of the human body; therefore, this class of Ti alloys plays an important role in the medical field that is unmatched by other materials. Conventionally, Ni-Ti SMAs are frequently used in orthopedics, dentistry, and cardiovascular stent treatments. NC Ni-Ti-based SMAs offer even better overall mechanical properties [41]. Ti-Zr-based alloys alloyed with Nb, Ta, Mo, and Sn are also another type of shape memory alloys. Sheremetyev et al. [42] reported that Ti-18Zr-15Nb alloy processed by ECAP at 250 • C for 7 passes showed a σ YS of 962 MPa, a σ UTS ultimate tensile strength of 988 MPa, and an elongation of 5.4%.
Yan et al. [43] found that compared to CG NiTi SMAs, the NC austenite NiTi (Ni-49.3Ti, at.%) SMA with a B2 (CsCl) type ordered structure showed a significantly enhanced compressive yield strength of 2552.1 MPa, and the value of stress-induced austenite transformation increased with decreasing grain size. Nie et al. [44] found that the HPT-processed NC Ni 50.2 Ti 49.8 alloy exhibited a hardness of 456.8 ± 14.9 HV. Sharifi and Kermanpur [45] performed hot rolling and annealing at 900 • C on Ni 50 Ti 50 alloy, followed by cold rolling with thickness reduction of 70% and annealing at 400 • C, and the resultant NC alloy exhibited superelastic properties including a recoverable strain of 12% and an upper plateau stress (σ SIM ) (i.e., the critical stress for stress-induced martensitic transformation) of 610 MPa, which is significantly higher than that of the CG alloy with a σ SIM of 160 MPa. Baigonakova et al. [46] found that NiTi0.1Ag wires provided optimal strength (1450 MPa) and ductility (33.4%) properties, due to the dislocation-free homogeneous nanocrystalline structure.
However, NiTi SMAs contain a large amount of Ni ions. NiTi alloy implants might release Ni ions into the human tissue and cause severe allergic reactions after implantation in the body [47]. Nanocrystallization not only significantly improved the mechanical and superelastic properties of NiTi SMAs, but also fundamentally solved the problem of rapid Ni ion release because NC NiTi SMAs improved corrosion resistance compared to their CG counterparts. Shri et al. [48] found that through severe plastic deformation of HPT, the corrosion behavior of NiTi SMAs was changed by grain refinement, the NC Ti-50Ni (at.%) exhibited a stable, protective layer on its surface in a cell culture medium and increased Nanomaterials 2022, 12, 2111 5 of 17 corrosion resistance, leading to decreased Ni ion release. Nie et al. [44] reported superiorly higher corrosion resistance of HPT-processed NC Ni 50.2 Ti 49.8 alloy with a substantially lower rate of Ni ion release than its microcrystalline counterpart in both Hanks' solution and artificial saliva, which was far below the threatening threshold of a daily diet. The results of murine fibroblast (L-929) and osteoblast cell lines (MG63) cultured and cell proliferation are shown in Figure 2. There is no cytotoxicity for nanocrystalline Ni 50.2 Ti 49.8 till 4 days culture with L-929 and MG63.
rapid Ni ion release because NC NiTi SMAs improved corrosion resistance compared to their CG counterparts. Shri et al. [48] found that through severe plastic deformation of HPT, the corrosion behavior of NiTi SMAs was changed by grain refinement, the NC Ti-50Ni (at.%) exhibited a stable, protective layer on its surface in a cell culture medium and increased corrosion resistance, leading to decreased Ni ion release. Nie et al. [44] reported superiorly higher corrosion resistance of HPT-processed NC Ni50.2Ti49.8 alloy with a substantially lower rate of Ni ion release than its microcrystalline counterpart in both Hanks' solution and artificial saliva, which was far below the threatening threshold of a daily diet. The results of murine fibroblast (L-929) and osteoblast cell lines (MG63) cultured and cell proliferation are shown in Figure 2. There is no cytotoxicity for nanocrystalline Ni50.2Ti49.8 till 4 days culture with L-929 and MG63. Li et al. [7] investigated the in vitro and in vivo biological properties of an ECAP-processed NC Ti49.2Ni50.8 alloy for orthopedic implant applications and indicated enhanced cell viability, adhesion, proliferation, ALP (alkaline phosphatase) activity, and mineralization than its CG counterpart.

Biomedical NC Stainless Steels (SSs)
Biomedical NC SSs have enhanced passivation behavior and corrosion resistance than their CG counterparts; furthermore, NC SSs also exhibit enhanced hydrophilic and protein adsorption properties, leading to improved biological properties including better cell attachment, spreading, and proliferation [49].
Previous studies have found that NC ASTM F-138 austenitic SSs have higher mechanical strength than their CG counterpart [15,50,51]. Heidari et al. found that the ASTM F2581 nanocrystalline stainless steels had 824 MPa yield strength, and the strength exceeded 1 GPa [51]. NC 304 SS fabricated by severe rolling showed a hardness of 480.0 ± 10.1 HV and improved corrosion resistance; Figure 3a shows the OCP curves of nanocrystalline 304 SS and microcrystalline 304 SS in artificial saliva. The result of OCP curve indicates that nanocrystalline stainless steels show more corrosion resistance than microcrystalline stainless steels in artificial saliva. Polarization studies (Figure 3b) revealed that NC 304 SS are more corrosion resistant than conventional CG 304 SS in an oral-like environment with higher corrosion potential [49]. Li et al. [7] investigated the in vitro and in vivo biological properties of an ECAPprocessed NC Ti 49.2 Ni 50.8 alloy for orthopedic implant applications and indicated enhanced cell viability, adhesion, proliferation, ALP (alkaline phosphatase) activity, and mineralization than its CG counterpart.

Biomedical NC Stainless Steels (SSs)
Biomedical NC SSs have enhanced passivation behavior and corrosion resistance than their CG counterparts; furthermore, NC SSs also exhibit enhanced hydrophilic and protein adsorption properties, leading to improved biological properties including better cell attachment, spreading, and proliferation [49].
Previous studies have found that NC ASTM F-138 austenitic SSs have higher mechanical strength than their CG counterpart [15,50,51]. Heidari et al. found that the ASTM F2581 nanocrystalline stainless steels had 824 MPa yield strength, and the strength exceeded 1 GPa [51]. NC 304 SS fabricated by severe rolling showed a hardness of 480.0 ± 10.1 HV and improved corrosion resistance; Figure 3a shows the OCP curves of nanocrystalline 304 SS and microcrystalline 304 SS in artificial saliva. The result of OCP curve indicates that nanocrystalline stainless steels show more corrosion resistance than microcrystalline stainless steels in artificial saliva. Polarization studies (Figure 3b) revealed that NC 304 SS are more corrosion resistant than conventional CG 304 SS in an oral-like environment with higher corrosion potential [49]. Nanomaterials 2022, 12, x FOR PEER REVIEW 6 of 17 Nie et al. using electrochemical measurement concluded a similar result that nanocrystalline stainless steel has higher corrosion resistance compared to microcrystalline stainless steel and the corrosion behavior of nanocrystalline 304 SS do not have significantly superior resistance to pitting corrosion compared to microcrystalline stainless steels [52].

Other Types of Bio-Inert NC Metallic Materials
There are also some other types of NC metallic materials for biomedical application. NC silver (Ag) prepared by spark plasma sintering (SPS) at 600 K for 5 min exhibited a mean grain size of 380 nm and showed a σYS 4.6 times higher than that of CG counterpart with a mean grain size of 49.65 µm and more than 30% uniform elongation [53]. The HPT-processed nanocrystalline CoCrMo exhibited improved tribocorrosion resistance but deteriorated corrosion resistance [54]. Huo et al. [55] fabricated an NC surface with an average grain size of ≤20 nm using sliding friction treatment (SFT) on CG pure Ta and comparatively studied the osteoblast cell responses to the CG and NC Ta using human osteoblastic hFOBl.19 cells. Their results showed that the NC surface exhibited higher surface hydrophilicity and enhanced corrosion resistance than the CG surface, thus leading to enhanced osteoblast adherence and spreading after 1 day's cell culturing and markedly improved cell proliferation, maturation, and mineralization after 14 days' cell culturing. Figure 4 shows the morphologies of hFOBl.19 cells on CG and NC pure Ta after 1, 3, and 7 days' of culturing. The results of this study indicated the superior cytocompatibility of the NC Ta surface compared to its CG counterpart [55]. Nie et al. using electrochemical measurement concluded a similar result that nanocrystalline stainless steel has higher corrosion resistance compared to microcrystalline stainless steel and the corrosion behavior of nanocrystalline 304 SS do not have significantly superior resistance to pitting corrosion compared to microcrystalline stainless steels [52].

Other Types of Bio-Inert NC Metallic Materials
There are also some other types of NC metallic materials for biomedical application. NC silver (Ag) prepared by spark plasma sintering (SPS) at 600 K for 5 min exhibited a mean grain size of 380 nm and showed a σ YS 4.6 times higher than that of CG counterpart with a mean grain size of 49.65 µm and more than 30% uniform elongation [53]. The HPT-processed nanocrystalline CoCrMo exhibited improved tribocorrosion resistance but deteriorated corrosion resistance [54]. Huo et al. [55] fabricated an NC surface with an average grain size of ≤20 nm using sliding friction treatment (SFT) on CG pure Ta and comparatively studied the osteoblast cell responses to the CG and NC Ta using human osteoblastic hFOBl.19 cells. Their results showed that the NC surface exhibited higher surface hydrophilicity and enhanced corrosion resistance than the CG surface, thus leading to enhanced osteoblast adherence and spreading after 1 day's cell culturing and markedly improved cell proliferation, maturation, and mineralization after 14 days' cell culturing. Figure 4 shows the morphologies of hFOBl.19 cells on CG and NC pure Ta after 1, 3, and 7 days' of culturing. The results of this study indicated the superior cytocompatibility of the NC Ta surface compared to its CG counterpart [55].
Some bio-inert NC metallic biomaterials published in the literature in recent years, their fabrication methods, grain sizes, mechanical properties, corrosion behaviors, biocompatibility, and potential applications are summarized in Table 1  Some bio-inert NC metallic biomaterials published in the literature in recent years, their fabrication methods, grain sizes, mechanical properties, corrosion behaviors, biocompatibility, and potential applications are summarized in Table 1.

Biodegradable NC Metallic Materials for Biomedical Applications
Biodegradable metals have been research hotspots for the last two decades [62]. Nanocrystallization of biodegradable metals can further enhance their comprehensive properties. Wang et al. [63] performed hot rolling on Mg-2Zn alloy and found that the alloy exhibited a grain size of~70 nm with a high σ YS of 223 MPa and σ UTS of 260 MPa, and a strong corrosion resistance with a corrosion rate of 0.2 mm/y in vivo when tested using Sprague Dawley rats. Nie et al. [9] demonstrated that ECAP-processed NC pure iron (Fe) exhibited higher corrosion resistance and improved hemocompatibility and cytocompatibility. Zhang et al. [64] investigated the corrosion resistance of an NC Mg-2Zn-0.24Ca (wt.%) alloy processed by HPT and annealing and reported that the grain size, number of (0002) oriented grains, second phase, and surface stress of the alloy changed with the annealing temperature and time, and these factors affected the corrosion rate. The HPT-processed alloy showed the best corrosion resistance with the maximum polarization resistance and lowest hydrogen evolution rate when annealed at 210 • C for 30 min.
The HPT-processed NC Mg-1Ca alloy showed at least 5-fold improvement in corrosion resistance than the CG alloy due to the separation of the second phase (Mg 2 Ca) particles and their continuous nanoprecipitation. Figure 5 shows the micrographs of CG and NC Mg-1Ca alloy. The microstructure of the homogenized CG Mg-1Ca alloy contained equiaxial grains (Figure 5a), while the NC alloy showed a much finer grain size of 100 ± 9 nm with a greater dislocation density and higher shear strength (Figure 5c) [65].

Biodegradable NC Metallic Materials for Biomedical Applications
Biodegradable metals have been research hotspots for the last two decades [62]. Nanocrystallization of biodegradable metals can further enhance their comprehensive properties. Wang et al. [63] performed hot rolling on Mg-2Zn alloy and found that the alloy exhibited a grain size of ~70 nm with a high σYS of 223 MPa and σUTS of 260 MPa, and a strong corrosion resistance with a corrosion rate of 0.2 mm/y in vivo when tested using Sprague Dawley rats. Nie et al. [9] demonstrated that ECAP-processed NC pure iron (Fe) exhibited higher corrosion resistance and improved hemocompatibility and cytocompatibility. Zhang et al. [64] investigated the corrosion resistance of an NC Mg-2Zn-0.24Ca (wt.%) alloy processed by HPT and annealing and reported that the grain size, number of (0002) oriented grains, second phase, and surface stress of the alloy changed with the annealing temperature and time, and these factors affected the corrosion rate. The HPT-processed alloy showed the best corrosion resistance with the maximum polarization resistance and lowest hydrogen evolution rate when annealed at 210 °C for 30 min.
The HPT-processed NC Mg-1Ca alloy showed at least 5-fold improvement in corrosion resistance than the CG alloy due to the separation of the second phase (Mg2Ca) particles and their continuous nanoprecipitation. Figure 5 shows the micrographs of CG and NC Mg-1Ca alloy. The microstructure of the homogenized CG Mg-1Ca alloy contained equiaxial grains (Figure 5a), while the NC alloy showed a much finer grain size of 100 ± 9 nm with a greater dislocation density and higher shear strength (Figure 5c) [65].  Gu et al. fabricated Mg-3Ca alloys with different grain sizes by melt-spinning at different wheel rotating speeds and their electrochemical test results showed that the corrosion rate of the NC Mg-3Ca in simulated body fluid was significantly reduced compared with CG Mg-3Ca, and the NC Mg-3Ca alloy showed a more uniform corrosion morphology. Further, the extract of the NC Mg-3Ca alloy showed no cytotoxicity in relation to L-929 cells, whereas the extract of CG Mg-3Ca alloy did. On the surface of the NC Mg-3Ca alloy, the L-929 cells exhibited improved adhesion than the CG Mg-3Ca alloy [66]. Table 2 summarizes some biodegradable NC metallic biomaterials published in the literature in recent years, their fabrication methods, grain sizes, and mechanical, corrosion, and biocompatibility properties. Most of the degradable nanocrystalline alloys reported thus far are Mg-based, with a small amount of Fe-based alloys; Zn-based alloys have not yet been publicly reported, and further development of Zn-based nanocrystalline alloys is needed in the future.

Conclusions
This article provides a review on the mechanical properties, corrosion behavior, biocompatibility, and clinical applications of different NC metallic biomaterials. The main conclusions are as follows: 1. Biomedical NC metallic materials, such as Ti and its alloys, TNTZ, NiTi SMAs, SS, and biodegradable Fe and Mg alloys have significantly improved tensile yield strength, ultimate tensile strength, and hardness without significant reduction in ductility. 2. Biomedical NC metallic materials, such as Ti and its alloys, TNTZ, NiTi SMAs, SS, and biodegradable Fe and Mg alloys have better corrosion resistance than their conventional CG metallic materials. 3. Nanocrystallization of metallic biomaterials can improve their biocompatibility due to the unique nanostructures on their surfaces. 4. In future research, the relationships between grain size, microstructural characteristics, and material properties of NC metallic materials should be systematically investigated. More research and development should be devoted to zinc-based degradable NC alloys, iron-based degradable NC alloys, and Mg and plural NC alloys.

Conflicts of Interest:
The authors declare no conflict of interest.