In vitrocellular biocompatibility andin vivodegradation behavior of calcium phosphate-coated ZK60 magnesium alloy.

Calcium phosphate (Ca-P) surface coating is a simple but effective way to enhance both corrosion resistance and biocompatibility of ZK60 magnesium alloy. However, cell compatibility on different Ca-P layers coated on ZK60 alloy has seldom been investigated. In this study, the effects of type, morphology and corrosion protection of several Ca-P coatings formed at pH 6.5, 7.8 and 10.2 on cell behavior were examined by using an osteoblastic cell line MC3T3-E1. Furthermore,in vivobehavior in rabbits of the alloy coated with the optimum Ca-P layer was also studied. It was found that the surface factors governed the cell morphology and density. The coating morphology plays a dominant role in these surface factors. The sample coated at pH 7.8 showed the best cellular biocompatibility, suggesting that the hydroxyapatite (HAp) layer formed at pH 7.8 was the optimum coating. In rabbits, this optimum coating enhanced remarkably the corrosion resistance of the alloy. During implantation, the outermost crystals of the HAp coating were shortened and thinned due to the dissolution of HAp caused by the body fluid of the rabbits. It is indicated that ZK60 alloy coated at pH 7.8 can be applied as a biodegradable implant.

Effect of pH of coating solution on the adhesion strength and corrosion resistance of hydroxyapatite and octacalcium phosphate coated WE43 alloy.

Hydroxyapatite (HAp) and octacalcium phosphate (OCP) layers were formed on Mg- 4mass% Y- 3mass% rare earth (WE43) alloy by a chemical solution deposition method at various pH values of pH 5.5, 6.2, 7.5, and 8.6. Adhesion strength of HAp and OCP layers was evaluated before and after immersing in a medium for 14 days by a pull-off test. The corrosion resistance of these coatings was measured by polarization tests performed in a simulated body fluid (SBF). XRD analysis demonstrated that HAp coating layers were formed at pH 7.5 and 8.6, while OCP coating layers were formed at pH 5.5 and 6.2. Adhesion test results showed that the as-coated pH7.5-HAp layer had the highest adhesion strength of 8.6 MPa, which was attributed to the very dense structure of the coating layer. The as-coated pH8.6-HAp layer showed the adhesion strength of 6.5 MPa. The adhesion strength of the as-coated pH5.5- and pH6.2-OCP layers was 3.9 and 7.1 MPa, respectively, that was governed by the thick and fragile property of the layers. After immersing in the medium for 14 days, the adhesion strength of pH7.5- and pH8.6-specimens decreased to 5.8 and 5.6 MPa, respectively. The pitting corrosion and formation of Mg(OH)2 under the HAp layers were responsible for the decrease of adhesion strength. The polarization tests in SBF at 37 °C showed that the corrosion current density decreased with the HAp and OCP coatings, indicating the improvement of the corrosion resistance of WE43 alloy. The HAp coatings improved the corrosion resistance more efficiently than the OCP coatings.

In vivo degradation and bone formation behaviors of hydroxyapatite-coated Mg alloys in rat femur.

Various coatings have been developed for biodegradable Mg alloys to control the degradation speed and to improve the bone conductivity. In this study, hydroxyapatite (HAp) coatings were formed on pure Mg, Mg-0.8mass% Ca (MgCa), Mg-4mass% Y-3mass% rare earth (RE) (WE43), Mg-3mass% RE-1mass% Y (EW31) and Mg-4mass% RE (RE4) alloy rods with a chemical solution deposition method. The HAp-coated and uncoated Mg/Mg alloy rods were implanted in the femurs of rats for 3-6 months, and the corrosion suppression and bone formation abilities of the HAp coating were examined using a scanning electron microscope. The corrosion rate of WE43 was suppressed by 1/3 with the HAp coating for 6 months, and the corrosion product showed very slow dissolution. The effect of the HAp coating for pure Mg and MgCa disappeared in 1-2 months with the thinning of the rods accompanying with the obvious dissolution of the corrosion products. The effect of the HAp coating for EW31 and RE4 was not stable due to the expansion and collapse of the corrosion products. The bone formation was enhanced on the HAp layers. Eventually, the HAp coating basically suppressed the corrosion initiation and corrosion progress of Mg substrates. The magnitude of the suppression effect depended mainly on the chemical and physical stability of the corrosion products.

Enhanced Corrosion Resistance and In Vitro Biocompatibility of Mg-Zn Alloys by Carbonate Apatite Coating.

B-type carbonate apatite (CAp) coatings were formed on as-cast and T4-treated Mg-xZn (x = 1, 5, and 7 wt %) alloys containing various sized Zn-rich second phase to improve the corrosion resistance and biocompatibility. The CAp coating grew uniformly on the alloys with a thickness of 1.1-1.3 µm and did not show cracks or pores on 30 µm-sized second-phase particles. The CAp coating retarded corrosion of Mg-Zn substrates for the first 3-5 days in Hanks' solution. Polarization resistance of the CAp-coated alloys was 10-90 and 1-70 times higher than the uncoated and hydroxyapatite (HAp)-coated alloys, respectively. The corrosion rate of CAp-coated alloys was greatly affected by the substrate alloys once the coatings were partly broken. The CAp-coated alloys showed 40-60 and 25-45% lower 14-day average corrosion rates than the uncoated and HAp-coated alloys, respectively, in the immersion test. The CAp coating significantly enhanced the viability of osteoblastic MC3T3-E1 cells on the Mg-Zn alloys for 72 h compared to the uncoated and HAp-coated alloys. The cell densities on CAp-coated alloys were similar for 72 h regardless of substrate alloys. Therefore, the CAp coating can be a superior coating candidate for corrosion-control and biocompatibility improvement for biodegradable Mg alloys.

Osteoclast and osteoblast responsive carbonate apatite coatings for biodegradable magnesium alloys.

Corrosion-control coatings which can enhance bone formation and be completely replaced by bone are attractive for biodegradable Mg alloys. Carbonate apatite (CAp) and hydroxyapatite (HAp) coatings were formed on Mg-4 wt% Y-3 wt% rare earth (WE43) alloy as a corrosion-control and bioabsorbable coating in the coating solution with various concentrations of NaHCO3. The incorporation of carbonate group in apatite structure was examined using X-ray diffraction and Fourier transform infrared spectroscopy. Rat osteoclast precursor and MC3T3-E1 osteoblast cells were cultured on the CAp- and HAp-coated WE43 to examine the osteoclastic resorption and the alkaline phosphatase (ALP) activity, respectively. Mg ions in the used medium were quantified to examine the corrosion-control ability. The NaHCO3 addition in the solution resulted in the formation of B-type CAp in which the phosphate group of apatite structure was substituted with the carbonate group. The osteoclastic resorption was observed only for the CAp coatings as the cracking of the coatings and the corrosion of substrate WE43 strongly localized under osteoclast cell bodies. The CAp and HAp coatings significantly enhanced the ALP activity of osteoblasts. The CAp-coated WE43 specimens showed 1/5 smaller amount of Mg ion release than the uncoated WE43 on the first day of culturing osteoblasts. For the subsequent 22 days, the Mg ion release was reduced to 1/2 by the CAp coatings. In the presence of osteoclasts, the CAp coatings showed slightly lower corrosion protectiveness than the HAp coating. It was demonstrated that the CAp coatings can be a bioabsorbable and corrosion-control coating for biodegradable Mg alloys.

In Vitro Corrosion and Cell Response of Hydroxyapatite Coated Mg Matrix in Situ Composites for Biodegradable Material Applications.

In this study, hydroxyapatite (HAp) coated Mg matrix composites were fabricated for biodegradable implant applications. Spark plasma sintering was employed to fabricate the Mg-10 wt% ZnO composite substrates. HAp was coated on the surface of the sintered composites and pure Mg by a chemical solution treatment. SEM and optical micrographs of coated samples showed that HAp grew homogeneously and formed a layer on the entire surface of both pure sintered Mg and Mg composites. The immersion and polarization test results demonstrated that the HAp coating significantly improved the corrosion resistance of the sintered composites. While the HAp coating layer is not effective in the improvement of the pure Mg substrate, cell culture test results revealed that the HAp coating improved cell adhesion and proliferation on the composites effectively through 72 h, while no cell could survive on the uncoated composites after 72 h. In addition, the corrosion tests and cell culture test results indicated that the composite with longer sintering time has better corrosion resistance and cell viability than those of the composite with shorter sintering time. The findings suggested that the HAp-coated Mg-10 wt% ZnO-2.5 h + 10 min composite is a high-potential candidate for biodegradable implant applications.

Macrophage phagocytosis of biomedical Mg alloy degradation products prepared by electrochemical method.

Biomedical Mg alloy is promising for its widespread use clinically. In vitro and in vivo studies showed that the degradation products of biomedical Mg alloy were composed of O, P, Ca, Mg and other alloying elements. However, little is known about the metabolism of the degradation products. In this study, the in vitro macrophage phagocytosis of the degradation products of a biomedical Mg-Nd-Zn-Zr alloy was directly observed. This result affirms the necessity to investigate the long-term fate of Mg alloy degradation products in physiological environments. Besides, an electrochemical method was proposed to prepare enough amount of degradation products in vitro efficiently.

Micromorphological effect of calcium phosphate coating on compatibility of magnesium alloy with osteoblast.

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were developed to control the degradation speed and to improve the biocompatibility of biodegradable magnesium alloys. Osteoblast MG-63 was cultured directly on OCP- and HAp-coated Mg-3Al-1Zn (wt%, AZ31) alloy (OCP- and HAp-AZ31) to evaluate cell compatibility. Cell proliferation was remarkably improved with OCP and HAp coatings which reduced the corrosion and prevented the H2O2 generation on Mg alloy substrate. OCP-AZ31 showed sparse distribution of living cell colonies and dead cells. HAp-AZ31 showed dense and homogeneous distribution of living cells, with dead cells localized over and around corrosion pits, some of which were formed underneath the coating. These results demonstrated that cells were dead due to changes in the local environment, and it is necessary to evaluate the local biocompatibility of magnesium alloys. Cell density on HAp-AZ31 was higher than that on OCP-AZ31 although there was not a significant difference in the amount of Mg ions released in medium between OCP- and HAp-AZ31. The outer layer of OCP and HAp coatings consisted of plate-like crystal with a thickness of around 0.1 µm and rod-like crystals with a diameter of around 0.1 µm, respectively, which grew from a continuous inner layer. Osteoblasts formed focal contacts on the tips of plate-like OCP and rod-like HAp crystals, with heights of 2-5 µm. The spacing between OCP tips of 0.8-1.1 µm was wider than that between HAp tips of 0.2-0.3 µm. These results demonstrated that cell proliferation depended on the micromorphology of the coatings which governed spacing of focal contacts. Consequently, HAp coating is suitable for improving cell compatibility and bone-forming ability of the Mg alloy.

Effect of macrophages on in vitro corrosion behavior of magnesium alloy.

The influence of cells on the corrosion behavior of biomedical magnesium alloy is an important but less studied topic, which is helpful for understanding the inconsistent corrosion rates between in vitro and in vivo experiments. In this work, macrophages were directly cultured on Mg-2.1Nd-0.2Zn-0.5Zr (wt %, abbreviated as JDBM) alloy surface for 72 or 168 hours. Macrophages retained good viability and the generation of reactive oxygen species (ROS) was greatly promoted on the alloy. Weight loss, Mg(2+) concentration, and cross-section observation results demonstrated that macrophages accelerated the in vitro corrosion of JDBM. The coverage of cell body did not affect the local thickness of corrosion product layer. The corrosion product layer had a porous inner Mg(OH)2 layer and a dense outer layer mainly composed of O, P, Mg, and Ca. The uniform acceleration of JDBM corrosion was attributed to the omnidirection diffusion of ROS from macrophages. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2476-2487, 2016.

In Vivo Evaluation of Bulk Metallic Glasses for Osteosynthesis Devices.

Bulk metallic glasses (BMGs) show higher strength and lower Young's modulus than Ti-6Al-4V alloy and SUS 316L stainless steel. This study aimed to perform in vivo evaluations of Zr65Al7.5Ni10Cu17.5 BMGs for osteosynthesis devices. In the study for intramedullary implants, osteotomies of the femoral bones were performed in male Wistar rats and were stabilized with Zr65Al7.5Ni10Cu17.5 BMGs, Ti-6Al-4V alloy, or 316L stainless steel intramedullary nails for 12 weeks. In the study for bone surface implants, Zr65Al7.5Ni10Cu17.5 BMGs ribbons were implanted on the femur surface for 6 weeks. Local effects on the surrounding soft tissues of the implanted BMGs were assessed by histological observation. Implanted materials' surfaces were examined using scanning electron microscopy equipped with energy dispersive X-ray spectroscopy (SEM-EDS). In the study for intramedullary implants, bone healing after osteotomy was assessed by peripheral quantitative computed tomography (QCT) and mechanical tests. Histological observation showed no findings of the biological effects. SEM-EDS showed no noticeable change on the surface of BMGs, while Ca and P deposition was seen on the Ti-6Al-4V alloy surface, and irregularities were seen on the 316L stainless steel surface. Mechanical test and peripheral QCT showed that, although there was no significant difference, bone healing of BMGs was more than that of Ti-6Al-4V alloy. The results indicated that Zr-based BMGs can lead to bone healing equal to or greater than Ti-6Al-4V alloy. Zr-based BMGs exhibited the advantage of less bone bonding and easier implant removal compared with Ti-6Al-4V alloy. In conclusion, Zr-based BMGs are promising for osteosynthesis devices that are eventually removed.

In vitro and in vivo biocompatibility and corrosion behaviour of a bioabsorbable magnesium alloy coated with octacalcium phosphate and hydroxyapatite.

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were formed on Mg-3 mass% Al-1 mass% Zn (AZ31) magnesium alloy by a single-step chemical solution deposition method. Chemically polished AZ31 (Cpol-AZ31) and HAp- and OCP-coated AZ31 (HAp- and OCP-AZ31) were immersed in a medium for 52 weeks or implanted in transgenic mice for 16 weeks to examine the long-term corrosion behaviour and in situ inflammation behaviour. In the medium, Mg-ion release was restricted for the initial several days and the corrosion rate thereafter was suppressed by approximately one-half with the HAp and OCP coatings. HAp-AZ31 showed a ∼20% lower corrosion rate than OCP-AZ31. Tissues of the transgenic mouse emit fluorescence in proportion to the degree of inflammation in situ. The luminescence intensity level was too low to be a problem regardless of the coatings. A thinner fibrous tissue layer was formed around OCP- and HAp-AZ31 than around Cpol-AZ31, indicating that the HAp and OCP coatings suppressed corrosion and foreign-body reaction in vivo. Visible pits were formed in filiform and round shapes in vitro and in vivo, respectively. Corrosion was observed underneath the coatings, and almost uniform corrosion took place in vitro, while local corrosion was predominant in vivo. These differences in corrosion morphology are attributed to the adhesion of tissues and the lower diffusivity on the surface in vivo than that in vitro. Dissolution behaviour of OCP crystals in vivo was different from that in vitro. It was demonstrated that the HAp and OCP coatings developed have great potential for a biocompatible and corrosion protection coating.

In vivo evaluation of Zr-based bulk metallic glass alloy intramedullary nails in rat femora.

Zr-based bulk metallic glasses (BMG) show high corrosion resistance in vitro and higher strength and lower Young's modulus than crystalline alloys with the similar composition. This study aimed to perform an in vivo evaluation of Zr65Al7.5Ni10Cu17.5 BMG. Osteotomy of the femur was done in rats and stabilized with intramedullary nails made of Zr65Al7.5Ni10Cu17.5 BMG, Ti-6Al-4V alloy, or 316L stainless steel. Systemic and local effects of each type of nail were evaluated by measuring the levels of Cu and Ni in the blood and the surrounding soft tissue. Changes of the surface of each nail were examined by scanning electron microscopy (SEM). Healing of the osteotomy was evaluated by peripheral quantitative computed tomography and mechanical testing. No increase of Cu and Ni levels was recognized. Surface of the BMG showed no noticeable change, while Ti-6Al-4V alloy showed Ca and P deposition and 316L stainless steel showed surface irregularities and pitting by SEM observation. The stress strain index, maximum torque, torsional stiffness, and energy absorption values were larger for the BMG than those for Ti-6Al-4V alloy, although there was no significant difference. The Zr-based BMG can promote osteotomy healing as fast as Ti-6Al-4V alloy, with the possible advantage of the Zr-based BMG that bone bonding is less likely, allowing easier nail removal compared with Ti-6Al-4V alloy. The Zr-based BMG is promising for the use in osteosynthetic devices that are eventually removed.

Fatigue property of a bioabsorbable magnesium alloy with a hydroxyapatite coating formed by a chemical solution deposition.

A hydroxyapatite (HAp) coating was directly formed on an extruded AZ31 magnesium alloy by a single-step chemical solution deposition. The HAp coating consists of an outer porous HAp layer, an inner continuous HAp layer, and a thin intermediate MgO layer, and the inner HAp and MgO layers are composed of nanocrystals. Tensile and fatigue tests were performed on the HAp-coated AZ31 in air. The HAp coating microscopically showed neither crack nor detachment at 5% static elongation (1.5% residual strain). With further elongation under tensile stress, cracks were formed perpendicularly to the tensile direction, and fragments of the coating detached with a fracture inside the inner continuous HAp layer. The fatigue strengths at 10(7) cycles (fatigue limit) of HAp-coated and mechanically polished AZ31 were ca. 80 MPa and ca. 90 MPa, respectively. The slight decrease in the fatigue limit with the HAp coating is attributed to small pits with a depth of ca. 10 µm formed on the substrate during the HAp-coating treatment. The HAp coating remained on the specimen without cracks after 10(7) cycles at the fatigue limit, which provides ca. 3% cyclic elongation.

Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid.

Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS) with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-)). For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR)) with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR) although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR) both in air and in PBS(-). A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR) in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR). The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.

Preparation and biological evaluation of hydroxyapatite-coated nickel-free high-nitrogen stainless steel.

Calcium phosphate was formed on nickel-free high-nitrogen stainless steel (HNS) by chemical solution deposition. The calcium phosphate deposition was enhanced by glutamic acid covalently immobilized on the surface of HNS with trisuccinimidyl citrate as a linker. X-ray diffraction patterns and Fourier transform infrared spectra showed that the material deposited on glutamic acid-immobilized HNS within 24 h was low-crystallinity calcium-deficient carbonate-containing hydroxyapatite (HAp). The biological activity of the resulting HAp-coated HNS was investigated by using a human osteoblast-like MG-63 cell culture. The HAp-coated HNS stimulated the alkaline-phosphate activity of the MG-63 culture after 7 days. Therefore, HAp-coated HNS is suitable for orthopedic devices and soft tissue adhesion materials.

Electrochemical study of Type 304 and 316L stainless steels in simulated body fluids and cell cultures.

The electrochemical corrosion behaviour of Type 304 and 316L stainless steels was studied in Hanks' solution, Eagle's minimum essential medium (MEM), serum containing medium (MEM with 10% of fetal bovine serum) without cells, and serum containing medium with cells over a 1-week period. Polarization resistance measurements indicated that the stainless steels were resistant to Hanks' and MEM solutions. Type 304 was more susceptible to pitting corrosion than Type 316L in Hanks' and MEM solutions. The uniform corrosion resistance of stainless steels, determined by R(p), was lower in culturing medium than in Hanks' and MEM. The low corrosion resistance was due to surface passive film with less protective to reveal high anodic dissolution rate. When cells were present, the initial corrosion resistance was low, but gradually increased after 3 days, consistent with the trend of cell coverage. The presence of cells was found to suppress the cathodic reaction, that is, oxygen reduction, and increase the uniform corrosion resistance as a consequence. On the other hand, both Type 304 and 316L stainless steels became more susceptible to pitting corrosion when they were covered with cells.

Electrochemical properties of 316L stainless steel with culturing L929 fibroblasts.

Potentiodynamic polarization and impedance tests were carried out on 316L stainless steel with culturing murine fibroblast L929 cells to elucidate the corrosion behaviour of 316L steel with L929 cells and to understand the electrochemical interface between 316L steel and cells, respectively. Potential step test was carried out on 316L steel with type I collagen coating and culturing L929 cells to compare the effects of collagen and L929 cells. The open-circuit potential of 316L steel slightly shifted in a negative manner and passive current density increased with cells, indicating a decrease in the protective ability of passive oxide film. The pitting potential decreased with cells, indicating a decrease in the pitting corrosion resistance. In addition, a decrease in diffusivity at the interface was indicated from the decrease in the cathodic current density and the increase in the diffusion resistance parameter in the impedance test. The anodic peak current in the potential step test decreased with cells and collagen. Consequently, the corrosion resistance of 316L steel decreases with L929 cells. In addition, collagen coating would provide an environment for anodic reaction similar to that with culturing cells.

Microstructure and corrosion behaviour in biological environments of the new forged low-Ni Co-Cr-Mo alloys.

Corrosion behaviour and microstructure of developed low-Ni Co-29Cr-(6, 8)Mo (mass%) alloys and a conventional Co-29Cr-6Mo-1Ni alloy (ASTM F75-92) were investigated in saline solution (saline), Hanks' solution (Hanks), and cell culture medium (E-MEM + FBS). The forging ratios of the Co-29Cr-6Mo alloy were 50% and 88% and that of the Co-29Cr-8Mo alloy was 88%. Ni content in the air-formed surface oxide film of the low-Ni alloys was under the detection limit of XPS. The passive current densities of the low-Ni alloys were of the same order of magnitude as that of the ASTM alloy in all the solutions. The passive current densities of all the alloys did not significantly change with the inorganic ions and the biomolecules. The anodic current densities in the secondary passive region of the low-Ni alloys were lower than that of the ASTM alloy in the E-MEM + FBS. Consequently, the low-Ni alloys are expected to show as high corrosion resistance as the ASTM alloy. On the other hand, the passive current density of the Co-29Cr-6Mo alloy with a forging ratio of 50% was slightly lower than that with a forging ratio of 88% in the saline. The refining of grains by further forging causes the increase in the passive current density of the low-Ni alloy.

Composition of surface oxide film of titanium with culturing murine fibroblasts L929.

Changes in the composition of surface oxide film on titanium specimens in the presence of amino acids, serum proteins, and cells were characterized using X-ray photoelectron spectroscopy. The surface oxide film on titanium formed in the air is so protective that the further oxidation of titanium is prevented in various circumstances. During immersion of the specimen in Hanks' solution, Eagle's minimum essential medium (MEM), and MEM with the addition of fetal bovine serum (MEM+FBS), calcium phosphate precipitated, causing the increase in thickness of the surface oxide film. Calcium phosphate was also precipitated with culturing murine fibroblast L929, but the amount of the calcium phosphate was smaller than those in Hanks' solution, MEM, and MEM+FBS. The relative concentration ratio of calcium to phosphorous, [Ca]/[P], increased with proteins charging negatively, while the ratio decreased with the cells whose extracellular matrix charging positively. In addition, sulfur precipitated as S(0) and/or S(2-) only with culturing the cells. Sulfate ions in the MEM+FBS are reduced at the interface between titanium and the solution with the existence of cells.