P Doped Silk Fibroin Films on Mg-1Ca Alloy with

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Characterization, Synthesis, and Modifications

Biomimetic Ca, Sr/P Doped Silk Fibroin Films on Mg-1Ca Alloy with Dramatic Corrosion Resistance and Osteogenic Activities Pan Xiong, Zhaojun Jia, Ming Li, Wenhao Zhou, Jianglong Yan, Yuanhao Wu, Yan Cheng, and Yufeng Zheng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00787 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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ACS Biomaterials Science & Engineering

Biomimetic Ca, Sr/P Doped Silk Fibroin Films on Mg-1Ca Alloy with Dramatic Corrosion Resistance and Osteogenic Activities Pan Xiong†, Zhaojun Jia†, Ming Li‡, Wenhao Zhou†, JiangLong Yan†, Yuanhao Wu†, Yan Cheng**, †

, Yufeng Zheng*,§

†

Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China

‡

China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University,

Beijing, 100053, China §

Department of Materials Science and Engineering, College of Engineering, Peking University,

Beijing 100871, China

（Manuscript Submitted to ACS Biomaterials Science & Engineering））

*Corresponding author E-mail: [email protected] **Co-Corresponding author E-mail: [email protected]

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ABSTRACT: Magnesium and its alloys have emerged as one of the most promising biodegradable metals for temporary bone implants, but challenges remain in controlling their corrosion and biocompatibility and endowing them with bioactivity and osteogenic functionality. Herein we presented newly developed bioactive Ca, Sr/P contained silk fibroin films (the Ca, Sr/P-silk) on top of Mg-1Ca alloy to simultaneously improve the corrosion resistance, osteocompatibility and osteogenic activities important in maintaining mechanical integrity and stimulating bone formation, respectively. Briefly, extracellular matrix (ECM) mimicking Ca, Sr/P-silk fibroin films were constructed layer upon layer on fluoridized Mg-1Ca alloy via simple spinning assembly. The corrosion resistance property of different samples was studied in vitro by immersion experiments and electrochemistry measurements in Hanks’ solution, with the silk-coated ones showing over one order of magnitude increase in corrosion resistance compared to the uncoated. Particularly, the Ca, Sr/P-silk had the best anticorrosion performance, presumably due to better retaining of the β-sheet silk conformation and ion-induced structural conversion from random coils to silk I and α-helices. Furthermore, the preliminary study of the corrosion behavior of the Ca, Sr/P-silk was confirmed the availability of the films for corrosion resistance improvement. The osteocompatibility and osteogenic activities were evaluated by the multiple osteoblast (MC3T3-E1) responses, i.e. proliferation, adherence, spreading, and differentiation in vitro. The Ca, Sr/P-silk exhibited the optimal osteogenic activity among all experimental groups. These preliminary results comprehensively confirmed the validity of the coating strategy and they implicated the great potential of the modified Mg alloys as degradable bone implants. KEYWORDS: Biomedical Mg alloy, surface modification, silk fibroin films, Ca, Sr/P, degradation, corrosion resistance, biocompatibility, osteogenesis


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1. Introduction With unique physicochemical properties, biodegradability and biocompatibility, magnesium and its alloys have drawn increasing research attention, especially in the field of cardiovascular and orthopedic biomaterials1-7. It is suggested that as biodegradable bone implant materials, magnesium and its alloys should remain their mechanical integrity over a period of 12–18 weeks to allow bone healing1, 8. However, the overwhelmingly high corrosion rate in body fluids in contrast to the tissue healing rate can lead to rapid deterioration of the mechanical integrity, greatly limiting their clinical application9. In addition, heavy corrosion results in local alkalosis and continuing release of copious hydrogen that would separate implants from host tissue layers6, 8

. Mg-1Ca alloy is one of the newly-designed promising magnesium alloys. It displays excellent

mechanical property and beneficial effects on bone healing with co-released Mg and Ca ions, but on the other hand, poor corrosion resistance8. Thus, improving the corrosion resistance of Mg-1Ca alloy is urgent and of foremost importance. In this end, a number of surface modification methods have been developed10-11, among which the fluoride treatment12-14 and polymeric coatings15-17 are most common. However, the fluoride treated surfaces tend to possess undesired cracks, thus needing further handling18. Despite their potential to load functional drugs and biomolecules, polymeric coatings are generally porous, and the bio-safety of both the polymers and their degradation products is still under debate19. Silk fibroin is a natural protein extracted from Bombyx mori silkworms20, which has excellent mechanical properties21, good biocompatibility22, absent immunogenicity22, limited bacterial adhesion23 and controllable biodegradability22-24. It owns the unique characteristic with the variable second structures including random coils, α-helices, β-sheets and β-turns. The transition



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among the second structure is thought to be largely brought by extensional shearing, although variations in pH, metallic ion contents and water content are thought to make a contribution as well25. Silk fibroin films have been researched for many years, in comparison to other polymeric films researched in biomedical materials, and they show better mechanical property than collagen, slighter foreign body response than the synthetic polymers Poly (3-hydroxybutyrate) (PHB), poly(caprolactone), polyamide and PLA22, 26. Notably, the particular second structure of silk fibroin endow the films with tunable mechanical properties, biocompatibility and biodegradation rate27. As we know, natural bone is a composite of mineral phase (mainly the apatite) and an organic matrix28. Thus, the fabrication of inorganic–organic hybrid films on bone implants sounds appealing. Especially, silk fibroin has an intrinsic resemblance to the arranged collagen in bone matrix that builds a 3D framework where minerals are deposited29. Through biomimetic mineralization30, mechanochemical route31 or co-precipitation methods32, silk fibroin can be easily complexed with calcium phosphates to form organic/inorganic hybrids, which have excellent mechanical properties and osteogenesis activity33-34 and improved biocompatibility and bioactivity35-36. On the other hand, strontium is one of the trace elements in natural bone. It is confirmed that Sr addition into calcium phosphate biomaterials is shown to facilitate the attachment and proliferation of osteo-precursor cells37-38. Ke Yang et al found that coating Mg-Sr alloy with a Sr–Ca–P containing MAO film encouraged new bone formation in vivo39. Up to now, strontium-doped Ca/P-silk fibroin materials have been rarely reported regardless of the numerous studies on Ca/P-silk fibroin films. In this work, we are the first to demonstrate a bioactive ECM-like films on Mg-1Ca alloy by facile spinning assembly of Ca, Sr/P inorganic salts/silk fibroin hybrids, as generated from in situ


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co-precipitation, aiming to improve the corrosion resistance and surface bioactivity and osteogenic capacity of the substrate. The fabricate process is illustrated in Figure 1. The fluoride treatment served as a pretreatment, which was generally used to improve film/substrate adhesion and allow for further functionalization17. Notably, the addition of Ca, Sr/P components into silk fibroin can aid in maintaining appropriate conformation of β-sheets and reducing the number of random coils, which in return might enhance the corrosion resistance and improve the biocompatibility and bioactivity of the surface. This work presents a new simple and efficient method for the surface modification of biodegradable magnesium alloys and it provides some new insights into the physicochemical and biological properties of silk fibroin protein materials for biomedical applications. 2. Materials and Methods The preparation of extruded Mg-1Ca(wt.%) alloy samples was detailed in our previous work18. The chemicals, including NaOH, HF, Ca(NO3)2, Sr(NO3)2, and K2HPO4 are purchased from Beijing Chemical Reagent Co., Ltd, China. All other reagents, unless otherwise specified, were in the reagent grade. 2.1. Specimens preparation The extruded Mg-1Ca (wt.%) alloy with a diameter of 12 mm was cut into 2 mm thick slices and then ground by SiC paper up to 2000 grit, subsequently dipped into acetone, ethanol and deionized water with ultrasonic washer for 10 minutes respectively, and then dried by cold-air blowing. 2.2. Fluoride pretreatment



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A two-step method was used to complete the fluoride pretreatment, the basic reaction was shown as follows: + 2 =

(1)

+ 2 = + 2

(2)

At the first step, all samples were immersed in the boiling NaOH solution (200 g/L) for 3 h, the volume of NaOH solution was related to the number of samples with the rate of 10 ml/cm2. To react uniformly, the solution was stirred. After the reaction completed, samples were rinsed with deionized water for three times and dried with cold air. 10 mL plastic centrifuge tubes were filled with 9 mL HF. Each specimen was immersed into one centrifuge tube. Then, the tubes were incubated in an oven at 60 °C for 6 h. Finally, all samples were dried at room temperature. 2.3. Preparation of silk fibroin films Silk fibroin solution was extracted as the previous study40. Bombyx mori. cocoons were cut into pieces and boiled in 2 L 0.02 M Na2CO3 solution for 30 min to remove the sericin fibroin, which was reported to cause the inflammatory41. The degummed silk was rinsed with deionized water for three times and dried in air overnight. The dried silk fibroin was solubilized in 9.3 M LiBr solution at 60 °C for 4 hours and dialyzed against water for 3 days. The final concentration of silk fibroin solution was 8 w/v%. The silk fibroin solution was diluted to 2 w/v%. A certain amount of Ca(NO3)2 (0.1mol/L), Sr(NO3)2 (0.1mol/L) and K2HPO4 (0.1mol/L) were added into the silk fibroin solution, and all


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the solutions were buffered to pH=10 with 1 mol/L NaOH. The composition of solutions used for films were listed in Table 1. To get better mechanical properties, PEG200 was also added into the solution, according to the previous work42. A Model KW-4A spin coater (Si-you-yen®, Beijing, China) was employed with a low rotation speed of 1500 rpm for 15 s as well as a high rotation speed of 3500 rpm for 10 s at room temperature. 50 µL of each silk fibroin solution as above was added to the specimen for a cycle, and there were 5 repeated times for each side of the samples. All the spin-coated samples were dried in a vacuum oven at room temperature. 2.4. Material characterization The surface and cross-section morphologies of the samples were investigated by S-4800 field emission scanning electron microscope (FE-SEM; Hitachi) equipped with an energy-dispersive spectrometer (EDS) attachment. The chemical states of all groups were determined by X-ray photoelectron spectroscopy (XPS; AXIS Ultra, Kratos) with Al Kα irradiation (hυ = 1486.71 eV). The functional groups of the silk fibroin films were recognized by Attenuated Total Reflectance-Fourier Transform Infrared analysis (ATR-FTIR, Thermo Fisher). The spectra were recorded from 4000 cm-1 to 400 cm-1. Peak Fit software was used to fit and calculate the characteristic peak of functional groups43. The static contact angle of silk fibroin-coated samples was measured using a contact angle meter equipped with a high-resolution camera (SL200B, Kino, USA) at room temperature (25°C). The static contact angle was measured by dropwise addition of distilled water (2 mL) onto the sample surface. Each sample was measured in triplicate. 2.5. In vitro corrosion behavior studies



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2.5.1. Immersion experiments The immersion tests were carried out in Hanks’ solution (NaCl 8.00 g/L, KCl 0.40 g/L, CaCl2 0.14 g/L, NaHCO3 0.35 g/L, MgSO4·7H2O 0.20 g/L, Na2HPO4·12H2O, 0.12 g/L, KH2PO4 0.06 g/L, pH = 7.4) at 37 °C according to ASTM-G31-72. The pH value and hydrogen evolution rate of the solution were monitored during the immersion tests. Three parallel samples were taken in every test. After being immersed for 10 and 30 days, respectively, samples were taken out of the solution, and rinsed with deionized water, then dried in air. The changes of surface morphologies before and after immersion for 10 and 30 days were checked by SEM. 2.5.2. Electrochemical measurements Electrochemical measurements were carried out in a standard three-electrode cell in Hanks’ solution at 37 °C using an electrochemistry workstation an electrochemistry workstation (PGSTAT 302N, Metrohm Autolab). The samples were served as the working electrode while a saturated calomel electrode (SCE) and a platinum piece were adopted as the reference and counter electrodes respectively. The exposed area of the working electrode to the electrolyte was 0.41 cm2. The electrochemical impedance spectroscopy (EIS) measurement was done with a scanning frequency range from 105 to 10-2 Hz with a single amplitude of 10 mV. EIS spectra were shown by Nyquist plots, and were quantitatively simulated using corresponding equivalent circuits. Potentiodynamic polarization scans were performed at a scanning rate of 1 mV/s, and the initial potential was 500 mV below the open circuit potential (OCP). An average of three measurements was taken for each group. 2.6. Cytocompatibility


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2.6.1. Cell culture and seeding Mouse osteoblast-like cells (MC3T3-E1) were used for the in vitro biocompatibility measurements. Cells were cultured in α-MEM (minimum essential medium alpha), which contained 10% FBS (fetal bovine serum, HyClone, Beijing), 1% penicillin and streptomycin (HyClone, Beijing), in a humidified incubator with 5% CO2 at 37 °C. All samples were sterilized in UV radiation for at least 1 h for each side. In the direct cell adhesion, samples were settled in 24-well plates (TCPS; Corning, USA), and 500µL cell suspensions were seeded in each well at a cell density of 5 x 104 /ml. In the indirect cell experiments, extracts of the samples were used. Each specimen was immersed in non-FBS α-MEM for 3 days with a ratio of 1.25 mL/cm2 according to ISO10993-12 to obtain the extracts. The extracts were preserved at 4 °C prior to the experiments. The seeded cells were cultured in 96-well plates (2000 cells per well), 48-well plates (4000 cells per well), 24-well plates (25000 cells per well) respectively for 24 h to allow the attachment of cells. Then, the medium was replaced by extracts with equal volume. 2.6.2. Cell adhesion Cells were seeded on the samples directly. After culturing 24 h, the samples were rinsed with PBS for three times and then immersed in 2.5% glutaraldehyde solution for 1 h at room temperature. The samples were then dehydrated in a gradient ethanol/DI water (the concentration of ethanol was 50, 60, 70, 80, 90, 95 and 100%) and each concentration for 10 min. The morphologies of the cells adhered to the surfaces of samples were observed using SEM. 2.6.3. Cell proliferation assay



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Cell viability was evaluated with released extracts employing a mitochondrial activity-based cell counting kit (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions. The seeded cells (2000 cells per well) were cultured in 96-well plates. In the meantime, 100 µl of α-MEM alone used as the negative control and 100 µL α-MEM containing 10% DMSO (dimethyl sulfoxide) used as the positive control. Cells were cultured for 3 and 5 days to measurements. At scheduled time points, the medium was replaced by 100 µL of fresh α-MEM with 10% CCK-8 solution and incubated for another 2 h to generate formazan. The spectrophotometric absorbance ([A]) was determined at 450 nm on a microplate spectrophotometer (Bio-Rad, USA). The cell viability was calculated as follows: !"#$#%!

%cell viability = &!'$#%!"#$#%!( ×100%

(3)

2.6.4. Live/dead assay Cells were cultured in 48 well plates for 3 and 5 days with extracts respectively and subjected to live/dead assay. Briefly, cells were washed with PBS for several times and then incubated with certain amounts of fluorescent dye (2 mM Calcein AM and 4mM PI, Live/Dead Cell Stains, Dojindo, Japan) for 20 min in the humidified incubator. The cells were then rinsed twice using PBS, and visualized using LSCM (laser scanning confocal microscope, Nikon ALR-SI; identical apparatus throughout the experiments). 2.6.5. Cytoskeleton and cell spreading Sterilized glass plates (φ=10mm) were put in 24-well plates and the cells were cultured on the plates with the extracts for 12 h and 24 h, respectively. Cells were then washed with PBS, fixed using 4% (w/v) paraformaldehyde for 10 min and permeabilized with 0.1% (v/v) Triton X-100


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(Sigma) for 7 min. Following this, 1.0% (v/v) FITC-phalloidin (Sigma) was used to stain the cellular actin for 30 min and 1 mg/mL DAPI (Sigma) was used to stain the nuclei for 5 min. Cells were washed for many times and imaged by LSCM. 2.6.6. Osteogenic differentiation studies Alkaline phosphatase (ALP) activity was measured to evaluate the osteogenic differentiation by culturing cells with extracts for 7 and 14 days. Cells were lysed in 1% Triton X-100 using standard freeze thaw cycles. The ALP activity in the lysis was probed spectrophotometrically by measuring the colorimetrical production of p-nitrophenol (p-NP) via p-nitrophenyl phosphate (p-NPP)/endogenous ALP enzymatic reaction, as per manufacturer instructions (Jiancheng, Nanjing, China). The results were normalized against the total intracellular protein content measured by the Micro BCA protein kit and expressed as mM production of p-NP by each gram of protein (mM p-NP/gprot). α-MEM alone was added per well into TCPS as the control, and the cell differentiation activity was calculated with respect to the control as follows: 12 !

Cell differentiation activity % = 123"&$4" ( ×100%

(4)

2.7. Statistical analysis All data were expressed as the mean ± standard deviation with n=3 and analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests using SPSS 19.0. The p

P Doped Silk Fibroin Films on Mg-1Ca Alloy with

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