ZnP‐Coated Zn1Cu0.1Ti Membrane with High Strength‐Ductility, Antibacterial Ability, Cytocompatibility, and Osteogenesis for Biodegradable Guided Bone Regeneration Applications

Zinc (Zn) and its alloys have recently gained research interest due to their good biosafety, biological function, biodegradability, and formability. Zinc‐phosphate (ZnP) coating has been shown to improve the corrosion resistance and biocompatibility of Zn alloys. Here, a biodegradable ZnP‐coating on Zn1Cu0.1Ti (ZCT) membrane with high strength‐ductility and mechanical stability, suitable degradation rate, effective antibacterial ability, excellent in vitro and in vivo cytocompatibility, and osteogenesis is reported. The ZnP‐coated ZCT exhibits high strength‐ductility with a yield strength of 264 MPa, ultimate tensile strength of 312 MPa, elongation of 36.0%, and high mechanical stability before and after 30 d immersion in Hanks’ and AS solutions, all of which are higher than those of ZCT. The ZnP coating shows good deformation resistance, healing effect, and bond strength with the substrate, meeting the clinical contour shaping requirements. The ZnP‐coated ZCT membrane sample shows higher cell viability toward MC3T3‐E1 and MG‐63 cells, osteogenic and mitochondrial quality‐control properties in vitro than those of the ZCT sample. Using a rat calvarial defect model, the ZnP‐coated ZCT membrane shows complete biosafety and considerable osteogenesis performance. Overall, the ZnP‐coated ZCT membrane is recommended as a promising biodegradable implant material for oral guided bone regeneration application.

and polytetrafluoroethylene (PTFE) have evolved into materials that are used universally in the medical field due to their high strength, good biocompatibility, and strong corrosion resistance. [7,9] However, due to its non-degradability, the permanently implanted barrier membrane cannot be absorbed by the body, necessitating a second surgery for removal after the procedure. [10] The non-degradable barrier membrane carries a high infection risk, which increases the likelihood of complications in clinical settings such as mucosal dehiscence, membrane exposure, and infection, all of which ultimately lead to the failure of osteogenesis. [10] In addition, Ti and PTFE barrier membranes have limited clinical applications because of their complicated manufacturing process and high cost. [11] In contrast, materials for bioabsorbable barrier membranes, such as collagen derived from animals and organically synthesized polymers, can self-degrade and be absorbed through the interaction of body fluids and enzymes, avoiding secondary removal surgery and minimizing the discomfort and financial burden of patients. [9,12] In addition, biodegradable barrier membranes have good biocompatibility, weak immunogenicity, moderate coagulation, and good bone inducibility, leading to aid in the growth and healing of bone tissues, especially collagen membranes. [13] However, the existing degradable organic and collagen membranes have low tensile strength and poor spatial maintenance ability and are prone to membrane collapse and displacement, making it challenging to meet the long-term and stable spatial support function of growing bone tissue. [14] Additionally, as the membrane degrades, an acidic environment will form around it. This acidic environment may result in inflammation, reactions to foreign bodies, and even the need for surgical debridement to remove the membrane. [15] Moreover, the degradable barrier membrane degrades quickly, and most collagen membranes degrade within 1-4 weeks, limiting the application of bone increment. [16] Over the last two decades, biodegradable metal alloys, which combine the high mechanical properties of metals with the biodegradability of degradable polymers, are being developed and used more frequently in bone tissue repair, bone fixation, and interventional scaffolds. [17,18] At present, the research of biodegradable alloys mainly focuses on magnesium (Mg)-, zinc (Zn)-, and iron (Fe)-based alloys, all of which are essential nutritional elements for the human body. [19,20] Mg alloys have good mechanical properties, biocompatibility, and osteogenic properties, and can degrade and be absorbed in the body. [9,19,21] However, the degradation rate of Mg alloys is rapid, and a large amount of hydrogen is produced, making it impossible to maintain bone regeneration space during the in vivo service period. [22] At the same time, although Mg alloys have been reported to exhibit a certain antibacterial ability, its antibacterial performance was relatively weak, leading to the failure of GBR surgery when the infection is more serious. [23] Fe has the highest standard electrode potential (−0.447 V) among the three degradable metal alloys, which slows the degradation rate of most Fe-based alloys. [24] At the same time, the dense degradation products on the implant surface are difficult to completely degrade in vivo, which further reduces the degradation rate of the Fe-based medical implant, seriously hindering bone tissue proliferation and thereby losing the advantage of degradable metals. [25] Pure Zn and some of its alloy degrade at a moderate rate, which can be used to balance the implant's degradation rate and mechanical stability. At the same time, Zn is involved in the development and growth of human somatic cells, gene expression, the immune system, the nervous system, and many other physiological reaction processes. [26] In addition to maintaining normal physiological functions, Zn also has bonepromoting functions. [27] However, pure Zn has low mechanical strength, ductility, and rigidity, making it difficult to meet the purpose of stable support of barrier membranes. In addition, the local release concentration of Zn ions in the degradation process is too high, resulting in poor biological activity. [28] Therefore, in order to ensure normal cell migration, adhesion, proliferation, and differentiation of tissue cells around the implant at the initial implantation stage, it is necessary to control the initial corrosion of the Zn alloy and the initial release of Zn ions to improve the biocompatibility of Zn-based implant. Currently, the alloying process, such as germanium (Ge), [29] Mg, [30] strontium (Sr), [30] calcium (Ca), [30] lithium (Li), [31] copper (Cu), [32,33] silver (Ag), [32,34] manganese (Mn), [35] Fe, [36] Ti, [37] and zirconium (Zr), [38] deformation processing, such as rolling, [29][30][31] extruding, [30,33,35] drawing, [34] equal channel angular pressing, [39] and high-pressure torsion, [40] and surface modification such as zinc-phosphate (ZnP) [28,41] and surface stabilization treatment [42] are commonly used approaches to solve the above-mentioned problems.
The appropriate addition of Cu and Ti elements combined with hot-rolling treatment can significantly improve the mechanical properties, wear properties, corrosion resistance, and biocompatibility of pure Zn. [43,44] In addition, the surface zinc phosphating (ZnP) treatment was also reported to significantly improve the corrosion resistance and biocompatibility of pure Zn. [28,45] Due to the high incidence (≈20%) of GBR membrane exposure complications after oral GBR surgery, [46] the GBR membrane is corroded by saliva. Therefore, evaluating the degradation and mechanical stability of the GBR membrane in Hanks' and artificial saliva (AS) solutions is very important for the GBR membrane application. In this study, a Zn1Cu0.1Ti (denoted as ZCT hereafter) membrane with a ZnP coating with high strength-ductility performance, mechanical stability, suitable degradation rate, good antibacterial ability, cytocompatibility, and osteogenesis was successfully prepared by hot-rolling and phosphating for oral GBR membrane applications.

Microstructure of ZCT and ZnP-Coated ZCT Membrane Samples
The optical micrographs of the ZCT membrane sample on the TRD and NRD planes are shown in Figure 1. It can be seen that the black second phases were clustered along the rolling direction (RD) on the TRD plane, and the small black second phase was streamlined parallel to the RD on the NRD plane (Figure 1a,b). The SEM images and corresponding EDS maps of the ZCT membrane sample on TRD and NRD planes are shown in Figure 1c,d. A large number of fine second granular phases with a phase size of 1.2 ± 0.2 µm parallel to the RD are seen in the ZCT on the TRD plane, and the Cu and Ti elements are relatively uniformly distributed on the Zn matrix ( Figure 1c); whereas, a large number of second phases with irregular shapes   Figure 1e-j shows SEM images and EBSD analysis results of the ZCT membrane sample on the NRD plane. Under the rolling force, the α-Zn phase becomes flattened and is distributed along the RD with streamlined deformation bands (Figure 1e,f). In the [001] texture of the α-Zn phase, the preferred grain orientation of the ZCT sample is (0001). The grain size distribution of the α-Zn phase of the ZCT sample tended to meet the lognormal distribution with an average grain size of 5.7 ± 0.7 µm (Figure 1g). Figure 1h shows grain boundary (GB) map of the ZCT sample, and its corresponding grain misorientation angle distribution is shown in Figure 1j. The red, green, and blue lines in the GB map correspond to the low-angle grain boundaries (LAGBs), medial-angle grain boundaries (MAGBs), and high-angle grain boundaries (HAGBs) with misorientation angles of ≈2°-5°, ≈5°-15°, and > 15°, respectively, and the ZCT sample showed 69.8% HAGBs, 13.6% MAGBs, and 16.6% LAGBs. The ZCT sample also has a very small amount of tiny TiZn 16 phase with a phase size of 2.1 ± 0.1 µm (Figure 1i). Figure 2a shows the surface morphology of the ZnP-coated ZCT membrane sample. Needle-and flake-like ZnP coatings were uniformly coated on the ZCT sample. EDS analysis showed that ZnP contained O, Zn, and P elements, and the Zn/P atom ratio is close to 1.5. Figure 2b,b′,c shows cross-section morphologies and corresponding EDS maps of the ZnPcoated ZCT sample. The ZCT sample surface was covered with a homogeneous and dense ZnP coating with a thickness of 3.3 ± 0.4 µm with no obvious cracks. A large amount of Zn, P, and O elements are uniformly distributed in the ZnP coating, while a large amount of Zn and a small amount of Cu and Ti are observed in the matrix. Figure 2d shows X-ray diffractometry (XRD) patterns of the ZCT and ZnP-coated ZCT membrane samples. It can be seen that both samples were mainly composed of α-Zn and a small amount of the ε-CuZn 5 phases. After phosphating, many diffraction peaks of the ZnP phase and a small number of diffraction peaks of the GO phase appeared on the ZnP-coated ZCT sample surface. In addition, the intensity of the diffraction peaks of the α-Zn and ε-CuZn 5 phases in the ZnP-coated ZCT sample was significantly weaker than those in the ZCT sample, exhibiting that a large amount of ZnP coating is covered on the sample surface. Figure 2e shows Fourier transform infrared spectroscopy (FTIR) patterns of the ZCT and ZnP-coated ZCT membrane samples. The absorption peaks at ≈463 and 517 cm −1 are associated with the OZn group on the ZCT sample surface, [56][57][58] probably due to oxidation during the polishing process. At the same time, the HCH absorption peaks at 2850 and 2920 cm −1 may come from alcohol cleaning. [56][57][58] The absorption peaks at ≈496, 551, 624, 924, 1005, and 1095 cm −1 are ascribed to the PO groups from the ZnP coating in the outer layer of the ZnP-coated ZCT sample. [56][57][58] In addition, the absorption peaks at 1337 and 1630 cm −1 are associated with the HOH groups from the ZnP coating. [56][57][58] The absorption peaks at 1430, 1458, 1539, 1630, 3197, and 3534 cm −1 are associated with the OH groups derived from carboxyl groups in the ZnP coating. [56][57][58] The COC peak at 1046 cm −1 is derived from the GO in the ZnP coating. [59] Figure 2f shows X-ray photoelectron spectroscopy (XPS) full spectrums of the ZCT and ZnP-coated ZCT samples. Both samples contain O, Zn, and C elements. In addition, the ZnPcoated ZCT sample had higher P and Ca contents than ZCT, and the element content of the ZnP-coated ZCT sample was generally consistent with EDS results, except for C. It may be due to the adhesion of C pollution during alcohol cleaning of surface. The corresponding high-resolution narrow scans of P 2p, C 1s, Ca 2p, O 1s, and Zn 2p are shown in Figure 2g. The peak values by Gaussian fitting were located at 133.1 and 133.8 eV for P 2p binding energy from the PO 4 3− structure; [56][57][58] 285.0, 285.4, and 288.9 eV for C 1s binding energy from the CC, COR, and COOR structure; [56][57][58] 347.3 and 350.9 eV for Ca 2p binding energy from the Ca 10 (PO 4 ) 6 (OH) 2 , and Ca 3 (PO 4 ) 2 structure; [56][57][58] 530.5, 531.1, and 532.9 eV for the O 1s binding energy from the O 2− , OH − , and H 2 O; [56][57][58] and 1021.6 and 1044.8 eV for Zn 2p binding energy from the Zn 3 (PO 4 ) 2 4H 2 O structure. [56][57][58] Based on the EDS, XRD, FTIR, and XPS results, it can be inferred that the phases of the ZnP-coated ZCT sample are composed of α-Zn, Zn 3 (PO 4 ) 2 , GO, and ε-CuZn 5 ; while the phases of the ZCT sample are composed of α-Zn and ε-CuZn 5 . The Cu 2 TiZn 22 phases do not appear in XRD and are detected in EDS analysis, mainly because the bulk content of this phase is below the detection limit of XRD testing. Figure 2h shows the contact angles of ZCT and ZnP-coated ZCT samples. ZCT showed a contact angle of 78.7 ± 1.7°, indicating a low wettability. After surface phosphating, the ZnPcoated ZCT sample showed a contact angle of 21.9 ± 2.1°, indicating increased hydrophilicity. It can be concluded that ZnP coating improved the wettability of the ZCT sample. This is mainly due to the uniform distribution of the needle-and flake-like ZnP coating on the sample surface, which increases the surface roughness and thus improves the surface hydrophilicity. [60] At the same time, there are more hydroxyl groups and negatively charged PO 4 3− ions on the sample surface of ZnPcoated ZCT, which can powerfully attract the positively charged H + ions and contribute to the improvement in the hydrophilicity of the ZnP-coated ZCT. [60,61] According to the equilibrium ZnCu and ZnTi phase diagram, [62] the solid solubility of Cu in Zn is 1.7 wt.% at 425 °C, so most Cu elements in ZCT alloy are able to be dissolved into the Zn matrix. However, Zn alloys usually form a nonequilibrium solidification in the conventional solidification process. When the molten Zn is poured into the metal mold via gravity casting, the melt will be supercooled due to the fast cooling speed, leading to an uneven distribution of elements, thus a non-equilibrium peritectic reaction occurs and forms the CuZn 5 phase. [63] Meanwhile, the electronegativity of Zn, Cu, and Ti is 1.65, 1.90, and 1.54, respectively, [64] and the electronegativity difference between both Zn and Ti and Cu is 0.25 and 0.36, respectively. So the CuZn 5 and Cu 2 TiZn 22 phases are preferentially generated. During the hot-rolling process, since the recrystallization temperature of most Zn alloys is much lower than the hot-rolling temperature, the dislocation accumulation in Zn alloys leads to dynamic recovery and recrystallization, leading to the reduction of dislocation density and the refinement of α-Zn grains in HR alloys. [65] At the same time, the crushed fine second phase plays a certain hindrance role to the dynamic recrystallization of Zn alloy and inhibits the coarsening of recrystallized grains. [66] During the preparation of ZnP coating, Zn ions were released by ZCT alloy, and the addition of Zn ions in phosphate solution combined with PO 4 3− ions, forming ZnP coating on the surface of the alloy. The nucleation and growth of ZnP coating will directly affect the chemical properties, micro-structure, corrosion resistance, and biocompatibility of biomaterials in the physiological environment. [67] A previous study [41] has found that adding GO can accelerate the phosphatization process, increase the density of ZnP coating and reduce the particle size of ZnP coating.    Figure 3b, and their corresponding electrochemical parameters are given in Table 2. The E corr of the samples showed a similar trend compared with the OCP values, and the I corr and V PDP of the samples showed the opposite trend compared with E corr with an ascending order of ZnP-coated ZCT in Hanks' < ZnP-coated ZCT in AS < ZCT in Hanks' < ZCT in AS, indicating that the ZnP-coated ZCT sample showed improved corrosion resistance than the bare ZCT sample in the same solution and the corrosion resistance of the same samples in Hanks' solution was higher than in AS solution. ZnP-coated ZCT in Hanks' solution showed the best corrosion resistance among all the samples, with the highest E corr of −0.809 ± 0.029 V, and lowest I corr of 4.3 ± 0.4 µA cm −2 and V PDP of 60 ± 6 µm y −1 . Figure 3c displays the Nyquist plots of ZCT and ZnP-coated ZCT samples. Two capacitive loops and three capacitive loops are observed in the Nyquist plots for ZCT and ZnP-coated ZCT samples, respectively. Therefore, two equivalent circuit models ( Figure 3d) were used to fit the EIS results, and their fitted electrochemical parameters of the samples in Hanks' and AS solutions are listed in Table 3. In the two equivalent circuit diagrams, R s , R 2 , and R 3 are the solution resistance, electric double-layer resistance that is related to charging transfer and electrochemical double-layer/oxide film effects between the electrode surface and solution, and mass transport resistance that is related to the Zn ion diffusion through the corrosion layer. [71] The CPE 2 and CPE 3 correspond to the capacitance of the R 2 and R 3 , respectively. R 1 and CPE 1 contributed to the solution/ZnP coating interface. The R total = R 1 +R 2 +R 3 or R 2 +R 3 indicates the total resistance of the ZCT sample, which is used to compare the corrosion resistance of the samples. The R total of the samples in descending order is ranked ZnP-coated ZCT in Hanks' > ZnP-coated ZCT in AS > ZCT in Hanks' > ZCT in AS, indicating that the corrosion resistance of the above samples is gradually decreased, which is consistent with the law of the corrosion resistance measured by PDP testing. The ZnP-coated ZCT in Hanks' solution showed the best corrosion resistance among these samples with a R total of 1484 ± 24 Ω cm 2 . Figure 3e shows the Bode impedance modulus and phase angle diagrams of the ZCT and ZnP-coated ZCT samples. The impedance modulus of the ZnP-coated ZCT sample in Hanks' and AS solutions is higher than that of the ZCT sample in high-and mediumfrequency regions, respectively, indicating that the ZnP-coated ZCT sample has a higher corrosion resistance in both two solutions. In addition, the impedance modulus of the same sample in Hanks' solution is higher than that in AS solution, indicating that the ZCT and ZnP-coated ZCT samples have higher corrosion resistance in Hanks' solution than in AS solution.

Corrosion Behavior of ZCT and ZnP-Coated ZCT Samples
The degradation rates of the ZCT and ZNP-coated ZCT samples after 30 d immersion in Hanks' and AS solutions are shown in Figure 3f and Table 2. The variation in V w of the samples is consistent with the change law of V PDP measured by PDP testing, and the ZnP-coated ZCT in Hanks' solution showed the smallest V w of 27 ± 1 µm a −1 among these samples, less than 30.8% of that of ZCT in Hanks' solution, showing the best corrosion resistance. Based on the V PDP and V w , the ZnP coating improved the corrosion resistance of the ZCT samples, and the corrosion performance in AS solution is lower than that in Hanks' solution. Figure Figure 3h-k shows 3D surface morphology, 3D Volta potential map, corresponding surface height, and Volta potential line profiles of Kelvin probe force microscopy (KPFM) on cross-sections of ZnP-coated ZCT sample without immersion. In the 3D surface morphology, the large irregular bulk second phase with a phase size of ≈ 7.2 ± 0.6 µm in line 1 and the fine granular second phase with a phase size of ≈ 2.4 ± 0.5 µm in line 2 is Cu 2 TiZn 22 and CuZn 5 phases, respectively. In line 1, the height and Volta potential in descending order are Cu 2 TiZn 22 > α-Zn matrix > ZnP coating, and the Volta potential difference between α-Zn matrix and both ZnP coating and Cu 2 TiZn 22 phase are ≈ 50 and 75 mV, respectively ( Figure 3j). In line 2, the α-Zn matrix showed a lower height and higher Volta potential than those of the CuZn 5 phase, and the Volta potential difference between α-Zn matrix and CuZn 5 phase is ≈ 39 mV ( Figure 3k). Figure 4a-d shows SEM images of corrosion products on sample surfaces after immersion in Hanks' and AS solutions for 30 d. Varying amounts of granular, flocculent, and elliptic corrosion products were distributed on the sample surface. In the same solution, the surface of the ZCT sample has more corrosion products than that of the ZnP-coated ZCT sample, and the surface of the ZCT sample is mainly granular and clumped corrosion products, while the surface of the ZnP-coated ZCT sample is mainly flocculent corrosion products. At the same time, the corrosion products of the ZCT and ZnP-coated ZCT samples in AS solution are more than those in Hanks' solution. In addition, the ZnP coating on the ZnP-coated ZCT sample surface after immersion in Hanks' solution is still intact and without obvious shedding, showing a good corrosion resistance whereas, the ZnP coating in AS solution appears to fall off or degrade in a large area. The corresponding EDS profiles on the sample surface in Figure 4c,d are shown in Figure 4e. Spot 1 is an elliptic corrosion product that contains large amounts of O, Zn, C, P, and a small amount of Ca, and the atomic ratio of O to Zn is close to 1:1. Spot 2 is a flocculating corrosion product that contains large amounts of O, C, P, Ca, Zn, and a small amount of Cl, and the atomic ratio of P to O is close to 1.5:1. Spot 3 is the needle-like coating that contains a large amount of O, Zn, P, C and a small amount of Ca and Cl. Notably, the P content was the highest among all tested areas. Figure 4f shows the XRD patterns of the corrosion products on the sample surfaces after immersion in Hanks' and AS solutions for 30 d. After immersion in Hanks' solution, both two sample surfaces mainly contained α-Zn, ZnP, and a small amount of ZnO, CuZn 5 , Ca 3 (PO 4 ) 2 , and Ca 10 (PO 4 ) 6 (OH) 2 (HA) phases. The ZnP-coated ZCT sample has a higher ZnP content than the ZCT sample, mainly because ZnP coating is still primarily covered on the ZCT sample. After immersion in AS solution, both two sample surfaces mainly contained α-Zn, ZnP, HA, Zn 5 (CO 3 ) 2 (OH) 6 , Zn 5 (OH) 8 Cl 2 H 2 O, and a small amount of Ca/Zn(H 2 PO 4 ) 2 H 2 O. Meanwhile, the diffraction peak intensity of corrosion products of the two samples in AS solution is higher than that in Hanks' solution except for the ZnP phase. This indicates that the same samples in AS solution have undergone more corrosion reactions and formed more corrosion products than in Hanks' solution, showing lower corrosion resistance, which is consistent with the corrosion rate law shown in Figure 3. Figure 4g shows FTIR spectra of the corrosion products on the ZCT and ZnP-coated ZCT samples after 30 d immersion in Hanks' and AS solutions. The absorption peaks at wavenumbers of ≈425, 478, and 877 cm −1 are associated with the bending vibration absorption peak of the metallic oxide (ZnO) groups. [56][57][58] The absorption peaks at wavenumbers of ≈558, 600, 1005, 1039, 1061, 1086, and 1096 cm −1 are associated with the PO groups. [56][57][58] The absorption peaks at wavenumbers of ≈1346, 1379, 1415, and 1475 cm −1 are associated with the CO groups; [56][57][58] the absorption peaks at 3300 and 3632 cm −1 correspond to OH groups, [56][57][58] and the absorption peak at 1640 cm −1 are attributed to the stretching vibration absorption peaks of OH group. [56][57][58] Figure 4h shows the XPS full spectrums of the corrosion products on the ZCT and ZnP-coated ZCT samples after 30 d immersion in Hanks' and AS solutions. The corrosion product on the sample surfaces consists of O, C, Zn, P, and Ca elements in the survey spectra, which is consistent with the EDS analysis results (Figure 4e). At the same time, the Ca content on both two sample surfaces after immersion in AS solution was higher than that in Hanks' solution; while the Zn content showed an opposite trend, indicating that the sample surface was covered with more corrosion products and formed more HA after immersion in AS solution. Their corresponding high-resolution XPS narrow spectrums of the immersion samples are shown in Figure 4i. P 2p peak at 132.8 eV corresponds to the P chemical bond in HA and ZnP. [56][57][58] C 1s at 285.0, 285.4, 287.8, and 289.4 eV correspond to the C chemical bond in CC, COR, CO, and CO 3 2− structure. [56][57][58] Ca 2p peak at 347.3 and 350.9 eV might correspond to the Ca chemical bond in the HA and Ca 3 (PO 4 ) 2 structure. [56][57][58] O 1s at 530.5 and 531.1 eV can be attributed to the O chemical bond in OH, ZnO, OH, CO 3 2− , and PO 4 3− structure. [56][57][58] Zn 2p at 1021.6 and 1044.8 eV can be assigned to the Zn chemical bond in ZnO, ZnP, Zn 5 (CO 3 ) 2 (OH) 6 , and Zn(H 2 PO 4 ) 2 H 2 O. [56][57][58] Based on the EDS, XRD, FTIR, and XPS results, it can be inferred that Spots ≈1-3 marked in Figure 4c,d are Zn 5 (CO 3 ) 2 (OH) 6 , HA, and ZnP phases, respectively. The corrosion products on the sample surfaces are mainly derived from ZnP and ZnO, and a small amount of Zn 5 (CO 3 ) 2 (OH) 6 , HA, and Zn 5 (OH) 8  The standard electrode potentials of Zn, Cu, and Ti elements are −0.762 V, 0.342 V, and −1.63 V, [24] respectively. The standard electrode potential of Zn element is between Cu and Ti elements. Therefore, a significant corrosion potential difference exists between the α-Zn phase and CuZn 5 or Cu 2 TiZn 22 phases in the Hanks' and AS solutions containing anion, which causes local micro-galvanic corrosion. [72] ZnP-coated ZCT sample showed a better corrosion resistance than ZCT, indicating that ZnP coating can play a good corrosion protection role. This is consistent with the results reported in previous studies. [41,45] Although the ZnP coating has a relatively thin thickness (3.3 ± 0.4 µm), it can still block the entry of anions, especially Cl ions, in Hanks' and AS solutions into the ZCT matrix due to its dense structure and chemical stability, thus weakening the further spread of corrosion reaction. [73] The corrosion resistance of both two samples in AS solution is worse than that in Hanks' solution, which is mainly because the pH value of  AS solution (pH = 6.0) is lower than that of Hanks' solution (pH = 7.4), resulting in the dissolution of the corrosion product layer. In particular, the easily soluble Zn 5 (OH) 8 Cl 2 H 2 O and Zn 5 (CO 3 ) 2 (OH) 6 H 2 O, and even the insoluble Zn(OH) 2 and ZnP phases also degraded (Figure 4d), gradually losing their protection function. [70,72,73] In addition, although the concentration of the Cl ions in AS solution is lower than that in Hanks' solution, the thiocyanate ions (SCN − ) in AS solution will aggravate local corrosion during long-term immersion. [74] Therefore, the chemical complexity of AS solution leads to the rupture of the ZnP coating, the corrosion product layer, and the passivation film, causing further corrosion. Compared with the ZCT substrate, the ZnP coating has a lower Volta potential measured in the KPFM testing ( Figure 3i). Therefore, the ZnP coating as the anode constitutes galvanic corrosion with the ZCT substrate as the cathode in the solution containing Cl ions when the coating is damaged, thereby accelerating the corrosion of the sample and the peeling of the coating. The incomplete coverage and rupture of the surface passivation and corrosion product layers will also form galvanic corrosion with the ZCT substrate and accelerate corrosion. [75] Figure 5 shows the tensile properties, fracture surfaces, quasi-in situ bending morphologies, and nanoscratch properties of the ZCT and ZnP-coated ZCT samples. Their corresponding tensile yield strength (σ ys ), ultimate strength (σ uts ), and elongation (ε) are shown in Figure 5b and Table 4. In the stress and strain curves (Figure 5a), all of the ZCT and ZnP-coated ZCT samples showed simultaneously high strength and high ductility, exceeding the yield strength and the elongation requirements of biodegradable bone-implant materials with σ ys greater than 230 MPa and ε greater than 15%. [76] The tensile properties of all of the ZnP-coated ZCT samples are higher than those of the ZCT counterparts (Figure 5b), indicating that the ZnP coating improved the tensile properties of the ZCT membrane sample. At the same time, the ZCT and ZnP-coated ZCT samples showed decreased strength and elongation after 30 d immersion in Hanks' and AS solutions, and the decrease in tensile properties after immersion in Hanks' solution was smaller than that in AS solution (Figure 5b), indicating that the corrosion damage degree in AS solution was higher than that in Hanks' solution. This result is consistent with the change law of degradation rates of the samples ( Table 2). The ZnP-coated ZCT sample without immersion exhibited the highest σ ys of 264 ± 1 MPa, σ uts of 312 ± 1 MPa, and ε of 36.0 ± 0.9% among all the samples, which is 4.3%, 4.0%, and 9.4% higher than those after 30 d immersion in Hanks' solution, and 9.1%, 5.8%, and 16.9% higher than those after 30 d immersion in AS solution, respectively. Figure 5c-h′ shows SEM images of ZCT and ZnP-coated ZCT fracture surfaces without and with 30 d immersion in Hanks' and AS solutions. There were many dimples of different sizes and clear neck shrinkage deformation on the tensile fracture surface of all membrane samples, indicating a typical ductile fracture and good plastic deformation ability. Compared with the ZCT samples, there were more large and deep dimples on the fracture surfaces of the ZnP-coated ZCT sample before and after immersion in Hank's and AS solutions. This is especially more significant for the sample after immersion in AS solution, indicating its better plastic deformation ability. The plastic deformation ability of the fracture surfaces of the samples is consistent with the change law of their elongation. In addition, there were a large amount of fine ZnP coatings on the fracture surface edges of all ZnP-coated ZCT samples, showing good deformation ability and bonding ability of the ZnP coating. The tensile sample surface of ZnP-coated ZCT without immersion after tensile testing is shown in Figure 5i. The fracture plane of the ZnP coating on the sample is roughly parallel to the tensile fracture plane, and the length of the fracture zone is only 586.3 ± 19.7 µm. A large amount of ZnP coating still exists in the fracture zone, indicating that the ZnP coating has good bonding strength and deformation resistance. Figure 5j-l′ shows the quasi-in situ morphologies of the ZnP-coated ZCT sample without immersion after bending with 0°, 90°, and 90°-0° bending angles. Before bending deformation, there were almost no cracks, holes, or uncovered areas on the coating surface (Figure 5j,j′), indicating that a dense layer of ZnP coating completely covered the ZCT substrate. After bending to 90°, many horizontal cracks with different widths appeared in the ZnP coating, and the cracks gradually reduced and tapered along the maximum deformation area (the vertex of bending at 90°) to the planes on both sides ( Figure 5k); at the same time, the crack extended along the fine crack of the original ZnP coating and part of or completely run through the ZnP coating ( Figure 5k′). After bending to 90° and then returning to 0°, the crack width on the ZnP coating was significantly reduced without coating cracking and peeling, and even some small cracks appeared to heal, showing good deformation resistance and healing effects (Figure 5l,l′). Figure 5m shows an optical micrograph of the nanoscratch trace, variation in depth with displacement, and friction force with applied load. The test depth and friction force increased linearly with the increasing applied load and displacement, and the ZnP coating on the ZCT sample gradually fell off. The coating completely peeled off at ≈1.3 mm displacement and ≈0.65 N applied load, and the test depth was 4.23 µm, close to the coating thickness shown in Figure 2b′. The corresponding SEM images of the sample surface after nano-scratch testing at different areas are shown in Figure 5n-n 3 . At the initial scratch stage (Figure 5n 1 ), the coating surface was flattened by the nanoscratch measuring head with no obvious cracks and tears in the coating. In the intermediate deformation stage (Figure 5n 2 ), part of the metal matrix is exposed. There are many cracks perpendicular to the scratch direction on the coating surface without peeling off phenomenon. At the same time, the cracks on the coating do not extend further to the undeformed area, indicating that the coating has good deformation resistance. In the final deformation stage (Figure 5n 3 ), the metal matrix is completely exposed, and there are many furrows parallel to the movement direction of the scratch. This result indicated good bending deformation resistance of the ZnP coating, implying that the ZnP-coated ZCT membrane sample can meet the contour shaping requirements of a GBR membrane in oral bone defects.

Mechanical Properties of ZCT and ZnP-Coated ZCT Samples
As a degradable GBR barrier membrane, good mechanical stability during bone repair is of great significance for www.afm-journal.de www.advancedsciencenews.com 2214657 (11 of 26) Figure 5. Tensile properties, fracture surfaces, quasi-in situ bending morphologies, and nanoscratch properties of ZCT and ZnP-coated ZCT samples: a) tensile stress-strain curves without and with immersion in Hanks' and AS solutions for 30 d; b) tensile yield strength (σ ys ), ultimate strength (σ uts ), and elongation (ε); c-h') SEM images of ZCT and ZnP-coated ZCT fracture surface without and with immersion; i) tensile sample surface of ZnPcoated ZCT without immersion after tensile testing; j-l') quasi-in situ morphologies of ZnP-coated ZCT after bending with 0°, 90°, and 90°-0°; m) variation in depth against displacement and friction force against applied load; and SEM images of n) nanoscratch trace and n 1 -n 3 ) sample surface after nano-scratch testing at different stages. maintaining bone repair space. ZCT and ZnP-coated ZCT samples have relatively high strength and ductility, mainly attributed to the solid solution strengthening of Cu, GB strengthening due to fine grain size, and second-phase strengthening from CuZn 5 and Cu 2 TiZn 22 phases formed by reactions between Cu and Ti elements and Zn elements. The solid solution effect of Cu causes a lattice distortion and hinders dislocation movement, playing a significant solution-strengthening effect and improving the strength and hardness of the alloy. [43,44] The second phase, uniformly distributed along the rolling direction, can hinder dislocation movement and improve strength and hardness. [43,44,77] At the same time, the α-Zn grain refinement and the crushing refinement of the second phases caused by recovery and recrystallization during the hot-rolling process can further improve the strength and elongation of the alloy. [78] In addition, the hardness and tensile properties of the ZCT membrane are higher than those of the HR ZCT alloy reported in our previous study, [43] indicating that the increase in rolling reduction contributed to the improvement of tensile properties and hardness of the ZCT alloy. It is mainly due to the further reduction of sizes of the α-Zn grains and second phases under large deformation, which improves the mechanical properties of the alloy. [79,80] ZnP-coated ZCT sample has higher tensile properties and mechanical stability than the ZCT sample before and after 30 d immersion in Hanks' and AS solutions, mainly due to its good corrosion resistance and deformation resistance, and high hardness of the dense ZnP coating. In the immersion tests, the ZnP coating significantly prevented the corrosion effect of the Hanks' and AS solutions to the ZCT matrix, resulting in a less mechanical property attenuation of the ZCT matrix in ZnP-coated ZCT sample than that in ZCT sample. However, due to the faster degradation rates in AS solution, both the ZCT and ZnP-coated ZCT suffered more serious corrosion damage, resulting in a significant attenuation of mechanical properties.
ZnP coating has good bonding strength with the ZCT matrix, mainly due to the combination of phosphate ions and Zn ions dissolved in the matrix after acid etching during the preparation of ZnP coating, forming a fine ZnP coating with a high hardness on the surface of the ZCT matrix. [81] The uneven surface after acid etching provided a larger contact area for coating adhesion, thus generating a larger interface attraction and improving adhesion strength. [82] In addition, GO with high tensile strength and hardness can effectively reduce the internal stress of the ZnP coating and consume fracture energy, thereby improving the strength, ductility, and deformation resistance of the ZnP coating. [83] Therefore, the good bending deformation resistance of the ZnP coating could enable ZnP-coated ZCT membrane to meet the contour shaping requirements of GBR membrane in bone defects. In addition, the ZCT sample can be pre-shaped according to the actual dental profile and then surface-treated with a simple ZnP coating process. Finally, the ZnP-coated ZCT sample can be implanted before cleaning, disinfection, and sterilization. Figure 6a shows the cell viability of MC3T3-E1 and MG-63 cells and pH of the culture medium obtained from α-minimum essential medium (α-MEM) and Dulbecco's modified eagle medium (DMEM) after co-culturing with the ZCT and ZnPcoated ZCT samples for 1 d. The ZnP-coated ZCT sample showed a higher cell viability and lower pH than the ZCT sample, showing better cytocompatibility. In addition, both samples showed slightly higher cell viability and lower pH in the culture medium for MC3T3-E1 cells than those of the culture medium for MG 63 cells. Figure 6b (Figure 3g). Meanwhile, the media of the ZnP-coated ZCT sample showed a lower ion concentration and pH than those of the ZCT sample. In addition, the ion concentration and pH values of the two samples in the DMEM solution are higher than those in the α-MEM solution. Figure 6e,g shows the cell viability of MC3T3-E1 and MG 63 cells during culturing with different concentration extracts for 3 d, respectively. The cell viability of both cells in the ZnP-coated ZCT extract was higher than that of the ZCT sample, showing good cytocompatibility. At the same time, the cell viability of MG 63 cells was slightly higher than that of MC3T3-E1 cells in both metal extracts except ZnP-coated ZCT extract at 50% and 25% concentrations. In the undiluted extract, the ZCT sample had a low cell viability of less than 75% toward MC3T3-E1 and MG 63 cells, showing a grade 2-3 cytotoxicity that is mild to moderate cytotoxic according to ISO 10993−5: 2009, [51] and the ZnP-coated ZCT sample had a high cell viability of more than 95%, indicating a grade 1 cytotoxicity that is just slightly toxic. With the dilution of the extract concentration, the ZCT sample showed a gradually increasing cell viability, while the ZnP-coated ZCT sample showed a trend of increasing and then slightly decreasing. The 50% concentration extract of the ZnP-coated ZCT sample had the highest cell viability of 104.1 ± 2.4% for MC3T3-E1 cells and 105.3 ± 1.8% for MG 63 cells, exhibiting the best cell proliferation effect. The fluorescent live/dead and DAPI staining images of MC3T3-E1 and MG-63 cells after culturing with 25% concentration extracts of ZCT and ZnP-coated ZCT for 3 d are shown in Figure 6h-m′. For fluorescent live/dead staining images, both types of cells were found to be in good health condition with triangular or short spindle shape for MC3T3-E1 cells and spindle shape for MG 63 cells, and almost no dead cells were present, except for a small number of dead cells in the ZCT extract. The number of cells in each group was consistent with the law of cell viability. Fluorescent DAPI staining images showed blue nuclei and purple cytoskeleton with no obvious cell shrinkage in all cells, showing a good health condition. The number of nuclei was also consistent with cell viability.

Cytotoxicity of ZCT and ZnP-Coated ZCT Samples
ZnP-coated ZCT sample showed better cytocompatibility toward MC3T3-E1 and MG 63 cells than the ZCT sample in direct and indirect cell culture assessments. It is mainly because the culture medium in direct culture and extract in the indirect culture of ZnP-coated ZCT have lower Zn and Cu ion concentrations and pH values than those of ZCT sample. On the one hand, the corrosion protection of ZnP coating significantly reduced the metal ion release from the ZCT matrix, significantly decreasing the cell growth inhibition and even apoptosis caused by the excess Zn ion concentration in the culture medium or extract. [84] On the other hand, Most osteoblasts can maintain good growth activity and structural integrity when the pH value ranges from 7.5 to 7.8. [85] The pH value of the ZnP-coated ZCT sample was mostly within the optimal pH value range, which was more conducive to cell growth and proliferation. In indirect cell culture, with the decrease of the concentration of the extract, the cell viability of the ZCT sample showed a gradually increasing trend; while the ZnP-coated ZCT sample showed a trend of first increasing and then slowly decreasing, which was mainly due to that the metal ion concentration and pH value in the extract gradually decreased with the dilution of the extract, and gradually approached the suitable Zn and Cu ion concentrations and pH values, which promoted the cell growth and proliferation. [86,87] The biological behavior of bone cells on the implant surface is the basis of tissue repair, mainly affected by wettability, roughness, and surface morphology. [88][89][90][91][92][93][94][95][96] The wettability of the material surface directly affects the interaction between the material and cells; the hydrophilic surface can enhance cell adhesion and growth on its surface by adsorbing more proteins. [88,89] Hydrophilic surfaces can induce the adsorption of extracellular matrix (ECM) proteins [90] and bind to integrin receptors [91] due to the arginineglycine-aspartic acid (RGD) motif, thus accelerating cell adhesion. Therefore, the hydrophilic surface of the ZnP-coated ZCT sample (Figure 2h) showed better cell adhesion than that of the ZCT sample, leading to higher cell viability. In addition, the GO phase with hydroxyl oxidation groups on the ZnP-coated ZCT sample (Figure 2d) also improved the surface wetness, which in turn enhanced cell adhesion, proliferation, and osteogenic differentiation capacity and promoted cytocompatibility. [92] Further, the larger roughness and surface area of the ZnP coating surface (Figure 2a) increased the contact area between cells and the sample surface, which is also conducive to the adhesion, growth, and proliferation of MC3T3-E1 and MG 63 cells. [93] The micro-and nano-structures on the coating surface allow bone tissue to grow into them and form a strong mechanical locking, promoting the implant to achieve good retention in the early stage. [94] The micro-and nano-structured morphologies of the coating surface encourage the adhesion, proliferation, and differentiation of bone cells, which enhances the bone integration ability of the implant [95] and promotes the differentiation of mesenchymal stem cells into osteoblasts even in the absence of osteogenic inducers, [96] facilitating the formation of bone union and injury healing. Natural bone tissue and extracellular matrix have diverse micro-and nano-scale morphologies. Therefore, the microscopic and nanoscale features on the coated surface can more accurately replicate the extracellular matrix milieu, which supports the biological functioning of bone cells. [96]

In Vitro Osteogenic and Mitochondrial Quality-Control Properties of ZCT and ZnP-Coated ZCT Samples
Figure 7a-c shows the ALP staining of the ZCT and ZnP-coated ZCT extracts at 25% concentration and the control group. ZCT and ZnP-coated ZCT samples showed deeper and denser purple-black areas on the wall than those of the control group, indicating that the ZCT and ZnP-coated ZCT samples showed a good mineralization-promoting performance. Meanwhile, the ZnP-coated ZCT sample possessed significantly deeper and denser purple-black areas than those of the ZCT sample, indicating that the ZnP coating contributed to the improvement of mineralization performance. Figure 7d shows the ALP activity of ZCT, ZnP-coated ZCT, and the control group. The change law of ALP activity was consistent with ALP staining; the deeper and denser purple-black areas obtained from ALP staining showed a higher ALP activity. ZnP-coated ZCT sample showed the highest ALP activity among the three groups, ≈4.90fold higher than those of the control and 2.55-fold higher than those of the ZCT sample.
The relative expressions of osteogenic differentiation marker genes in MC3T3-E1 cells cultured in an osteogenic medium are shown in Figure 7e-h. The ALP and OCN expressions of MC3T3-E1 cells in ZnP-coated ZCT extract were significantly higher than those in the control group and ZCT sample, especially the OCN expression levels were 98.4-fold higher than the control and 2.4-fold higher than the ZCT sample, indicating that the ZnP-coated ZCT sample has stronger osteogenic mineralization ability. In addition, the ZnP-coated ZCT extract had significantly higher COL-1 and slightly lower Runx-2 (p > 0.5) expressions than the ZCT extract, indicating that the ZnPcoated ZCT sample up-regulated the mRNA level of COL-1 as a marker of early osteogenic differentiation. Figure 8 shows the in vitro mitochondrial quality-controlrelated genes including PARKIN, LC3B, PINK1, PGC1α, PGC1β, TFAM, DRP1, FIS1, MFN1, and MFN2 in relation to MC3T3-E1 cells after cultured in ZCT and ZnP-coated ZCT extracts at 25% concentration for 3 d. In all of the mitochondrial quality-control-related genes, the ZnP-coated ZCT sample showed a higher gene expression than the ZCT sample,  indicating that ZnP coating promoted mitochondrial biogenesis, fusion, and mitogenic response. Meanwhile, compared with the control group, the ZnP-coated ZCT sample had a significantly increased mitochondrial biogenesis marker (PGC1α, PGC1β, and TFAM), fusion (MFN1), fission (FIS1) expression, and a significantly decreased expression of mitophagy-related genes (LC3B and PINK1), fusion (MFN2), and cleavage (DRP1). This result indicates that the ZnP-coated ZCT sample is mainly responsible for the mitochondrial biogenesis in MC3T3-E1 cells.
Bone remodeling is a major metabolic process of the body to regulate bone structure and function, involving osteoclast resorption, osteoblast bone formation, and osteocyte coordination of osteoclast and osteogenic functions. [97] Mitochondria play a key role in cell energy metabolism, signal transduction, proliferation, differentiation, and apoptosis and are widely distributed in osteoblasts. [98] In addition to providing energy for osteoblasts to perform the synthesis functions, secretion, and mineralization of bone matrix, mitochondria play an essential role in regulating osteoblast survival, proliferation, and death. [99] Thus, a balanced mitochondrial quality control can promote the natural homeostasis of bone tissue function. Compared with the ZCT sample, the ZnP-coated ZCT sample showed an enhanced osteogenic gene expression, a better ability to maintain mitochondrial health, and can better participation in cell-mediated biological mineralization process through mitochondrial biogenesis, autophagy, fission, and fusion. This is mainly due to the effect of Zn and P elements in the ZnP coating on mitochondrial function. Similar to Ca ions, appropriate Zn ions can promote the osteoblast mineralization ability and activate the alkaline phosphatase (ALP), carbonic anhydrase, and collagenase in bone metabolism and bone formation. [100] However, the P element provides a P source for the synthesis of mitochondrial membrane, DNA, RNA, and other cell substrates, and participates in the maintenance, transcription, and translation of mitochondrial DNA; mitochondria can transport Ca and P to the calcification site to promote the occurrence of calcification and regulate bone metabolism. [101] Although the up-regulation of key genes of mitochondrial quality control in ZnP-coated ZCT sample may promote osteogenesis and mineralization, the enhancement of mitochondrial quality control has only been studied at the gene expression level, and more molecular biological tests are needed to verify this process in future work. Figure 9 shows the antibacterial effects of ZCT, ZnP-coated ZCT, and CP Ti samples after co-culturing with S. aureus for 24 h. Figure 9a-c shows the inhibition zone images of ZCT, ZnP-coated ZCT, and CP Ti samples. There was no discernible bacterial inhibition ring surrounding the CP Ti, indicating that it has no antibacterial ability. In contrast, there are clear translucent circular or oval bacterial inhibition rings of different sizes surrounding ZCT and ZnP-coated ZCT samples. The corresponding inhibition zone diameter (IZD) of ZCT and ZnP-coated ZCT samples are shown in Figure 9g, all of which are more than 1 mm, showing a significant antibacterial ability according to DIN EN ISO 20645-2004. [54] The ZCT sample showed a higher IZD (1.39 ± 0.05 mm) than that of the ZnP-coated ZCT sample (1.02 ± 0.07 mm), exhibiting a better antibacterial ability. Figure 9d-f shows the numbers of adherent colony-forming units (CFUs), and their CFU numbers are shown in Figure 9h. There were different amounts of spherical S. aureus on the well plates corresponding to bacterial suspensions of each sample, and the surface of CP Ti showed a partial agglomeration. The CFU numbers in descending order are ranked: CP Ti > ZnP-coated ZCT > ZCT, indicating that the antibacterial properties of the above sample gradually decreased. Meanwhile, the change rule of the CFU was correlated with the IZD; that is, the higher IZD value showed a smaller CFU. Figure 9i-k shows the SEM images of bacterial adhesion on sample surfaces. Many white globular S. aureus were attached to the surface of CP Ti, and the bacteria tended to be clustered. On the surface of ZCT samples, there were only a few bacteria, while more bacteria were embedded in the gaps and cracks of the ZnP coatings on the ZnP-coated ZCT sample surface with a bacterial number between CP Ti and ZCT samples.

Antibacterial Property of ZCT and ZnP-Coated ZCT Samples
Compared with CP Ti, the ZCT and ZnP-coated ZCT samples have better antibacterial performance, mainly due to the release of Zn 2+ ions which have a good antibacterial effect, and the increased pH value during the culturing process. The Zn 2+ may interact with negatively charged bacterial cell membranes, change the permeability of bacteria, destroy the integrity of cell membranes, and ultimately lead to leakage and death of bacterial solutes, thus acting as an antibacterial agent. [102] Zn ions can also bind to and inactivate bacterial proteins and interact with bacterial nucleic acids to prevent bacterial migration. [103] In addition, when excessive Zn ions come into contact with the cell membrane of bacteria, it will regulate the enzymatic reactions related to the cell membrane, destroy the absorption of Ca ions, change the permeability of the cell membrane, and thus achieve the sterilization effect. [104] Therefore, Zn ions have antibacterial effects on the S. aureus. [105,106] As a widespread antibacterial ion, Cu ions bind to negatively charged regions of bacteria through an electrostatic effect, causing rupture of the outer membrane of bacteria, damaging the nucleic acid structure, changing the bacteria osmotic pressure, and weakening the activity of cell synthetase. [107] The ZCT sample showed a better antibacterial effect than the ZnP-coated ZCT, which may be due to the higher Zn and Cu ion concentrations in the bacterial culture medium based on its higher corrosion rate (Figures 3g,6d,f), which improves the antibacterial performance. However, increased pH can also significantly reduce bacteria activity. [108] Compared with the ZnP-coated ZCT sample, the ZCT sample had a higher pH increase (Figures 3g  and 6a), which may be more conducive to improving antimicrobial properties. In addition, there is a small amount of ZnO phase (Figure 2e) on the ZCT sample surface, which can produce hydrogen peroxide and enter the cell membrane, thereby causing bacterial damage to inhibit the bacteria growth or kill bacteria. [109]

In Vivo Osteogenesis of ZCT and ZnP-Coated ZCT Samples
A schematic and photograph of a real GBR membrane, micro-CT images, and histological micrographs of in vivo skull regeneration in calvarial defects of ZCT and ZnP-coated ZCT membranes after 3 months of implantation are shown in Figure 10. The schematic and photograph of a real GBR membrane are shown in Figure 10a,b. All of the rats were in good health during the implant service and recovered smoothly. There were no obvious complications, such as inflammation and allergic reaction on the top of the head after GBR surgery. Figure 10c,d shows the 3D micro-CT images of skull regeneration after 3 months of surgery. Both experimental groups showed new bone (NB) formation at the edge of the skull defect. Compared with ZCT membrane, the ZnP-coated ZCT membrane showed a smaller bone defect size and more new bone regeneration and even formed mature high-density callus in the middle of the defect area. Figure 10e,f′ shows the crosssections of micro-CT images of skull regeneration. The bone defects of the ZnP-coated ZCT membrane had narrower bone defect gaps and more new bone mass than those of the ZCT membrane, which is consistent with the 3D micro-CT images (Figure 10c,d). In addition, there was a large amount of undegraded bone meal in both samples, indicating that the degradation cycle of commercial bone meal in vivo was more than 3 months. Figure 10g shows the quantitative analysis results of the osteogenesis indices using micro-CT. It can be seen that the ZnP-coated ZCT sample had a significantly higher BV/TV, Tb.N, and lower Tb.Sp than ZCT sample, indicating that the ZnP-coated ZCT sample was more conducive to increased bone mass and bone densification, showing a better bone induction and regeneration performance. Figure 10h-k′ shows the histological micrographs of skull regeneration in calvarial defects of ZCT and ZnP-coated ZCT with HE and Masson staining after 3 months of surgery. No necrotic tissue, inflammatory infiltration, and foreign body reaction were observed in the bone histological sections of ZCT and ZnP-coated ZCT samples, indicating that the two implants had good histocompatibility with the surrounding bone tissue. In the HE staining images, a large number of continuous fibrous connective tissue bands with more dark blue osteoblasts, a small number of newly formed trabeculae, and vascular red blood cells were observed between the ZCT sample and the skull (Figure 10h-h′); whereas, the ZnP-coated ZCT sample had narrower fibrous connective tissue bands and more bone trabeculae and even appeared dark red mature bone tissue similar to callus, as well as many osteoblasts in the trabecular bone and smaller intertrabecular marrow spaces (Figure 10i-i′). The results of Masson staining are consistent with those of the HE staining and micro-CT results. The ZCT sample was mainly composed of a large amount of collagen fibers and only a small amount of new bone (Figure 10j-j′). In the callus of ZnP-coated ZCT sample, there were a lot of new blue bone areas and red bone collagen tissue near the inner side of the skull (Figure 10k-k′). The ZnP-coated ZCT sample showed better osteogenic and osteoinductive properties than the ZCT sample, consistent with the micro-CT results. In addition, the ZnP-coated ZCT sample showed a better ability to resist the invasion of surrounding fibrous connective tissue into the bone defect area than the ZCT sample, which is beneficial to bone reconstruction in the oral defect site. Figure 11 shows the histological micrographs of various tissues including heart, liver, lungs, and kidneys with HE and Masson staining after 3 months of implantation of ZCT and ZnP-coated ZCT membranes. The HE staining of the four kinds of tissues for ZCT and ZnP-coated ZCT samples showed that the morphology of cardiomyocytes, hepatocytes, and lung tissues was normally arranged, the glomeruli and renal tubular cells were clear, and the cytoplasm was uniform, with no visible annulus, tissue edema, inflammatory cell infiltration, or other abnormal lesions (Figure 11a-d′). Masson staining of the four kinds of tissues for ZCT and ZnP-coated ZCT samples indicated no significant collagen fibers in the tissue cells (Figure 11e-h′). In addition, the average concentration of Zn 2+ in the serum of rats was 0.12 ± 0.03 mmol L −1 for ZCT and 0.10 ± 0.02 mmol L −1 for ZnP-coated ZCT, respectively, which are close to the normal value of ≈ 0.09 mmol L −1 . [110] The concentration of Zn 2+ ions in ZCT is slightly higher than that in ZnP-coated ZCT, but the difference is insignificant, which is consistent with the law of degradation rate in vitro. Histopathological analysis showed that the excess metal ions released from ZCT and ZnP-coated ZCT samples in rats could be excreted through renal regulation, indicating no adverse effect on the circulation, immune and urinary system of rats, and had ideal biosafety in vivo.
The ZnP-coated ZCT sample has better osteogenic performance than the ZCT sample, mainly due to the release of Zn and P ions from the ZnP coating during degradation process, which have osteogenic and mineralization functions. Zn ions can promote the proliferation and differentiation of osteoblasts, and P ions can provide a P source for chondrocyte mineralization and activate the bone morphogenetic protein 2 (BMP-2) signaling pathway, thereby promoting bone repair in bone defect areas. [111] ZnP coating has a chemical mechanism similar to hydroxyapatite (HA), playing a certain role in bone conduction, and can be used as a nucleation site for bone mineraliza-tion to promote bone matrix synthesis and mineralization by osteoblasts. [112] In addition, the ZnP-coated ZCT sample has a lower degradation rate than the ZCT sample, which can significantly reduce the concentration of Zn ions released from the ZCT substrate and decrease the pH value around the implant, improving the microenvironment for the growth of new bone tissue. [113] Further, improved wettability of the implant surface facilitates early bone deposition and bone-implant interface binding. [114] At the later stage of in vivo service, the blood vessel formation and bone mass increase at the bone defect site, and the ZCT sample gradually degrades. The biological toxicity of the degradation product that contains Zn, Cu, and Ti elements is critical to the body. The degradation rate of the ZCT sample after immersion in Hanks' and AS solutions for 30 d is 39 and 47 µm a −1 (Figure 3f and Table 2); and the released ion concentrations of each kind of the metal after implantation can be estimated according to the corresponding corrosion rate, which can be calculated to be 76 and 93 µg cm −2 d −1 , respectively. Considering the dimensions of a GBR implant with a surface area of approximately 2.0 cm 2 , the ion release concentrations of Zn, Cu, and Ti are estimated to be 75.16, 0.76, and 0.08 µg d −1 in Hanks' solution and 91.98, 0.93, and 0.09 µg d −1 in AS solution, respectively, which are significantly lower than their corresponding recommended daily intake (RDI) for adults, approximately ≈12-16 mg for Zn, ≈0.9-1.2 mg for Cu, and ≈0.3-2.0 mg for Ti, respectively. [115,116] Most Zn, Cu, and Ti ions absorbed into the systemic circulation are excreted through feces and urine. Even if all metals cannot be eliminated from the body, the total content of Zn, Cu, and Ti ions after full degradation is 137.7, 1.4, and 0.1 mg, which is substantially lower than their corresponding ion levels in humans of ≈2-4 g, ≈84-126 mg, and 15 mg, respectively. [115][116][117] Therefore, the metal ions released from the ZCT sample or enriched in corrosion products will not induce biological toxicity to the human body after complete degradation. Although the bone meal was not wholly degraded after the GBR operation, Hämmerle et al. [118] reported that the combination of bone replacement material and GBR membrane had better osteogenic performance than the single addition of bone replacement material. At the same time, the β-tricalcium phosphate (TCP) phase in bone meal, as the main component of natural bone tissue, has a regulatory effect on the early differentiation of osteoblasts and can promote the repair of bone defects. [119] Therefore, the combination of degradable bone meal and GBR membrane may be more beneficial to oral bone defect repair. The above process is also commonly used in the clinical treatment of dental defects.

Conclusions
In this study, we have developed a biodegradable ZnP-coated ZCT membrane with high strength-ductility and mechanical stability, appropriate corrosion and degradation rates, effective antibacterial ability, excellent in vitro and in vivo cytocompatibility and osteogenesis via hot-rolling and conversion phosphating for oral GBR membrane applications. The key conclusions are as follows: 1. The microstructure of the ZCT membrane mainly consisted of an α-Zn matrix phase and the second phases of CuZn 5 and Cu 2 TiZn 22 that are uniformly streamlined parallel to the RD. Surface conversion phosphating produced a homogeneous and dense ZnP coating with a thickness of ≈3.3 µm, and the ZnP-coated ZCT membrane showed significantly increased hydrophilicity with the water contact angle decreased from ≈78.7° to ≈21.9°. Overall, the ZnP-coated ZCT membrane is promising for biodegradable GBR membrane applications for oral bone defect repair due to its appropriate degradation rate and antibacterial property, excellent strength-ductility and contour shaping ability, good cytocompatibility, osteogenetic effect, and osteoinductivity.

Experimental Section
Preparation of ZCT and ZnP-Coated ZCT Membrane Samples: The as-cast (AC) ZCT alloy ingot was prepared using the method described in a previous study. [43] Subsequently, plates with dimensions of 60 mm length, 20 mm width, and 10 mm thickness were cut from the AC ingots using electrical discharge machining (EDM) to prepare 0.2 mm thickness ZCT membrane samples: the plates were hot-rolled (HR) to a thickness of 1 mm at a 1 mm reduction per pass using a two-high cogging mill and then hot-rolled to a final thickness of 0.2 mm at a 0.2 mm reduction per pass by a six-high cluster mill after being preheated at 250 °C for 5 min. An X-ray fluorescence spectrometer (XRF; S4 Pioneer, Bruker, Germany) determined the chemical compositions of the membranes to be 1.03 wt.% Cu, 0.08 wt.% Ti, and balanced Zn.
The ZCT membrane samples were ground with SiC papers up to 2000-grit and washed with ethanol and deionized water for surface coating. ZnP coating on the ZCT membrane samples was performed by immersing the membranes into a phosphating solution containing 0.07 m Zn(NO 3 ) 2 , 0.15 m H 3 PO 4 , and 1 g L −1 graphene oxide (GO) for 5 min at room temperature (RT) according to the previous report. [41] The ZnP-coated membrane (denoted as ZnP-coated ZCT hereafter) samples were then rinsed using deionized water and dried in air.
For the electrochemical, immersion, surface contact angle testing, nano-scratch testing, cytotoxicity, ALP analysis, and expression of osteogenic and mitochondrial dynamics genes, disc samples with 8 mm diameter and 0.2 mm thickness were cut from the ZCT membrane using EDM.
Characterization of Microstructure, Chemistry, and Surface Contact Angle of ZCT and ZnP-Coated ZCT Membranes: The microstructures and phases of the ZCT membrane samples on the transverse rolling direction (TRD) and normal to rolling direction (NRD) planes were determined using optical microscopy (OM; DM2500C, Leica, Germany) and scanning electron microscopy (SEM; Pro X FEI, Phenom, Netherlands) coupled with energy dispersive spectroscopy (EDS; X-Max, Oxford, England) at 15 keV. Before microstructure observation, all samples were polished with 0.5 µm diamond slurry and etched with 2% HNO 3 /ethanol solution. Electron backscattered diffraction (EBSD) was conducted to analyze the grain size and orientation distribution of the membrane samples on the TRD plane using field-emission scanning electron microscopy (FESEM; Quanta 450, Hillsboro, OR, USA) equipped with an EBSD system (EDAX-TSL, USA) at 20 keV. The samples for EBSD analysis were ground with SiC paper and polished with 40 nm colloidal silica suspension (OPS, Struers, Germany). The EBSD data was analyzed using TSL-OIMTM 5 software.
The microstructures and phases of ZnP-coated ZCT membrane surface and cross-section were determined using SEM coupled with EDS. The phases of the ZCT membrane and ZnP-coated membrane samples were measured using XRD (D/max 2500, Rigaku, Japan) with a Cu-Kα radiation source over the 2θ range of 5°-70° at 2° min −1 scan rate. The functional groups on the sample surfaces were characterized using FTIR (Nicolet 6700, Thermo Fisher, USA) in the wave number range of 4000-400 cm −1 at 4 cm −1 resolution. The chemistry of the sample surfaces was analyzed using XPS (K-Alpha+, Thermo Fisher Scientific, USA) with an Al-Kα radiation source and the total survey spectra was collected at 1200-0 eV binding energy range with a 1 eV step size. The high-resolution narrow spectra of P 2p, C 1s, Ca 2p, O 1s, and Zn 2p were collected to determine the precise chemical bonding information.
The surface contact angle of the ZCT and ZnP-coated ZCT membrane surfaces was measured using a contact angle goniometer (OCA15EC, Data Physics, Germany) with 2 µL droplet of deionized water as a testing medium at RT.
Electrochemical Corrosion Testing: Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) testing of the ZCT and ZnP-coated ZCT membrane samples were measured using an electrochemical workstation (ParStat 2273, Princeton Applied Research, USA) to investigate the electrochemical corrosion behaviors. A typical three-electrode cell system was conducted in the Hanks' (pH = 7.40) and AS solution (pH = 6.00) solutions after being adjusted using 1 m NaOH or HCl at 37.0 ± 0.5 °C. The chemical composition of the Hanks' solution [45] and AS solution [47] is listed in Table 5. The electrochemical samples with a 0.5 cm 2 exposure area, saturated calomel electrode (SCE), and platinum sheet were used as the working electrode, reference electrode, and counter electrode, respectively. The PDP testing was measured with a potential range of −0.4-1.1 V relative to the steady OCP at a scanning rate of 1 mV s −1 . The corrosion current density (I corr ) of the electrochemical samples was calculated using Tafel extrapolating method, and the corrosion rate (V PDP ) was calculated based on the I corr according to the ASTM G102−89. [48] EIS testing was measured in an applied frequency range of 10 5 -10 −2 Hz under steady OCP with an amplitude of 10 mV.
Immersion Testing of ZCT and ZnP-Coated ZCT Membranes: Immersion testing of the ZCT and ZnP-coated ZCT membrane samples was carried out in Hanks' and AS solutions at 37.0 ± 0.5 °C with a surface-to-volume ratio of 20 mL cm −2 for 30 d. After 30 d of immersion, the pH value and Zn and Cu ion concentrations of the Hanks' and AS solutions were measured by a pH meter (PB-21, Sartorius, Germany) and an inductively coupled plasma atomic emission spectrometer (ICP-AES; 720ES, Agilent, USA), respectively. The morphologies and chemical composition of the corrosion products on sample surfaces were characterized using SEM-EDS. The phase constituent of the corrosion products on sample surfaces was analyzed using XRD over the 2θ range of 10°-70°. The functional groups and chemical analyses of the corrosion products on the sample surface were characterized using FTIR and XPS under the same conditions. After the characterization of corrosion products on the sample surface, a 200 g L −1 chromic acid solution was used to remove the corrosion products completely and the sample weights were measured by an electronic balance before and after immersion, and the degradation rate (V w ) was calculated on the weight loss according to ASTM G31−12. [49] The 3D surface topography and contact potential difference (CPD) of the cross-section of ZnP-coated sample before immersion testing were measured using atomic force microscopy (AFM; Dimension Icon, Bruker, Germany) equipped with KPFM operated in the tapping mode with a 0.5 Hz scan frequency at RT, and the 3D images were taken in 256 × 256-pixel resolution over a 50 µm × 50 µm area.
Tensile, Bend, Hardness, and Nanoscratch Testing: The tensile properties of the ZCT and ZnP-coated ZCT membrane samples before and after immersion in Hanks' and AS solutions for 30 d were tested at a deformation rate of 1 mm min −1 at RT using a universal testing machine (Instron-3369, Instron, MA, USA) that does not require a clamping fixture (to avoid coating peeling). Tensile plate-type samples with 10 mm gauge length were cut by EDM, and their length direction was kept strictly parallel to the rolling direction according to ASTM E8/E 8M-16. [50] The fracture surfaces of the tensile samples and the tensile sample surface of the ZCT and ZnP-coated ZCT samples without and with 30 d immersion after tensile testing were observed by SEM. The quasi-in situ morphologies of the ZnP-coated ZCT sample after bending to different angles of 0°, 90°, and 90°-0° were observed using SEM.
The Vickers hardness of the ZCT membrane sample on the TRD plane was determined using a microhardness tester (MicroMet 6000, Buehler, USA) with 50 g load and 15 s loading time. The bonding properties between the coating and substrate of the ZnP-coated ZCT sample were evaluated via nanoscratch testing using a nanoindentation tester (UNHT3, Anton Paar, USA). Diamond indenters were inserted into the surface with an increasing force from 10 mN to 1 N at a scratch speed of 1.98 N min −1 , producing a scratch length of 2.0 mm on the ZnP-coated ZCT sample enough to penetrate the ZnP coating. The scratch morphology of the ZnP-coated ZCT sample after nano-scratch testing was observed by SEM.
Cytotoxicity Evaluation: Direct cytotoxicity, cell proliferation, and indirect cytotoxicity assay of disc samples were performed using MC3T3-E1 mouse pre-osteoblast cell line (CTCC GDC030, China) and MG 63 human osteosarcoma cells (CTCC GDC074, China) according to ISO 10993−5: 2009 [51] and the detailed procedure was described in a previous study. [45]  For the direct cytotoxicity, MC3T3-E1 cells with a density of 3 × 10 4 cells well −1 were cultured on disc samples, and 1 mL α-MEM culture medium was added into the 24-well plates. After 1 d incubation Table 5. Chemical compositions of Hanks' [45] and AS solutions [47] (g L −1 ). under a humidified atmosphere with 5% CO 2 at 37 °C, the disc samples were gently washed using PBS three times and fixed in 2.5% glutaraldehyde fixative for 6 h, followed by dehydration in ethanol gradient from 60-100% (10% steps for 15 min each) and dried in air. Finally, the surface morphologies were observed by SEM (JSM-7100F, JEOL, Japan). The pH values of the culture medium after co-culturing were measured using a pH meter. For the indirect cytotoxicity, disc samples were immersed in both two culture media with a ratio of 1.25 cm 2 mL −1 according to ISO 10993−12: 2009 [52] after autoclave sterilizing at 121 °C for 30 min and then placed in the same culture conditions for 2 d to prepare the extracts. The Zn and Cu ion concentrations and pH values of the undiluted extracts were measured using an ICP-AES and pH meter. MC3T3-E1 and MG 63 cells were seeded in 96-well plates with a cell density of 1 × 10 4 cells well −1 and then cultured in the same culturing conditions for 1 d. Subsequently, the cell culture medium was replaced by the extracts with differently diluted extracts (100%, 50%, and 25%) and then incubated with extracts for 3 d under the same conditions. The flush culture medium was used for the control group.
For the cell proliferation assay, fluorescence live/dead and 4',6-diamidino-2-phenylindole (DAPI) staining were used to assess the cell morphology. Cell suspension of 300 µL was added into 48-well plates at a density of 1 × 10 4 cells mL −1 and cultured for 1 d. After pre-incubation, 300 µL extracts with 25% concentration were used to replace the original cell culture medium and continuously cultured for 3 d under the same culturing conditions. For live/dead cell staining, the wells were filled with a 250 µL mixture of 2 µmol L −1 calcein-acetoxymethyl and 3 µmol L −1 propidium iodide (PI) (both Thermo Fisher Scientific, Germany) diluted with 0.01 m PBS and incubated for 15 min in the dark. For DAPI staining, samples were fixed with 4% paraformaldehyde for 30 min at RT. The cells were stained with phalloidin, fluorescein isothiocyanate labeled (Phalloidin-FITC) (Sigma-Aldrich, P8582) for 40 min and with DAPI (Sigma-Aldrich, P8582) for 15 min after washing with PBS. Finally, the cell morphology was observed using fluorescence microscopy (Axio Observer A1, Zeiss, Germany). The green fluorescence of acetoxymethyl (AM) and red fluorescence of PI were analyzed at 488 nm (excitation) and 520 nm (detection) and 488 nm (excitation) and 620 nm (detection), respectively. The blue fluorescence indicating nucleus was analyzed at 340 (excitation) and 488 nm (detection).
ALP Staining and Activity Assessment: MC3T3-E1 cells with a density of 3 × 10 4 cells well −1 were seeded on 24-well plates and stimulated with osteogenesis differentiation medium (Thermo Fisher Scientific, USA) and 25% concentration extracts for 7 d according to the protocol described previously. [53] For ALP staining, cells were fixed with 4% paraformaldehyde for 30 min at 4 °C and stained by a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) ALP color development kit (Beyotime, China). The photomicrographs of the well were taken using stereoscopic microscopy (SMZ800N, Nikon, Japan). According to the manufacturer's instructions, the ALP activity was measured using a commercial ALP assay kit (Beyotime, China). The absorbance of ALP activity was measured with an automatic microplate reader at 405 nm.
Expression of Osteogenic and Mitochondrial Dynamics Genes: MC3T3-E1 cells with a density of 3 × 10 4 cells well −1 were seeded onto 24-well plates and then co-cultured with the 25% concentration extracts under the same conditions for 7 d after being replaced every 2 d. The total RNA was isolated and precipitated using a mixture of guanidine thiocyanate and phenol (Trizol), followed by quantifying the total RNA using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA). The PrimeScript RT reagent kit (TaKaRa, Shiga, Japan) was used to reverse-transcribe the RNA into cDNA. Finally, the SYBR Premix Ex Taq II kit (TaKaRa, Japan) was used to quantitatively analyze the expressions of the various target genes with an ABI 7500 Fast machine (Applied Biosystems, Courtaboeuf, France). The genes of ALP, OCN, COL-1, and Runx2 were used to evaluate the osteogenic differentiation, and PARKIN, LC3B, PINK1, PGC1α, PGC1β, TFAM, DRP1, FIS1, MFN1, and MFN2 were used to evaluate the mitochondrial quality-control. The PCR primer sequence and annealing temperature are provided in Table 6.
Antibacterial Assessment: An IZD and CFU enumeration assay were used to evaluate the antibacterial properties of the disc samples according to DIN EN ISO 20645-2004, [54] EN ISO 6888-1-2021, [55] and the detailed procedure described in the previous study. [45] For the IZD assay, Staphylococcus aureus (S. aureus; CMCC26003, China) with a concentration of approximately 1 × 10 8 CFU mL −1 was used as the bacteria model for antibacterial assessment. For sterilization purposes, the disc samples with 10 mm diameter and 0.2 mm thickness were autoclave sterilized at 121 °C for 20 min and followed UV-sterilized for 30 min before antibacterial assessment. Finally, the IZD values were observed and measured after incubation at 37 °C for 1 d. For the CFU enumeration assay, bacterial suspension with a concentration of 1 × 10 6 CFU mL −1 was added to a 24-well plate, and then the ZCT and ZnP-coated ZCT membrane discs were placed into the wells after sterilization. After 1 d of incubation under the sample conditions, 100 µL of the bacterial suspension was pipetted after sonicating the tube for 20 min, plated evenly on a tryptic soy agar (TSA) plate, and then incubated for 1 d. Finally, the number of CFUs on each plate was counted with ImageJ software (version 2.0.0, NIH). After 1 d incubation under the sample conditions, the disc samples co-cultured with S. aureus were gently washed using PBS three times and fixed in 2.5% glutaraldehyde fixative for 6 h, followed by dehydration in ethanol gradient and dried in air. Finally, the surface morphologies were then observed using SEM (JSM-7100F). For comparison, commercially available pure Ti (CP Ti, 99.99%) with the same size was used as the control group.
Surgical Procedure of In Vivo Testing Using Rats: All experiments involving live animals were carried out in accordance with the laws and regulations of the animal ethics committee of Wenzhou Medical University. Male Sprague-Dawley (SD) rats aged 8 weeks were used for the in vivo study, and a limited cylindrical bone defect repair model for rat skull was established. The rats were anesthetized by intraperitoneal injection with pentobarbital sodium (40 mg kg −1 ). Articaine (LanBi, China) with 1:100 000 epinephrine was injected subcutaneously for analgesia during and after the operation. After shaving, two horizontally distributed circular holes with a diameter of 5 mm for limiting bone defects without damaging the meninges were prepared using a trephine. The hole's edge was 1 mm away from the mid-sagittal suture of the skull. Each circular hole was uniformly filled with 5 mg of degradable dental bone powder (β-TCP, Cerasorb M, Curasan, Germany) with a particle size of 500 µm to simulate clinical GBR surgery. The bone defect surface was covered with ZCT and ZnP-coated ZCT membranes with 0.5 mm pores prepared by laser drilling. After wound irrigation with saline irrigation and tension-free reduction of the periosteum, the surgical site was closed with 5-0 Ethicon sutures, and antibiotics (penicillin, 80 000 U d −1 ) were intraperitoneally administered for 3 consecutive d. The rats were euthanized and the skulls and organs were collected, and the Zn ion concentrations in the whole blood were measured using ICP-AES after 3 months of sterile feeding.

Micro-Computed Tomography (Micro-CT) Assay:
The rat skull was completely removed and fixed with 4% paraformaldehyde after 3 months of implantation and sacrificing, then the implants were carefully removed to preserve the newly formed bone around the implants. The micro-CT (Skyscan1176, Bruker, Germany) was used to quantify the local osteogenic ability at 80 kV and 300 uA. After the scan was completed, 3D reconstructions were performed using Data Viewer, CTVox, CTAn, and CTVol (Bruker, Germany) software. The osteogenesis indices of bone volume fraction (BV/TV; the number of mineralized bone voxels divided by the total number of voxels), trabecular thickness (Tb.Th), trabecular numbers (Tb.N), and trabecular separation (Tb.Sp) were quantitatively analyzed using Image-pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
Histomorphometric Evaluation: After micro-CT analysis, hard tissue was subjected to decalcification by ethylenediaminetetraacetic acid (EDTA, 12%) for 30 d. All bone samples were dehydrated in a graduated ethanol solution followed by paraffin fixation and section. For the soft tissue staining, the soft tissue (heart, liver, lung, and kidney) were fixed, embedded, and sectioned after euthanasia. For histomorphometric evaluation of hard and soft tissue, hematoxylin and eosin (HE) and Masson's trichrome staining were employed. The tissue sections were observed using a Pannoramic MIDI scanner (3DHistech, Budapest, Hungary).
Statistical Analysis: All results were presented as means±standard deviation (n = 3 separate experiments). Statistical analysis was carried out using SPSS V18.0 software (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance (ANOVA) with Tukey's post hoc analysis was performed to determine the significant differences between groups. The statistically significant (hereafter denoted as *) and highly statistically significant (**) between groups were defined as p < 0.05 and p < 0.01, respectively.