Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Regulating Degradation Behavior by Incorporating Mesoporous Silica for Mg Bone Implants Youwen Yang,†,‡,§ Xiaoning Guo,†,⊥ Chongxian He,§ Chengde Gao,§ and Cijun Shuai*,‡,§,∥ ‡
Jiangxi University of Science and Technology, Ganzhou 341000, China State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha 410083, China ⊥ The Second Xiangya Hospital, Central South University, Changsha 410011, China ∥ Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha 410008, China §
ABSTRACT: Magnesium (Mg) alloys are potential bone implant materials because of their natural biodegradability, good biocompatibility and suitable mechanical properties. However, the too rapid degradation in physiological environment has delayed their introduction for orthopedic applications to date. To improve the degradation behavior, mesoporous silica (MS) was incorporated into ZK60 (Mg-6Zn-0.5Zr, wt %) via selective laser melting technology. Results showed that MS homogeneously incorporated in Mg matrix with good bonding interface. MS was chemical inert against Mg and shifted the corrosion potential positively, indicating an enhanced corrosion resistance. Moreover, MS promoted the deposition of apatite on surface and formed a compact protection layer, thus effectively preventing the further corrosion of Mg matrix. As a result, the degradation rate was reduced by 57%, with MS containing up to 8 wt %. In addition, ZK60/8MS composite exhibited improved biocompatibility. It was suggested the ZK60/8MS composite with improved degradation behavior and good biocompatibility was a potential candidate biomaterial for the bone implants. KEYWORDS: mesoporous silica, ZK60, degradation behavior, biocompatibility, microstructure
■
INTRODUCTION Mg and its alloys have been proposed as promising bone implant materials because of their natural biodegradability, good biocompatibility, and suitable mechanical properties.1,2 Mg can degrade in the human body in the form of corrosion, thus avoiding the second surgery.3 Meanwhile, Mg is necessary element in human body with a daily intake of 250−300 mg and is naturally stored in the bones.4 Furthermore, Mg and its alloys possess similar Young’s modulus of elasticity (41−55 GPa) and density (1.7−1.9 g cm−3) to that of human bone (15−25 GPa and 1.8−2.1 g cm−3), which effectively prevents the stress shielding effect.5 Nevertheless, Mg alloys urgently demand an adjustable degradation to meet the requirement for bone repairing application, because their degradation rates are too rapid to provide enough time for bone healing (at least 12 weeks). The main reasons for the rapid degradation of Mg alloys can be ascribe to (1) the low corrosion potential (standard corrosion potential −2.37 V) makes it extremely easy to be corroded in the physiological environment;6 (2) the incompact corrosion product layer mainly consisted of Mg(OH)2 results in continuous corrosion.7 To address these issues, extensive efforts have been devoted to enhance the corrosion resistance of Mg by alloying with inert noble elements or surface modifications.8−11 However, most of the alloying elements have very limit solid solution in α-Mg and their intermetallic © XXXX American Chemical Society
compounds prefer to form negative galvanic corrosion against α-Mg. On the other hand, surface modification only offer a solution for delaying the start of corrosion and rapid corrosion usually takes place once the modified surface breaks down. Recently, mesoporous silica (MS) has received intensive attentions in bone tissue engineering because of its unique intrinsic structure with large specific surface area and pore volume.12,13 Previous studies revealed that increasing the specific surface area and pore volume significantly accelerated the kinetic process of apatite deposition.14,15 This motivates us to hypothesize that an enhanced apatite deposition rate may be achieved if MS was incorporated into Mg alloys. And the apatite layer covered on Mg matrix may offer more effective protection as compared with Mg(OH)2 layer. On the other hand, it is possible to enhance the surface passivation through compositing Mg alloys with electrochemical inactive MS. Furthermore, silicon is a necessary element in human body.16 It plays a key role in aiding the bone healing process, as well as the construction of the immune system.17 In this work, for the first time, MS was incorporated in Mg6Zn-0.5Zr (ZK60) alloy by using selective laser melting (SLM) technology. SLM was a typical additive manufacturing Received: January 5, 2018 Accepted: February 15, 2018 Published: February 15, 2018 A
DOI: 10.1021/acsbiomaterials.8b00020 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 1. Typical optical microstructures of the SLM processed ZK60 composited with different nominal MS contents. (a) ZK60, (b) ZK60/4MS, (c) ZK60/8MS, and (d) ZK60/12MS. As the addition amounts of MS reached 12 wt %, segregation occurred, resulting in the formation of pores in matrix. fabricated samples.8 Moreover, laser beam spot and hatch spacing had a significant effect on the accuracy of the fabricated samples.22 Therefore, it was important to get the optimal parameters. After a series of preliminary experiments, cubic samples (6 × 6 × 6 mm3) were fabricated using the SLM system with optimal processing parameters: laser power 120 W, spot size 50 μm, layer thickness 100 μm, hatch spacing 40 μm, and scan speed 20 mm•s−1. Ar gas was used to provide a protective atmosphere during SLM. The SLM processed Mg-based composites in this study had a nominal MS contents with 4, 8, or 12 wt %. These composites were designated as ZK60/4MS, ZK60/8MS, and ZK60/12MS, respectively, with increasing MS content. Microstructural Characterizations. The SLM processed samples were polished with SiC abrasive paper and chemically etched using a picric acid solution. Then the microstructures were observed utilizing an optical microscope (Leica DM500, Germany) and a scanning electron microscopy (SEM, QUANTA FEG250, USA) equipped with energy dispersive spectroscopy (EDS, JSM-5910LV, Japan). The relative density of the SLM processed samples was investigated by measuring the porosity.23 The phase compositions were analyzed by an X-ray diffractometer (XRD, D8 Advance, Bruker AXS Inc., Germany) in a small angle range from 0 to 6° with a scan rate 2° min−1. Surface hydrophilicity of SLM processed ZK60/MS was investigated using a contact angle analyzer (DSA100, Germany). Electrochemical Tests. The electrochemical properties of SLM processed ZK60/MS in simulated body fluid (SBF, 37 °C, pH 7.4) were investigated using an electrochemical workstation (CHI604D, China). The SBF consisted of 8.035 g L−1 NaCl, 0.355 g L−1 NaHCO3, 0.225 g L−1 KCl, 0.231 g L−1 K2HPO4·3H2O, 0.311 g L−1 MgCl2·6H2O, and 0.292 g L−1 CaCl2. A flat cell and ZK60/MS samples were used as the standard setup and working electrode, respectively. While a platinum sheet and Ag/AgCl/in saturated KCl electrode were used as counter electrode and reference electrode,
technology, which could fabricate complicated parts that was difficult to produce using traditional manufacturing process. Meanwhile, SLM involved a rapid cooling rate over 1 × 105 K/s during solidification, thus preventing the growth of grains and reducing the composition segregation.18 And the finer grains and homogeneous microstructure were believed to be favorable for enhancing the mechanical properties and degradation resistance.19 ZK60 was selected basing on the considerations of its excellent mechanical strength and good cytocompatibility.20 The degradation behavior, microstructures evolution and in vitro cells responses of SLM processed Mg-based composites were studied in depth.
■
MATERIALS AND METHODS
Material and Sample Fabrication. The original materials included spherical ZK60 powder (30−60 μm, 99.9% purity) and the stick shaped MS (1−5 μm). ZK60 powder was purchased from Tangshan Weihao Materials Co., Ltd. (Tangshan, China). And MS with pore diameter of 5−10 nm and specific surface area of 550−600 m2/g was obtained from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). These two kinds of powder were mixed homogeneously using a ball mill at a rotation speed 260 rpm for 4 h. Ar gas was used during milling to avoid the oxidation. The homemade SLM system consisted of a fiber laser with a maximal power of 500 W and a spot diameter of 50 μm, a working platform and a computer system.21 The samples were fabricated by a layer-by-layer method. As a layer was deposited via high-energy laser melting, another layer was created just on top of the previous one. The SLM process parameters including laser powder and scanning rate determined the laser energy density. And too high a laser energy density caused the burning of Mg, whereas low laser energy caused the partial melting of the Mg powder, thus reducing the density of the B
DOI: 10.1021/acsbiomaterials.8b00020 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering respectively. An open circuit potential of ZK60/MS samples were achieved before recording the potential dynamic polarization curves. Three replicas were tested for each group. Immersion Tests. Immersion tests were performed in SBF (37 °C, pH 7.4) at a surface area to volume ratio of 0.1 cm2•mL−1 without changing the SBF. At specific immersion time, soaked specimens were removed from SBF and the corresponding ion concentrations (Ca, P) in SBF were determined utilizing inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Thermo Elemental, USA). Meanwhile, the pH values of the SBF were detected during immersion. Meanwhile, the corrosion surfaces were studied by SEM after soaking for 1 week. Weight loss method was used to quantitatively assess the corrosion rate. After immersion for 1 week, samples were removed from SBF. The weight losses were measured after removing the corrosion products. The corrosion rates were calculated by1 C = W /(DAt )
For SLM processed Mg-based parts, the densification rate was a key factor which influenced the degradation behavior and mechanical properties.26 Therefore, the relative density of SLM processed ZK60/MS composites was studied by measuring the porosity, with results depicted in Figure 2. SLM processed
(1)
Where C referred to the corrosion rate (mm year−1), W the weight loss of the samples (g), D the standard density (g mm−3), A the exposure area (mm2). and t the soaking time (year). In Vitro Cell Tests. Human osteoblast-like MG-63 cells (ATCC, Rockville, MD, USA) were used for the in vitro cell tests. DMEM supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin and 100 mg mL−1 streptomycin were used as culture medium. SLM processed ZK60/MS composites were soaked in culture medium for 3 days to prepare the extracts with the exposure area to solution volume ratio of 1.25 cm2 mL−1. An indirect contact method was used to study the cell viability. MG63 cells were seeded in a 96-well plate at a density of 5 × 103 cells/mL with 100 μL of culture medium for each well and then cultured for 24 h. The culture medium was then substituted with 100 μL of prepared extracts. After culture for 1, 3, and 5 days, respectively, 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well and incubated for 2 h at 37 °C. Afterward, the absorbance was determined at 450 nm by a paradigm detection platform (BECK MAN, CA). Direct Cell Adhesion Assay. MG-63 cells were directly seeded on the specimens, which were set in 24-well plates, with a density of 1 × 104 cells per well. Then the cells were cultured for 6 h, 1 and 3 days, respectively. Subsequently, the cells were rinsed gently with phosphate buffered solution (PBS, Grand Island Biological Co., USA) and then stained using Calcein-AM and Ethidium homodimer-1 reagents for 15 min. Afterward, the samples were rinsed gently with PBS twice, and then fixed on glass slides to observe the cells morphologies using a fluorescence microscopy (BX60, Olympus, Japan). Statistical Analysis. Each group of experiments was repeated at least three times. And the obtained data were expressed as the mean ± standard error. Student’s t test was utilized to determine the level of significance. Differences were considered significant with a p-value
