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Metal-phenolic networks as a promising platform for pH-controlled release of bioactive divalent metal ions
Applied Surface Science 511 (2020) 145569
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Full Length Article
Metal-phenolic networks as a promising platform for pH-controlled release of bioactive divalent metal ions ⁎
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Kui Xua, , Mi Zhoua, Ming Lib, Weizhen Chenc, Yabin Zhua, , Kaiyong Caid a
Biomedical Engineering Research Center, Medical School of Ningbo University, Ningbo, Zhejiang 315211, PR China Joint Surgery Department, Ningbo No.6 Hospital, Ningbo, Zhejiang 315040, PR China c Center of Clinical Laboratory & the Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, PR China d Key Laboratory of Biorheological Science and Technology, Ministry of Education College of Bioengineering, Chongqing University, Chongqing 400044, PR China b
A R T I C LE I N FO
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Keywords: Titanium-based materials Metal-phenolic networks Tannic acid Bioactive divalent metal ions pH-controlled release
Titanium (Ti)-based implants have to undergo many severe ordeals under the complex in vivo microenvironments, which makes greater demands on the biological function of their surfaces. As a result, it is particularly important to develop the intelligent surfaces of Ti-based implants. In this study, metal-phenolic networks (MPNs) were prepared on pure Ti surfaces to achieve the pH-controlled release of bioactive divalent metal ions (e.g., Sr2+, Cu2+, Zn2+, and Mn2+). The practical potential of MPNs-based coatings in biomedical applications was further highlighted by the controllable release amount and species of metal ions. MPNs coated Ti surfaces displayed excellent biological properties, including enhanced protein adsorption, good biocompatibility as well as the promoted adhesion and proliferation of mesenchymal stem cells (MSCs). What’s more, a continuous release of bioactive divalent metal ions (e.g., Sr2+ and/or Cu2+) significantly accelerated the osteogenic differentiation of MSCs. The surface modification method based on MPNs provides an extremely feasible solution for preparing Ti-based implants that can adapt to different in vivo microenvironments.
1. Introduction As a common kind of implant materials in orthopedics clinic, titanium (Ti) and its alloys are always expected to take the complex in vivo microenvironments in their strides, which requires them to have a series of biological properties (e.g., osteogenesis, angiogenesis, anti-infection, anti-cancer, immunomodulatory, hemocompatibility) [1–4]. Up to now surface biofunctionalization has been considered to be the most feasible method to improve the bioactivity of their surfaces. For developing the satisfying Ti-based implants, in recent years, biofunctional coatings with the capacity of sustainedly releasing bioactive substances have received considerable attention due to concerns with flexibly deploying the released substances. Also, the coatings have an ability that turns the tunable dosage of released drug into reality under physiological conditions. For example, a high local concentration of antimicrobial agents released from the implants can effectively prevent the proliferation of bacteria in a short time after implantation [5], while bioactive ions have relatively low risk of systemic toxicity only at low concentration [6]. By contrast, making multifunctional surfaces
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possible is the greatest driving force for the rapid development of Tibased implants with the ability of sustainedly releasing bioactive substances. Among all the bioactive substances, metal ions (Mn+) have demonstrated tremendous potential in infection resistance, inflammation down-regulation, osteogenic differentiation acceleration, and therefore are commonly used to modify the surfaces of Ti-based implants [7–9]. Several methods have been used to fabricate controlled release coatings of metal ions, such as hydrothermal treatment [10], ion exchange [11], ion implantation [12], and so on. However, many of these strategies do not allow the modified surfaces to voluntarily adjust the release rate of metal ions in different in vivo microenvironments. The same biological indicators may be distinct in different parts of the body. For instance, there are significant differences on local pH value in different cell or tissue microenvironments. Weakly alkaline condition is conducive to the proliferation and differentiation of osteoblasts [13,14]. In the osteoporosis microenvironment, biological events of bone resorption caused by osteoclasts prompte a local pH of about 4.5 [15]. What′s more, the accumulation of acidic substances produced by bacteria
Corresponding author at: Medical School of Ningbo University, Zhejiang 315211, PR China. Corresponding author at: Medical School of Ningbo University, Zhejiang 315211, PR China. E-mail addresses: [email protected] (K. Xu), [email protected] (Y. Zhu).
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https://doi.org/10.1016/j.apsusc.2020.145569 Received 7 December 2019; Received in revised form 19 January 2020; Accepted 28 January 2020 Available online 03 February 2020 0169-4332/ © 2020 Elsevier B.V. All rights reserved.
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incorporating single and multiple metals [19,20], monometallic and multimetallic MPNs (Fig. 1, a) can enable Ti-based implants to perform a variety of expected biological functions.
creates an acidic biofilm microenvironment at local infection sites [16]. Therefore, pH-controlled release coatings of metal ions needs to be developed to meet the requirements of these in vivo microenvironments for Ti-based implants. For these reasons, in this work, we report a simple, low-cost, and rapid strategy to achieve the pH-controlled release of common divalent metal ions (e.g., Sr2+, Cu2+, Zn2+, and Mn2+). This approach is based on metal-phenolic networks (MPNs) that are formed by the reversible complexation between plant polyphenol (e.g., tannic acid (TA) used in this study) and metal ions. In 2013, MPNs were first discovered as a kind of simple, safe, and inexpensive coating material [17]. The most striking feature of MPNs-based coatings is that they are suitable for a wide variety of substrates. They can cover all sorts of nanoscale and micron-scale objects, such as gold nanoparticles, calcium carbonate and silica microparticles, and even bacteria. And this process is not affected by surface charge and surface topography. Importantly, Caruso and his co-workers have recently showed that TA, a natural polyphenol extracted from gallnut, may be coordinated to about 20 different metal ions to generate robust MPNs with the specialty of pH-responsive disassembly on the surfaces of many materials [18]. Due to the capacity of
2. Experimental section 2.1. Materials Northwest Institute for Nonferrous Metal Research (Xi’an, China) provided us with 15 mm diameter and 3 mm thickness Ti materials (medical grade, Ti > 99.5%). Tannic acid (ACS reagent), 3-(N-morpholino) propanesulfonic acid (MOPS; ≥ 99.5%), strontium chloride hexahydrate (SrCl2·6H2O; ACS reagent, ≥ 99.0%), copper (II) sulfate pentahydrate (CuSO4·5H2O; ACS reagent, ≥ 98.0%), zinc chloride (ZnCl2; ACS reagent, ≥ 97.0%), calcium chloride (CaCl2; ACS reagent, ≥ 96.0%), magnesium chloride hexahydrate (MgCl2·6H2O; ACS reagent, ≥ 99.0%), Manganese (II) sulfate monohydrate (MnSO4·H2O; ACS reagent, ≥ 98.0%), sodium dodecyl sulfate (SDS; ACS reagent, ≥ 99.0%), 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride, streptomycin, penicillin, 4% paraformaldehyde, fluorescein diacetate (FDA),
Fig. 1. Surface physical characteristics of monometallic and multimetallic MPNs: (a) illustration of the fabrication of MPNs on pure titanium surfaces; representative SEM images of Sr-MPNs (b & b1) and Sr/Cu-MPNs (c & c1) substrates; representative sectional SEM images of Sr-MPNs (d) and Sr/Cu-MPNs (e) samples; (f) the thickness of different MPNs (n = 10); (g) water contact angles of different MPNs (n = 5); (h) XPS survey spectra of Sr-MPNs and Sr/Cu-MPNs surfaces; (i) elemental composition for the surfaces of Sr-MPNs and Sr/Cu-MPNs plates by XPS. All the quantitative data were indicated as mean ± SD. 2
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propidium iodide (PI), bovine serum albumin (BSA), fibrinogen (FN), lglutamine (L-Gln), triton X-100, and sodium pyruvate were provided by Sigma Aldrich Co. (St. Louis, USA). Low glucose Dulbecco’s Modified Eagle’s Medium (L-DMEM), fetal bovine serum (FBS) and fluorescein isothiocyanate (FITC)-labeled phalloidin were bought from Invitrogen Co. (Carlsbad, USA). Beyotime Biotechnology Co. (Jiangsu, China) provided us with bicinchoninic acid (BCA) assay kit, cell counting kit-8 (CCK-8) kit, lactate dehydrogenase (LDH) cytotoxicity assay kit, 0.25% trypsin solution, BCIP/NBT alkaline phosphatase color development kit, and p-nitrophenyl phosphate assay kit. Picro Sirius Red solution was obtained from Aladdin Industrial Co. (Shanghai, China). Alizarin red sodium salt was bought from Alfa Aesar Co. (Tianjin, China). Phosphate buffer solution (PBS) was obtained from Dingguo Biotechnology Co. (Beijing, China). Other chemicals were supplied by Sinopharm Chemical Reagent Co. (Shanghai, China).
2.5. Release detection of metal ions At 37 °C, different MPNs coated Ti substrates (n = 3) were immersed into 1.0 mL PBS solution (pH = 4.5, 6.5, and 7.4). After incubation for different time (3, 6, 9, 12, 15, 18, 21, and 30 days), the solution containing metal ions was collected completely and the release amount of metal ions from MPNs modified Ti surfaces was measured by a inductively coupled plasma - optical emission spectroscopy (ICP-OES, Vista AX, USA).
2.6. Protein adhesion measurement BSA/PBS solution (200 μL, 1 mg/mL) was firstly added onto the Ti samples (n = 6). After incubation at 37 °C for 4 h, the plates were washed with PBS to remove the non-adherent proteins. Then, 2% SDS solution (200 μL) was pipetted onto the surfaces of different Ti foils. At 37 °C, the samples were incubated for 12 h. After that, determining the adsorbed protein content was performed with a BCA assay kit. The absorbance of the detected solution at λ = 570 nm was measured using a spectrophotometric microplate reader (Multiskan GO 1510, Thermo Fisher Scientific Inc., USA). The protein content on the TA or MPNs coated Ti substrates was normalized to that on the pure Ti plates. FN adsorption measurement was carried out with the same processes.
2.2. Pretreatment of Ti materials All Ti disks were firstly burnished with different grades of sandpapers (No. 400, 1000, 2000, and 7000), followed by ultrasonic cleaning in distilled water (18.25 MΩ·cm), acetone, and ethanol. After drying at 60 °C for 24 h, the foils underwent an alkali-heat treatment in 5 M sodium hydroxide (NaOH) aqueous solution at 80 °C for 24 h according to our previous studies [8,21]. Subsequently, the treated samples were immersed in hydrochloric acid (HCl) solution (100 mM) at 40 °C for 2 h [8,21]. After being calcinated at 600 °C for 2 h, the samples were washed and named as AHTi.
2.7. Cell culture In this study, mesenchymal stem cells (MSCs) were obtained from bone marrow of the tibia and femur of male Sprague Dawley rats with 4–5 weeks old (about 100 g). The cells were cultured at 37 °C under 5% CO2 atmosphere with L-DMEM supplementing with 10% FBS, 4 mM LGln, 0.1 mg/mL streptomycin, 110 mg/mL sodium pyruvate, and 100 U/mL penicillin. A subculture was performed when the confluence of cells reached about 90–95%. MSCs at the 3th or 4th passage were used for in vitro investigations. The process was carried out referring to the guidelines of the Institutional Animal Care and Use Committee of China. All rats were applied with approval of the Animal Ethics Committee of Ningbo University.
2.3. Preparation of MPNs on Ti substrates All solutions were freshly prepared when they would be used. Standard preparation process of monometallic MPNs on Ti substrates is described as follows: In a beaker, 5 mL 23.5 mM TA solution and 5 mL 37 mM metal solution (e.g., SrCl2, CuSO4, ZnCl2, or MnSO4 solution) were added, and then well mixed. The pH of the mixture was then raised by adding 10 mL MOPS-HCl buffer (10 mM, pH = 8.0). After that, the mixed solutions of TA and M2+ (TA-M2+ solutions) were centrifuged at 12000 rpm for 5 min to remove precipitates from the solutions. Subsequently, the pretreated Ti foils were soaked into the purified TA-M2+ solutions for the time required (20, 40, or 80 h). The MPNs coated Ti samples were washed to remove excess TA and metal ions. To strongly cross-link the metal ions with TA molecules, the obtained Ti foils were subsequently soaked into MOPS-HCl buffer (10 mM, pH = 8.0) for 30 min [22], and were named as M−MPNs (e.g., SrMPNs, Cu-MPNs, Zn-MPNs, and Mn-MPNs). The preparation of multimetallic MPNs was similar to the protocol described above. The initial concentration of TA was kept constant at 23.5 mM, while sum of metal ion initial concentration was also kept constant at 37 mM. For the 5Sr/ Cu-MPNs, Sr/Cu-MPNs, Sr/5Cu-MPNs, and Cu/Zn/Mn-MPNs preparations, a solution of strontium and copper ions ([Sr2+] : [Cu2+] = 5 : 1, [Sr2+] : [Cu2+] = 1 : 1, or [Sr2+] : [Cu2+] = 1 : 5) and a mixed solution containing copper, zinc, and manganese ions ([Cu2+] : [Zn2+] : [Mn2+] = 1 : 1 : 1) were prepared in advance, respectively. The control group of AHTi-TA substrates was fabricated by soaking AHTi substrates into 5.88 mM TA for the time required (20, 40, or 80 h).
2.8. Cell morphology observation The morphologies of MSCs cultured on pure Ti and TA or MPNs coated Ti surfaces was observed with a laser scanning confocal microscope (LSCM; TCS SP8, Leica, Germany). After seeding for 48 h (an initial density of 0.5 × 104 cells/cm2), the cells were gently rinsed, fixed, and permeabilized. Then, they were stained with FITC-labeled phalloidin (200 μL in 24-well plates, 5 U/mL) at 4 °C for 24 h. Subsequently, a counterstain was carried out with DAPI (200 μL in 24well plates, 10 mg/mL) for MSCs adhered to pure Ti and TA or MPNs coated Ti surfaces. After that, cell morphologies were obtained by LSCM. Cell area and number were calculated by analyzing LSCM images with Image Pro Plus 6.0 (IPP 6.0).
2.9. Determination of cell adhesion force 2.4. Observation of surface physical and chemical properties After culturing for 12 and 24 h, the floating cells were firstly removed and the adhesion cells were stained with DAPI. Subsequently, cell counting was carried out under an inverted fluorescence microscope (Olympus Co., Japan). After that, the Ti specimens were respectively centrifuged at 500 and 1000 rpm for 5 min. The fraction of adhesion cells was calculated according to the number of remained cells on the plates. According to our previous study [8], the mean cell adhesion force could be obtained on the strength of an assumption that the distribution of cell adhesion force obeys normal distribution.
Surface microtopographies of different Ti samples were investigated by scanning electron microscopy (SEM; SU70, Hitachi, Japan). Water contact angles (WCAs) of different Ti foils were measured by a model 200 video-based optical system (Future Scientific Co., Tai Wan, China). X-ray photoelectron spectroscopy (XPS; Phi-5000 Versa Probe, ULVCAPHI, USA) was used to analyze the surface chemistry of the samples. The peak fitting of high-resolution C1s and O1s was carried out with XPS Peak (version 4.1). 3
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Fig. 2. Ion release behaviours and SEM observation of MPNs pH-disassembly: cumulative release profiles of Sr2+ from Sr-MPNs (a) and Sr/Cu-MPNs (b) surfaces as well as Cu2+ from Sr/Cu-MPNs (c) foils after incubation in PBS (pH = 4.5, 6.5 or 7.4) at 37 °C for 30 days; release rates of Sr2+ from Sr-MPNs (a1) and Sr/Cu-MPNs (b1) plates as well as Cu2+ from Sr/Cu-MPNs (c1) substrates; SEM observation of MPNs pH-disassembly on the Sr-MPNs (d) and Sr/Cu-MPNs (e) surfaces. The quantitative data were indicated as mean ± SD for n = 3.
and 3 days. After collecting and centrifuging the culture media, supernatant was obtained for measuring LDH activity. In brief, the supernatant (120 μL) and LDH working liquid (60 μL) was orderly added into a well of 96-well plate. Subsequently, the plate was incubated at room temperature for 30 min, followed by LDH activity detection at λ = 490 nm. As for CCK-8 assay, the MSCs (1 × 104 cells/cm2 of initial density) were pretreated after incubation for 7 and 14 days (washing with PBS). 200 μL of new medium (no FBS) supplementing with 20 μL CCK-8 was added into each well and subsequently incubated in a cell incubator for another 30 min. The absorbance of obtained solution was measured at λ = 450 nm.
2.10. Cytocompatibility assays In this study, the cytocompatibility of Ti, AHTi, AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs were evaluated (n = 6). The investigation was firstly performed by FDA/PI staining. At 3 days after incubation, MSCs (1 × 104 cells/cm2 of initial density) were washed with PBS for 3 times, and then 500 mL PBS supplementing with 10 μL FDA (5 mg/mL) and 8 μL PI (2 mg/mL) were added to stain the living and dead cells. After that, the representative fluorescence images were obtained by LSCM. In addition, the cell number of dead cells was quantified by analyzing LSCM images with IPP 6.0. For quantitative evaluation, LDH activity and CCK-8 assays were carried out one after another. In 24-well plates, 2 × 104 MSCs were seeded, and then the plates were incubated for 1 4
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2.12. Qualitative and quantitative investigation of collagen I (Col I) secretion Firstly, MSCs (an initial density of 1 × 104 cells/cm2) were seeded onto different Ti surfaces in 24-well plates. Subsequently, when the cells were culturedat 37 °C for 7 and 14 days, the secreted Col I were stained with Picro Sirius Red solution. Representative optical photographs were recorded using a stereoscopic microscope. To quantify Col I secretion of MSCs, the stained cells were treated with 200 μL 0.1 M NaOH solution. Finally, the absorbance of 120 μL solution was measured at λ = 540 nm. 2.13. Qualitative and quantitative investigation of extracellular matrix (ECM) mineralization To stain the formed calcium deposition of MSCs cultured on different Ti substrates, 2 × 104 MSCs were seeded onto a Ti sample in 24well plates. After 14 and 21 days, the cells were rinsed and fixed orderly. Subsequently, ARS staining was performed with 0.1% ARS/TrisHCl solution (pH = 4.1). Representative optical photographs were recorded using a stereoscopic microscope. For quantification of mineralization, 300 μL of 10 v% acetic acid (HAc) was added into each well. After incubation for 30 min, the cells were scraped from the surfaces of Ti materials, followed by heating at 85 °C for 10 min and centrifuging at 12000 rpm for 15 min. The acid in the supernatant was then neutralized by 10 v% ammonium hydroxide (NH4OH). The quantitative ECM mineralization was obtained by measuring the absorbance of solution at λ = 405 nm. 2.14. Statistical analysis All quantitative data were indicated as mean ± standard deviation (SD). The statistical analysis was performed with GraphPad Prism 8.0 via one-way analysis of variance (ANOVA) and unpaired t-test. The confidence levels were set as 95%, 99%, and 99.9%. 3. Results and discussion Our results of SEM observation showed that when the final concentrations of TA and M2+ were constant (5.88 mM for TA and 9.25 mM for M2+), the surface topography and thickness of monometallic MPNs could be controlled by the immersion time. As shown in Figs. S1 and S2a, after being soaked in the mixed solutions of TA and M2+ for 20 h, the microtopography of AHTi surfaces was not covered completely by a thin layer (orange arrow in Fig. S2a). As the soaking time increased further (40 h), many TA-M2+ nanoparticles appeared on the surfaces (green arrow in Fig. S2a), while then the surfaces became smoother (80 h). The potential mechanisms of MPNs microtopographies’ change with the increase of soaking time were proposed as follows: (1) Microporous mesh-like structure of AHTi substrates caused the uneven deposition of TA-M2+ complex, leading to the form of aggregates. But with the increase of the thickness, the effect weakened gradually. (2) During the generation of MPNs on the surfaces, metal-phenolic nuclei of TA-M2+ nanoparticles were simultaneously formed and continuously grew into nanoparticles in the solution [23]. They would deposit, fuse, and grow into the MPNs [23]. (3) Due to the formation of MPNs and TA-M2+ nanoparticles, metal ions were consumed after soaking for a period of time, and then only TA was deposited uniformly on the MPNs surfaces (Fig. S2, b). Nevertheless, many cracks had appeared on the surfaces of MPNs coated Ti substrates, and even the underlying microstructure was clearly visible (green arrow in Fig. 1b & c). This should be attributed to the fact that free water molecules enclosed into the hydrophilic MPNs and the microporous meshlike structure in the process of MPNs formation were evaporated rapidly during the drying process, which brought about the non-uniform contraction of MPNs. The thickness of the resulting monometallic MPNs
Fig. 3. Protein and cell adhesion on the 5 kinds of Ti samples: the determination of adsorbed BSA (a)and FN (b) after incubation at 37 °C for 4 h; (c) representative LSCM images of MSCs; (d) the cell area (n = 60); (e) the cell number (n = 10); (f) the average cell adhesion forces (n = 6). The relative values were normalized to corresponding data of Ti substrates. All the quantitative datawere indicated as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
2.11. Qualitative and quantitative determination of alkaline phosphatase (ALP) For ALP qualitative determination, MSCs (1 × 104 cells/cm2 of initial density) were seeded onto different Ti surfaces in 24-well plates and then incubated in a cell incubator for 7 days. After being rinsed and fixed, the cells were stained with BCIP/NBT ALP color development kit. Representative images were obtained by a stereoscopic microscope (Leica, Germany). For ALP quantitative investigation, the cells grown on different Ti substrates were firstly incubated for 7 and 14 days. Then, the cells were lysed by 1% triton X-100. The cell lysate was obtained and centrifuged. After that, total intracellular protein and ALP activity were determined with BCA kit and p-nitrophenyl phosphate assaykit, respectively.
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Fig. 4. Cell compatibility evaluation of 5 kinds of Ti samples: (a) Live/Dead cell staining with FDA/PI for mesenchymal stem cells (MSCs) grown on the Ti, AHTi, AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs substrates, scale bar: 200 μm; (b) dead cell number (n = 10); (c) LDH activity (n = 5); (d) Cell viability (n = 5). ABS means absorbance. All the quantitative data were indicated as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
monometallic MPNs, two results could make a strong case that the four metal ions were successfully combined with TA to generate MPNs on the Ti plates: (1) There were significant differences on C and O signals between the AHTi-TA and MPNs coated Ti surfaces. Take the Sr-MPNs substrates as an example, compared with the AHTi-TA surfaces, the elemental content of C significantly increased to 62.10 at% after being soaked in the mixed solutions of TA and Sr2+ for 40 h (Fig. S9). Nevertheless, at 80 h after immersion, the content of C had almost no change (Fig. 1, i). The C1s spectra of AHTi-TA and Sr-MPNs surfaces could be fitted with 4 peaks assigned to C-C/C = C/C-H at 284.54 eV, C-O/C-OH at 286.17 eV, C = O at 288.57 eV, and O-C = O at 291.21 eV (Fig. S7 and S8a). By contrast, O1s spectra are more reflective of the coating process. The O1s spectrum detected from the AHTi-TA surfaces was fitted with three peaks assigned to HO-C at 529.60 eV, O-Ti at 531.50 eV, and O = C at 532.81 eV (Fig. S7) [18,25]. In the fitted O1s spectrum of Sr-MPNs surfaces, however, part of the HO-C peak shifted from 529.60 eV to 531.19 eV, mainly because of electron transfer from TA to Sr2+ [18]. (2) The characteristic peaks of metallic elements, including Sr3d, Cu2p, Zn2p, and Mn2p, were observed in the XPS survey spectra of their corresponding MPNs. After being soaked in the mixed solutions of TA and M2+ for 80 h, the contents of Sr2+, Cu2+, Zn2+, and Mn2+ on the modified Ti surfaces were 2.46, 2.71, 2.58, and 2.52 at%, respectively (Fig. 1i and Fig. S10). It was also important to note that, variation trend of Sr element content on the Sr-MPNs surfaces was similar to that of WCA as the soaking time increased from 20 to 80 h. The same reason was responsible for the result, that is, almost only TA was deposited uniformly for the last 40 h of the 80 h soaking process. As for Sr/Cu-MPNs substrates, the characteristic peaks of Sr and Cu appeared simultaneously in the XPS survey spectra (Fig. 1, h). However, the strontium-to-copper signal ratio (Sr/ Cu) of 0.781 was significantly lower than the concentration ratio of Sr2+ to Cu2+ (1 : 1) in the mixed solution of Sr2+, Cu2+, and TA (Fig. 1, i). So did the Zn/Cu/Mn-MPNs substrates (Fig. S12). We suspect this may be because different metal ions display different chelating abilities with TA [26]. Nevertheless, the content ratio of strontium and copper in
increased up to about 300 nm when the immersion time was increased to 80 h (Fig. 1d & f and Fig. S3). Interestingly, extremely similar results could be observed for the surface topography and thickness of multimetallic MPNs (Fig. 1c, c1, e & f and Figs. S4 & S5). The results also indicate that, for the multimetallic MPNs system of divalent metal ions, the interactions among different metal ions have little influence on the formation of MPNs. The change of surface wettability also confirmed the successful preparation of MPNs on Ti surfaces. Water contact angles (WCAs) of pure Ti and AHTi plates were similar to our previous studies (Fig. S1, c) [8,24]. After immersion in 5.88 mM TA solution for 80 h, the WCA of AHTi-TA substrates significantly went up to 12 ± 1.9° (Fig. S3, c). It was probably because the microporous structure of AHTi substrates was covered by TA (Fig. S2) and there were plenty of hydrophobic phenyl rings in TA molecules. Hydrophilic phenolic hydroxyl groups would be reduced once part of them complexed with metal ions, which directly led to a significant increase in the WCAs of MPNs coated Ti surfaces (Fig. 1g and Figs. S1 & S3–S5). It was worth noting that, however, the WCAs of MPNs coated Ti surfaces showed the characteristic of climbing up and then declining when the soaking time increased from 20 to 80 h (Fig. S3, c). In addition, there was no ionic effect on the MPNs wettability, and all the MPNs coated Ti surfaces (being soaked for 80 h) showed extremely similar wettability (Fig. 1g and Figs. S1 & S3–S5). The result was almost completely identical with the study of Yun et al [22]. We then explored the formation of MPNs on the Ti foils from a chemical point of view via XPS measurement. As shown in Fig. 1h and Fig. S6-S12, the characteristic peaks of carbon (C), argon (Ar), and oxygen (O) appeared in all the XPS survey spectra. In general, Ar signal was believed to have originated in the detection atmosphere [24]. On the surfaces of Ti and AHTi substrates, C signal was due to the hydrocarbon of environmental contamination [24]. The carbon-to-oxygen signal ratio (C/O) on the AHTi-TA and MPNs coated Ti surfaces was close to that of the theoretical value for TA (C/O = 1.65), indicating that TA or MPNs was successfully coated onto the AHTi surfaces. For 6
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Fig. 5. In vitro osteogenic differentiation: (a) ALP, Col I, and ECM mineralization staining of MSCs grown onto the Ti, AHTi, AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs surfaces after incubation for 7, 14, or 21 days (scale bar: 1 mm); quantitative evaluations of ALP activity (b), Col I secretion (c), and ECM mineralization (d). ABS means absorbance. All the quantitative data were indicated as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
also been observed on the surfaces of Sr/Cu-MPNs plates, indicating that there was nometal ion effecton the disassembly of multimetallic MPNs (Fig. 2, b & b1). Regardless of the conditions, Cu2+ exhibited extremely similar releasing behaviours to Sr2+ (Fig. 2, c & c1). We had also observed the microtopography of Sr-MPNs and Sr/Cu-MPNs surfaces after being soaked in PBS with different pH for 1 and 14 days. As shown in Fig. 2d and e, at 1 day after immersion, the degree of membrane dissociation varied significantly: In pH = 7.4 and 6.5 PBS, relatively intact MPNs could be observed, while only a thin layer remained in an acidic solution of pH = 4.5. At 14 days, there was stilla small area of continuous MPNs on the substrates soaked in pH = 7.4 PBS, whereas coatings treated by acidic solution had degraded completely. These results confirm the results of ion release behaviours well. All in all, the features described above of MPNs opportunely meet the requirements of different in vivo conditions for Ti-based implants: In normal bone tissue (pH = 7.4), the implants are asked to continuously stimulate osteogenesis-related cells for a long time to promote new bone formation; in weakly acidic bone resorption microenvironment
the MPNs could be adjusted by controlling the concentration ratio of Sr2+ to Cu2+ in the mixed solution of Sr2+, Cu2+, and TA (Fig. S11). In a word, we may regulate the amount and species of the introduced metal ions with ease and precision, which lays the foundation for obtaining multifunctional Ti-based implants. Fig. 2 displayed the release behaviours of Sr2+ and/or Cu2+ from Sr-MPNs and Sr/Cu-MPNs surfaces, which also reflected the pH-disassembly kinetics of the MPNs. Obviously, the stability of the investigated MPNs reduced gradually when the pH decreased from 7.4 to 4.5. At pH = 7.4, Sr2+ was released from Sr-MPNs substrates for up to 15 days (Fig. 2, a). During the first 3 days, the average rate of ion release reached a maximum of 1.1 μg/cm2/day (Fig. 2, a1). In pH = 6.5 PBS, the average release rate for the first three days increased dramatically to 1.8 μg/cm2/day (Fig. 2, a1), and strontium ions were released completely at the initial 7 days (Fig. 2, a). At a pH of 4.5, the MPNs became unstable and were rapidly disassembled for the first three days. Seventy-two hours later, it was difficult todetect Sr2+ in the recovered PBS (Fig. 2, a1). Almost identical release characteristics of Sr2+ had 7
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7 days, MSCs grown on the MPNs coated Ti substrates showed the highest cell viability when comparing with the other Ti plates (p < 0.01 or p < 0.001). Noteworthily, TA also promoted the proliferation of MSCs. Similar tendencies were observed at 14 days after incubation. The reason of TA or MPNs displaying a good biocompatibility may lie in the free radical scavenging capability of phenolic hydroxyl in TA molecules [31,32]. All the above results indicated the excellent biocompatibility of MPNs coated Ti substrates. As displayed in Fig. 5, after incubation for 4 or 7 days, the alkaline phosphatase (ALP) activity on the surfaces of MPNs coated Ti foils was significantly higher than that on the Ti, AHTi, and AHTi-TA substrates (p < 0.05 or p < 0.001), which was confirmed by the ALP staining (Fig. 5, a). In bone tissue, Col I is the predominant structural protein and is usually secreted in the later stages of osteogenic differentiation [8]. Herein, we could observe that whether at 2 or 3 weeks after culturing, MSCs grown on the Sr-MPNs and Sr/Cu-MPNs samples secreted more Col I than those cultured on the Ti, AHTi, and AHTi-TA substrates (p < 0.05, p < 0.01 or p < 0.001) (Fig. 5, a & c). Extremely similar results could be observed for the ECM mineralization of MSCs seeded on these 5 kinds of Ti surfaces (Fig. 5, a & d). Nevertheless, there was no significant effect of TA on the cell osteogenic differentiation of MSCs (Fig. 5). Taken together, the MPNs coated Ti substrates (i.e., Sr-MPNs and Sr/Cu-MPNs substrates) were confirmed topromote the expressions of ALP and Col I in MSCs, in turn accelerating their ECM mineralization.
(pH = 6.5), the accelerated ion release can rapidly inhibit osteoclast activity; in the infection sites (pH = 4.5), the faster ion release makes the bactericidal ions locally accumulate in high concentrations over a short period of time, playing a rapid bactericidal effect. Also, we had briefly evaluated the biological properties of MPNs coated Ti substrates (i.e., Sr-MPNs and Sr/Cu-MPNs substrates), including protein adhesion, cell adhesion, cytotoxicity, cell proliferation and osteogenic differentiation of MSCs. For protein adhesion investigation, BSA and FN were respectively used as anionic and cationic model proteins. Fig. 3a showed that although all samples were negatively charged, positively charged BSA could be effectively adsorbed on the surfaces of each substrate. Nevertheless, the amount of BSA adsorbed on the TA and MPNs coated Ti substrates was more than twice that on the Ti and AHTi surfaces. There was no significant difference of BSA adsorption among the AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs substrates. Similar results of FN adsorption were observed (Fig. 3, b). All of the above observation was also consistent with the work of Yang et al [27]. TA plays a crucial role in protein adsorption as same as dopamine coating of implants [28]. Phenyl rings and phenolic hydroxyls in TA molecules serve as the adhesion sites of hydrophobic and hydrophilic regions in protein molecules, respectively. It ensures minimal conformational changes in the adsorbed proteins and maintains their native structures, which facilitates the retention of their activity [29]. As shown in Fig. 3c, most of cells adhered to pure Ti surfaces displayed triangle-like morphology. Incomplete spreading state was more pronounced on the surfaces of AHTi substrates, even cells with reduced spreading and less actin filaments on the verge of apoptosis could be clearly observed (white arrow in Fig. 3c). Therefore, the cell area of MSCs grown on the AHTi surfaces was significantly smaller than that of cells on the pure Ti substrates (p < 0.001) (Fig. 3, d). Obviously, TA coatings greatly improved the living microenvironment of cells. The cells showed more pseudopodia and favourable spreading. On the surfaces of Sr-MPNs and Sr/Cu-MPNs substrates, MSCs exhibited better spreading and more cytoskeleton formation than those adhered to the others. Moreover, MPNs coatings significantly increased cell number of MSCs (p < 0.001) (Fig. 3, e). These phenomena maybe due to the positive stimulatory effect of released metal ions (Sr2+ and/or Cu2+) on the MSCs [8,11,24,30]. In addition, the average cell adhesion forces of MSCs adhered to the different Ti samples were detected to investigate the influences of TA or MPNs on the initial cell adhesion. At 12 h after seeding, the forces obtained from AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs substrates were 291.76 ± 43.46, 287.87 ± 44.24, and 284.38 ± 40.44 pN, which were significantly higher than those measured from pure Ti and AHTi surfaces (p < 0.001) (Fig. 3, f). Twelve hours later, the average cell adhesion forces of MSCs grown on these 3 kinds of Ti surfaces were dramatically increased to 446.06 ± 43.25, 453.39 ± 83.78, and 461.46 ± 46.97 pN (Fig. 3, f). The results indicated that TA or MPNs could effectively enhance cell anchorage. However, no effect of metal ions on cell adhesion was observed. Live/Dead cell staining was then applied to observe the living (green fluorescent) and dead cells (red fluorescent; white dotted circle in Fig. 4a). Compared to other substrates, more dead cells were observed on the AHTi surfaces. TA, especially MPNs, significantly improved the biocompatibility of the substrates. It could be seen that the number of dead cells decreased dramatically from 854.2 ± 266.8 cells/cm2 of AHTi surfaces to 229.3 ± 205.8 cells/cm2 of Sr-MPNs surfaces and 120.4 ± 95.4 cells/cm2 of Sr/Cu-MPNs surfaces (p < 0.001) (Fig. 4, b). Furthermore, we used a lactate dehydrogenase (LDH) assay to quantitatively evaluate the cytotoxicity of different Ti surfaces via measuring LDH released from damaged cells. As shown in Fig. 4c, the highest LDH activity was detected on the AHTi substrates after incubation for 1 or 3 days, which was significantly reduced by TA or MPNs (p < 0.05, p < 0.01, or p < 0.001). The proliferation of MSCs cultured on Ti, AHTi, AHTi-TA, and MPNs coated Ti surfaces were then evaluated by the CCK-8 method (Fig. 4, d). After seeding for
4. Conclusions In summary, in this study, we had successfully prepared MPNs coatings on the Ti-based implants surfaces to achieve the pH-controlled release of common divalent metal ions (e.g., Sr2+, Cu2+, Zn2+, and Mn2+). When the final concentrations of TA and M2+ were constant (5.88 mM for TA and 9.25 mM for M2+), the thickness of MPNs and the amount of introduced metal ions in MPNs showed a strong immersion time dependence. The highlight of this study lies in the pH-responsive disassembly of MPNs. In PBS, the release rate of metal ions increased rapidly as the pH decreased from 7.4 to 4.5. No metal ion effect was observed on the formation of MPNs and the release behaviours of metal ions. The evaluation of MPNs’ biological properties confirmed that MPNs coated Ti surfaces displayed excellent biocompatibility and could promote the adhesion and proliferation of MSCs. What’s more, a continuous release of bioactive divalent metal ions (Sr2+ and/or Cu2+) significantly accelerated the osteogenic differentiation of MSCs grown on the Sr-MPNs and Sr/Cu-MPNs surfaces. Therefore, the surface modification method based on MPNs provides an extremely feasible solution for preparing Ti-based implants that can adapt to different in vivo microenvironments. CRediT authorship contribution statement Kui Xu: Conceptualization, Methodology, Data curation, Software, Writing - original draft, Writing - review & editing, Supervision. Mi Zhou: Data curation, Writing - original draft. Ming Li: Writing - original draft. Weizhen Chen: Data curation, Software. Yabin Zhu: Writing - review & editing, Supervision. Kaiyong Cai: Writing - review & editing. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgments The work was financially sponsored by K.C. Wong Magna Fund in Ningbo University and Zhejiang Key Laboratory of Pathophysiology (No. 201911). K. Xu really appreciated the financial support from the Talent Introduction Fund of Ningbo University (Nos. 421700020, 8
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421709530, and 421804880). In addition, we would like to gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 81802133), the Project of Scientific Innovation Team of Ningbo (No. 2015B11050), and the Ningbo Natural Science Foundation (No. 2018A610261).
(2012) 8662–8670. [14] W. Liu, T. Wang, C. Yang, B.W. Darvell, J. Wu, K. Lin, et al., Alkaline biodegradable implants for osteoporotic bone defects-importance of microenvironment pH, Osteoporos. Int. 27 (2016) 93–104. [15] S.L. Teitelbaum, Bone resorption by osteoclasts, Science 289 (2000) 1504–1508. [16] T. Wang, C. Wang, S. Zhou, J.H. Xu, W. Jiang, L.H. Tan, et al., Nanovalves-based bacteria-triggered, self-defensive antibacterial coating: Using combination therapy, dual stimuli-responsiveness, and multiple release modes for treatment of implantassociated infections, Chem. Mater. 29 (2017) 8325–8337. [17] H. Ejima, J.J. Richardson, K. Liang, J.P. Best, M.P. van Koeverden, G.K. Such, et al., One-step assembly of coordination complexes for versatile film and particle engineering, Science 341 (6142) (2013) 154–157. [18] J. Guo, Y. Ping, H. Ejima, K. Alt, M. Meissner, J.J. Richardson, et al., Engineering multifunctional capsules through the assembly of metal-phenolic networks, Angew. Chem. Int. Ed. 53 (2014) 5546–5551. [19] H. Ejima, J.J. Richardson, F. Caruso, Metal-phenolic networks as a versatile platform to engineer nanomaterials and biointerfaces, Nano Today 12 (2017) 136–148. [20] G. Yun, J.J. Richardson, M. Biviano, F. Caruso, Tuning the mechanical behavior of metal-phenolic networks through building block composition, ACS Appl. Mater. Interfaces 11 (2019) 6404–6410. [21] K. Xu, X. Shen, W. Chen, C. Mu, C. Jiang, Y. Zhao, et al., Nanosheet-pore topographical titanium substrates: a biophysical regulator of the fate of mesenchymal stem cells, J. Mater. Chem. B 4 (2016) 1797–1810. [22] G. Yun, Q.A. Besford, S.T. Johnston, J.J. Richardson, S. Pan, M. Biviano, et al., Selfassembly of nano- to macroscopic metal-phenolic materials, Chem. Mater. 30 (2018) 5750–5758. [23] Q.Z. Zhang, S. Li, J. Chen, K. Xie, S. Pan, J.J. Richardson, et al., Oxidation-mediated kinetic strategies for engineering metal-phenolic networks, Angew. Chem. Int. Ed. 58 (2019) 12563–12568. [24] K. Xu, W. Chen, C. Mu, Y. Yu, K. Cai, Strontium folic acid derivative functionalized titanium surfaces for enhanced osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo, J. Mater. Chem. B 5 (2017) 6811–6826. [25] S.L. Gaw, G. Sakala, S. Nir, A. Saha, Z.J. Xu, P.S. Lee, et al., Rational design of amphiphilic peptides and its effect on antifouling performance, Biomacromolecules 19 (2018) 3620–3627. [26] W. Luo, G. Xiao, F. Tian, J.J. Richardson, Y. Wang, J. Zhou, et al., Engineering robust metal-phenolic network membranes for uranium extraction from seawater, Energy Environ. Sci. 12 (2019) 607–614. [27] L. Yang, L. Han, Q. Liu, Y. Xu, L. Jia, Galloyl groups-regulated fibrinogen conformation: understanding antiplatelet adhesion on tannic acid coating, Acta Biomater. 64 (2017) 187–199. [28] W.B. Tsai, W.T. Chen, H.W. Chien, W.H. Kuo, M.J. Wang, Poly(dopamine) coating of scaffolds for articular cartilage tissue engineering, Acta Biomater. 7 (2011) 4187–4194. [29] H. Lee, J. Rho, P.B. Messersmith, Facile conjugation of biomolecules onto surfacesvia mussel adhesive protein inspired coatings, Adv. Mater. 21 (2009) 431–434. [30] I. Burghardt, F. Luthen, C. Prinz, B. Kreikemeyer, C. Zietz, H. Neumann, et al., A dual function of copper in designing regenerative implants, Biomaterials 44 (2015) 36–44. [31] X. Zhang, M. Liu, X. Zhang, F. Deng, C. Zhou, J. Hui, et al., Interaction of tannic acid with carbon nanotubes: enhancement of dispersibility and biocompatibility, Toxicol. Res. 4 (2015) 160–168. [32] W. Chen, X. Shen, Y. Hu, K. Xu, Q. Ran, Y. Yu, et al., Surface functionalization of titanium implants with chitosan-catechol conjugate for suppression of ROS-induced cells damage and improvement of osteogenesis, Biomaterials 114 (2017) 82–96.
Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsusc.2020.145569. References [1] W. Chen, K. Xu, B. Tao, L. Dai, Y. Yu, C. Mu, et al., Multilayered coating of titanium implants promotes coupled osteogenesis and angiogenesis in vitro and in vivo, Acta Biomater. 74 (2018) 489–504. [2] D. Wang, F. Peng, J. Li, Y. Qiao, Q. Li, X. Liu, Butyrate-inserted Ni-Ti layered double hydroxide film for H2O2-mediated tumor and bacteria killing, Mater. Today 20 (2017) 238–257. [3] K. Gulati, S.M. Hamlet, S. Ivanovski, Tailoring the immuno-responsiveness of anodized nano-engineered titanium implants, J. Mater. Chem. B 6 (2018) 2677–2689. [4] K. Xu, K. Cai, Self-assembled phase-transited lysozyme/heparin coating on titanium surfaces for improved hemocompatibility, Mater. Lett. 247 (2019) 95–98. [5] Q. Chen, W. Li, O.M. Goudouri, Y.P. Ding, S. Cabanas-Polo, A.R. Boccaccini, Electrophoretic deposition of antibiotic loaded PHBV microsphere-alginate composite coating with controlled delivery potential, Colloid. Surface. B 130 (2015) 199–206. [6] D.M. Vasconcelos, S.G. Santos, M. Lamghari, M.A. Barbosa, The Two faces of metal ions: from implants rejection to tissue, Biomaterials 84 (2016) 262–275. [7] A. Cochis, S. Ferraris, R. Sorrentino, B. Azzimonti, C. Novara, F. Geobaldo, et al., Silver-doped keratin nanofibers preserve a titanium surface from biofilm contamination and favor soft-tissue healing, J. Mater. Chem. B 5 (2017) 8366–8377. [8] K. Xu, W. Chen, G. Fu, X. Mou, R. Hou, Y. Zhu, et al., In situ self-assembly of graphene oxide/polydopamine/Sr2+ nanosheets on titanium surfaces for enhanced osteogenic differentiation of mesenchymal stem cells, Carbon 142 (2019) 567–579. [9] T. Hu, H. Xu, C. Wang, H. Qin, Z. An, Magnesium enhances the chondrogenic differentiation of mesenchymal stem cells by inhibiting activated macrophage-induced inflammation, Sci. Rep. 8 (2018) 3406. [10] K. Kapat, P.P. Maity, A.P. Rameshbabu, P.K. Srivas, P. Majumdar, S. Dhara, Simultaneous hydrothermal bioactivation with nano-topographic modulation of porous titanium alloys towards enhanced osteogenic and antimicrobial responses, J. Mater. Chem. B 6 (2018) 2877–2893. [11] K. Xu, W. Chen, Y. Hu, X. Shen, G. Xu, Q. Ran, et al., Influence of strontium ions incorporated into nanosheet-pore topographical titanium substrates on osteogenic differentiation of mesenchymal stem cells in vitro and on osseointegration in vivo, J. Mater. Chem. B 4 (2016) 4549–4564. [12] B.S. Kim, J.S. Kim, Y.M. Park, B.Y. Choi, J. Lee, Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell, Mater. Sci. Eng. C Mater. Biol. Appl. 33 (2013) 1554–1560. [13] Y. Shen, W. Liu, C. Wen, H. Pan, T. Wang, B.W. Darvell, et al., Bone regeneration: importance of local pH-strontium-doped borosilicate scaffold, J. Mater. Chem. 22
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Full Length Article
Metal-phenolic networks as a promising platform for pH-controlled release of bioactive divalent metal ions ⁎
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Kui Xua, , Mi Zhoua, Ming Lib, Weizhen Chenc, Yabin Zhua, , Kaiyong Caid a
Biomedical Engineering Research Center, Medical School of Ningbo University, Ningbo, Zhejiang 315211, PR China Joint Surgery Department, Ningbo No.6 Hospital, Ningbo, Zhejiang 315040, PR China c Center of Clinical Laboratory & the Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, PR China d Key Laboratory of Biorheological Science and Technology, Ministry of Education College of Bioengineering, Chongqing University, Chongqing 400044, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Titanium-based materials Metal-phenolic networks Tannic acid Bioactive divalent metal ions pH-controlled release
Titanium (Ti)-based implants have to undergo many severe ordeals under the complex in vivo microenvironments, which makes greater demands on the biological function of their surfaces. As a result, it is particularly important to develop the intelligent surfaces of Ti-based implants. In this study, metal-phenolic networks (MPNs) were prepared on pure Ti surfaces to achieve the pH-controlled release of bioactive divalent metal ions (e.g., Sr2+, Cu2+, Zn2+, and Mn2+). The practical potential of MPNs-based coatings in biomedical applications was further highlighted by the controllable release amount and species of metal ions. MPNs coated Ti surfaces displayed excellent biological properties, including enhanced protein adsorption, good biocompatibility as well as the promoted adhesion and proliferation of mesenchymal stem cells (MSCs). What’s more, a continuous release of bioactive divalent metal ions (e.g., Sr2+ and/or Cu2+) significantly accelerated the osteogenic differentiation of MSCs. The surface modification method based on MPNs provides an extremely feasible solution for preparing Ti-based implants that can adapt to different in vivo microenvironments.
1. Introduction As a common kind of implant materials in orthopedics clinic, titanium (Ti) and its alloys are always expected to take the complex in vivo microenvironments in their strides, which requires them to have a series of biological properties (e.g., osteogenesis, angiogenesis, anti-infection, anti-cancer, immunomodulatory, hemocompatibility) [1–4]. Up to now surface biofunctionalization has been considered to be the most feasible method to improve the bioactivity of their surfaces. For developing the satisfying Ti-based implants, in recent years, biofunctional coatings with the capacity of sustainedly releasing bioactive substances have received considerable attention due to concerns with flexibly deploying the released substances. Also, the coatings have an ability that turns the tunable dosage of released drug into reality under physiological conditions. For example, a high local concentration of antimicrobial agents released from the implants can effectively prevent the proliferation of bacteria in a short time after implantation [5], while bioactive ions have relatively low risk of systemic toxicity only at low concentration [6]. By contrast, making multifunctional surfaces
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possible is the greatest driving force for the rapid development of Tibased implants with the ability of sustainedly releasing bioactive substances. Among all the bioactive substances, metal ions (Mn+) have demonstrated tremendous potential in infection resistance, inflammation down-regulation, osteogenic differentiation acceleration, and therefore are commonly used to modify the surfaces of Ti-based implants [7–9]. Several methods have been used to fabricate controlled release coatings of metal ions, such as hydrothermal treatment [10], ion exchange [11], ion implantation [12], and so on. However, many of these strategies do not allow the modified surfaces to voluntarily adjust the release rate of metal ions in different in vivo microenvironments. The same biological indicators may be distinct in different parts of the body. For instance, there are significant differences on local pH value in different cell or tissue microenvironments. Weakly alkaline condition is conducive to the proliferation and differentiation of osteoblasts [13,14]. In the osteoporosis microenvironment, biological events of bone resorption caused by osteoclasts prompte a local pH of about 4.5 [15]. What′s more, the accumulation of acidic substances produced by bacteria
Corresponding author at: Medical School of Ningbo University, Zhejiang 315211, PR China. Corresponding author at: Medical School of Ningbo University, Zhejiang 315211, PR China. E-mail addresses: [email protected] (K. Xu), [email protected] (Y. Zhu).
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https://doi.org/10.1016/j.apsusc.2020.145569 Received 7 December 2019; Received in revised form 19 January 2020; Accepted 28 January 2020 Available online 03 February 2020 0169-4332/ © 2020 Elsevier B.V. All rights reserved.
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incorporating single and multiple metals [19,20], monometallic and multimetallic MPNs (Fig. 1, a) can enable Ti-based implants to perform a variety of expected biological functions.
creates an acidic biofilm microenvironment at local infection sites [16]. Therefore, pH-controlled release coatings of metal ions needs to be developed to meet the requirements of these in vivo microenvironments for Ti-based implants. For these reasons, in this work, we report a simple, low-cost, and rapid strategy to achieve the pH-controlled release of common divalent metal ions (e.g., Sr2+, Cu2+, Zn2+, and Mn2+). This approach is based on metal-phenolic networks (MPNs) that are formed by the reversible complexation between plant polyphenol (e.g., tannic acid (TA) used in this study) and metal ions. In 2013, MPNs were first discovered as a kind of simple, safe, and inexpensive coating material [17]. The most striking feature of MPNs-based coatings is that they are suitable for a wide variety of substrates. They can cover all sorts of nanoscale and micron-scale objects, such as gold nanoparticles, calcium carbonate and silica microparticles, and even bacteria. And this process is not affected by surface charge and surface topography. Importantly, Caruso and his co-workers have recently showed that TA, a natural polyphenol extracted from gallnut, may be coordinated to about 20 different metal ions to generate robust MPNs with the specialty of pH-responsive disassembly on the surfaces of many materials [18]. Due to the capacity of
2. Experimental section 2.1. Materials Northwest Institute for Nonferrous Metal Research (Xi’an, China) provided us with 15 mm diameter and 3 mm thickness Ti materials (medical grade, Ti > 99.5%). Tannic acid (ACS reagent), 3-(N-morpholino) propanesulfonic acid (MOPS; ≥ 99.5%), strontium chloride hexahydrate (SrCl2·6H2O; ACS reagent, ≥ 99.0%), copper (II) sulfate pentahydrate (CuSO4·5H2O; ACS reagent, ≥ 98.0%), zinc chloride (ZnCl2; ACS reagent, ≥ 97.0%), calcium chloride (CaCl2; ACS reagent, ≥ 96.0%), magnesium chloride hexahydrate (MgCl2·6H2O; ACS reagent, ≥ 99.0%), Manganese (II) sulfate monohydrate (MnSO4·H2O; ACS reagent, ≥ 98.0%), sodium dodecyl sulfate (SDS; ACS reagent, ≥ 99.0%), 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride, streptomycin, penicillin, 4% paraformaldehyde, fluorescein diacetate (FDA),
Fig. 1. Surface physical characteristics of monometallic and multimetallic MPNs: (a) illustration of the fabrication of MPNs on pure titanium surfaces; representative SEM images of Sr-MPNs (b & b1) and Sr/Cu-MPNs (c & c1) substrates; representative sectional SEM images of Sr-MPNs (d) and Sr/Cu-MPNs (e) samples; (f) the thickness of different MPNs (n = 10); (g) water contact angles of different MPNs (n = 5); (h) XPS survey spectra of Sr-MPNs and Sr/Cu-MPNs surfaces; (i) elemental composition for the surfaces of Sr-MPNs and Sr/Cu-MPNs plates by XPS. All the quantitative data were indicated as mean ± SD. 2
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propidium iodide (PI), bovine serum albumin (BSA), fibrinogen (FN), lglutamine (L-Gln), triton X-100, and sodium pyruvate were provided by Sigma Aldrich Co. (St. Louis, USA). Low glucose Dulbecco’s Modified Eagle’s Medium (L-DMEM), fetal bovine serum (FBS) and fluorescein isothiocyanate (FITC)-labeled phalloidin were bought from Invitrogen Co. (Carlsbad, USA). Beyotime Biotechnology Co. (Jiangsu, China) provided us with bicinchoninic acid (BCA) assay kit, cell counting kit-8 (CCK-8) kit, lactate dehydrogenase (LDH) cytotoxicity assay kit, 0.25% trypsin solution, BCIP/NBT alkaline phosphatase color development kit, and p-nitrophenyl phosphate assay kit. Picro Sirius Red solution was obtained from Aladdin Industrial Co. (Shanghai, China). Alizarin red sodium salt was bought from Alfa Aesar Co. (Tianjin, China). Phosphate buffer solution (PBS) was obtained from Dingguo Biotechnology Co. (Beijing, China). Other chemicals were supplied by Sinopharm Chemical Reagent Co. (Shanghai, China).
2.5. Release detection of metal ions At 37 °C, different MPNs coated Ti substrates (n = 3) were immersed into 1.0 mL PBS solution (pH = 4.5, 6.5, and 7.4). After incubation for different time (3, 6, 9, 12, 15, 18, 21, and 30 days), the solution containing metal ions was collected completely and the release amount of metal ions from MPNs modified Ti surfaces was measured by a inductively coupled plasma - optical emission spectroscopy (ICP-OES, Vista AX, USA).
2.6. Protein adhesion measurement BSA/PBS solution (200 μL, 1 mg/mL) was firstly added onto the Ti samples (n = 6). After incubation at 37 °C for 4 h, the plates were washed with PBS to remove the non-adherent proteins. Then, 2% SDS solution (200 μL) was pipetted onto the surfaces of different Ti foils. At 37 °C, the samples were incubated for 12 h. After that, determining the adsorbed protein content was performed with a BCA assay kit. The absorbance of the detected solution at λ = 570 nm was measured using a spectrophotometric microplate reader (Multiskan GO 1510, Thermo Fisher Scientific Inc., USA). The protein content on the TA or MPNs coated Ti substrates was normalized to that on the pure Ti plates. FN adsorption measurement was carried out with the same processes.
2.2. Pretreatment of Ti materials All Ti disks were firstly burnished with different grades of sandpapers (No. 400, 1000, 2000, and 7000), followed by ultrasonic cleaning in distilled water (18.25 MΩ·cm), acetone, and ethanol. After drying at 60 °C for 24 h, the foils underwent an alkali-heat treatment in 5 M sodium hydroxide (NaOH) aqueous solution at 80 °C for 24 h according to our previous studies [8,21]. Subsequently, the treated samples were immersed in hydrochloric acid (HCl) solution (100 mM) at 40 °C for 2 h [8,21]. After being calcinated at 600 °C for 2 h, the samples were washed and named as AHTi.
2.7. Cell culture In this study, mesenchymal stem cells (MSCs) were obtained from bone marrow of the tibia and femur of male Sprague Dawley rats with 4–5 weeks old (about 100 g). The cells were cultured at 37 °C under 5% CO2 atmosphere with L-DMEM supplementing with 10% FBS, 4 mM LGln, 0.1 mg/mL streptomycin, 110 mg/mL sodium pyruvate, and 100 U/mL penicillin. A subculture was performed when the confluence of cells reached about 90–95%. MSCs at the 3th or 4th passage were used for in vitro investigations. The process was carried out referring to the guidelines of the Institutional Animal Care and Use Committee of China. All rats were applied with approval of the Animal Ethics Committee of Ningbo University.
2.3. Preparation of MPNs on Ti substrates All solutions were freshly prepared when they would be used. Standard preparation process of monometallic MPNs on Ti substrates is described as follows: In a beaker, 5 mL 23.5 mM TA solution and 5 mL 37 mM metal solution (e.g., SrCl2, CuSO4, ZnCl2, or MnSO4 solution) were added, and then well mixed. The pH of the mixture was then raised by adding 10 mL MOPS-HCl buffer (10 mM, pH = 8.0). After that, the mixed solutions of TA and M2+ (TA-M2+ solutions) were centrifuged at 12000 rpm for 5 min to remove precipitates from the solutions. Subsequently, the pretreated Ti foils were soaked into the purified TA-M2+ solutions for the time required (20, 40, or 80 h). The MPNs coated Ti samples were washed to remove excess TA and metal ions. To strongly cross-link the metal ions with TA molecules, the obtained Ti foils were subsequently soaked into MOPS-HCl buffer (10 mM, pH = 8.0) for 30 min [22], and were named as M−MPNs (e.g., SrMPNs, Cu-MPNs, Zn-MPNs, and Mn-MPNs). The preparation of multimetallic MPNs was similar to the protocol described above. The initial concentration of TA was kept constant at 23.5 mM, while sum of metal ion initial concentration was also kept constant at 37 mM. For the 5Sr/ Cu-MPNs, Sr/Cu-MPNs, Sr/5Cu-MPNs, and Cu/Zn/Mn-MPNs preparations, a solution of strontium and copper ions ([Sr2+] : [Cu2+] = 5 : 1, [Sr2+] : [Cu2+] = 1 : 1, or [Sr2+] : [Cu2+] = 1 : 5) and a mixed solution containing copper, zinc, and manganese ions ([Cu2+] : [Zn2+] : [Mn2+] = 1 : 1 : 1) were prepared in advance, respectively. The control group of AHTi-TA substrates was fabricated by soaking AHTi substrates into 5.88 mM TA for the time required (20, 40, or 80 h).
2.8. Cell morphology observation The morphologies of MSCs cultured on pure Ti and TA or MPNs coated Ti surfaces was observed with a laser scanning confocal microscope (LSCM; TCS SP8, Leica, Germany). After seeding for 48 h (an initial density of 0.5 × 104 cells/cm2), the cells were gently rinsed, fixed, and permeabilized. Then, they were stained with FITC-labeled phalloidin (200 μL in 24-well plates, 5 U/mL) at 4 °C for 24 h. Subsequently, a counterstain was carried out with DAPI (200 μL in 24well plates, 10 mg/mL) for MSCs adhered to pure Ti and TA or MPNs coated Ti surfaces. After that, cell morphologies were obtained by LSCM. Cell area and number were calculated by analyzing LSCM images with Image Pro Plus 6.0 (IPP 6.0).
2.9. Determination of cell adhesion force 2.4. Observation of surface physical and chemical properties After culturing for 12 and 24 h, the floating cells were firstly removed and the adhesion cells were stained with DAPI. Subsequently, cell counting was carried out under an inverted fluorescence microscope (Olympus Co., Japan). After that, the Ti specimens were respectively centrifuged at 500 and 1000 rpm for 5 min. The fraction of adhesion cells was calculated according to the number of remained cells on the plates. According to our previous study [8], the mean cell adhesion force could be obtained on the strength of an assumption that the distribution of cell adhesion force obeys normal distribution.
Surface microtopographies of different Ti samples were investigated by scanning electron microscopy (SEM; SU70, Hitachi, Japan). Water contact angles (WCAs) of different Ti foils were measured by a model 200 video-based optical system (Future Scientific Co., Tai Wan, China). X-ray photoelectron spectroscopy (XPS; Phi-5000 Versa Probe, ULVCAPHI, USA) was used to analyze the surface chemistry of the samples. The peak fitting of high-resolution C1s and O1s was carried out with XPS Peak (version 4.1). 3
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Fig. 2. Ion release behaviours and SEM observation of MPNs pH-disassembly: cumulative release profiles of Sr2+ from Sr-MPNs (a) and Sr/Cu-MPNs (b) surfaces as well as Cu2+ from Sr/Cu-MPNs (c) foils after incubation in PBS (pH = 4.5, 6.5 or 7.4) at 37 °C for 30 days; release rates of Sr2+ from Sr-MPNs (a1) and Sr/Cu-MPNs (b1) plates as well as Cu2+ from Sr/Cu-MPNs (c1) substrates; SEM observation of MPNs pH-disassembly on the Sr-MPNs (d) and Sr/Cu-MPNs (e) surfaces. The quantitative data were indicated as mean ± SD for n = 3.
and 3 days. After collecting and centrifuging the culture media, supernatant was obtained for measuring LDH activity. In brief, the supernatant (120 μL) and LDH working liquid (60 μL) was orderly added into a well of 96-well plate. Subsequently, the plate was incubated at room temperature for 30 min, followed by LDH activity detection at λ = 490 nm. As for CCK-8 assay, the MSCs (1 × 104 cells/cm2 of initial density) were pretreated after incubation for 7 and 14 days (washing with PBS). 200 μL of new medium (no FBS) supplementing with 20 μL CCK-8 was added into each well and subsequently incubated in a cell incubator for another 30 min. The absorbance of obtained solution was measured at λ = 450 nm.
2.10. Cytocompatibility assays In this study, the cytocompatibility of Ti, AHTi, AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs were evaluated (n = 6). The investigation was firstly performed by FDA/PI staining. At 3 days after incubation, MSCs (1 × 104 cells/cm2 of initial density) were washed with PBS for 3 times, and then 500 mL PBS supplementing with 10 μL FDA (5 mg/mL) and 8 μL PI (2 mg/mL) were added to stain the living and dead cells. After that, the representative fluorescence images were obtained by LSCM. In addition, the cell number of dead cells was quantified by analyzing LSCM images with IPP 6.0. For quantitative evaluation, LDH activity and CCK-8 assays were carried out one after another. In 24-well plates, 2 × 104 MSCs were seeded, and then the plates were incubated for 1 4
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2.12. Qualitative and quantitative investigation of collagen I (Col I) secretion Firstly, MSCs (an initial density of 1 × 104 cells/cm2) were seeded onto different Ti surfaces in 24-well plates. Subsequently, when the cells were culturedat 37 °C for 7 and 14 days, the secreted Col I were stained with Picro Sirius Red solution. Representative optical photographs were recorded using a stereoscopic microscope. To quantify Col I secretion of MSCs, the stained cells were treated with 200 μL 0.1 M NaOH solution. Finally, the absorbance of 120 μL solution was measured at λ = 540 nm. 2.13. Qualitative and quantitative investigation of extracellular matrix (ECM) mineralization To stain the formed calcium deposition of MSCs cultured on different Ti substrates, 2 × 104 MSCs were seeded onto a Ti sample in 24well plates. After 14 and 21 days, the cells were rinsed and fixed orderly. Subsequently, ARS staining was performed with 0.1% ARS/TrisHCl solution (pH = 4.1). Representative optical photographs were recorded using a stereoscopic microscope. For quantification of mineralization, 300 μL of 10 v% acetic acid (HAc) was added into each well. After incubation for 30 min, the cells were scraped from the surfaces of Ti materials, followed by heating at 85 °C for 10 min and centrifuging at 12000 rpm for 15 min. The acid in the supernatant was then neutralized by 10 v% ammonium hydroxide (NH4OH). The quantitative ECM mineralization was obtained by measuring the absorbance of solution at λ = 405 nm. 2.14. Statistical analysis All quantitative data were indicated as mean ± standard deviation (SD). The statistical analysis was performed with GraphPad Prism 8.0 via one-way analysis of variance (ANOVA) and unpaired t-test. The confidence levels were set as 95%, 99%, and 99.9%. 3. Results and discussion Our results of SEM observation showed that when the final concentrations of TA and M2+ were constant (5.88 mM for TA and 9.25 mM for M2+), the surface topography and thickness of monometallic MPNs could be controlled by the immersion time. As shown in Figs. S1 and S2a, after being soaked in the mixed solutions of TA and M2+ for 20 h, the microtopography of AHTi surfaces was not covered completely by a thin layer (orange arrow in Fig. S2a). As the soaking time increased further (40 h), many TA-M2+ nanoparticles appeared on the surfaces (green arrow in Fig. S2a), while then the surfaces became smoother (80 h). The potential mechanisms of MPNs microtopographies’ change with the increase of soaking time were proposed as follows: (1) Microporous mesh-like structure of AHTi substrates caused the uneven deposition of TA-M2+ complex, leading to the form of aggregates. But with the increase of the thickness, the effect weakened gradually. (2) During the generation of MPNs on the surfaces, metal-phenolic nuclei of TA-M2+ nanoparticles were simultaneously formed and continuously grew into nanoparticles in the solution [23]. They would deposit, fuse, and grow into the MPNs [23]. (3) Due to the formation of MPNs and TA-M2+ nanoparticles, metal ions were consumed after soaking for a period of time, and then only TA was deposited uniformly on the MPNs surfaces (Fig. S2, b). Nevertheless, many cracks had appeared on the surfaces of MPNs coated Ti substrates, and even the underlying microstructure was clearly visible (green arrow in Fig. 1b & c). This should be attributed to the fact that free water molecules enclosed into the hydrophilic MPNs and the microporous meshlike structure in the process of MPNs formation were evaporated rapidly during the drying process, which brought about the non-uniform contraction of MPNs. The thickness of the resulting monometallic MPNs
Fig. 3. Protein and cell adhesion on the 5 kinds of Ti samples: the determination of adsorbed BSA (a)and FN (b) after incubation at 37 °C for 4 h; (c) representative LSCM images of MSCs; (d) the cell area (n = 60); (e) the cell number (n = 10); (f) the average cell adhesion forces (n = 6). The relative values were normalized to corresponding data of Ti substrates. All the quantitative datawere indicated as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
2.11. Qualitative and quantitative determination of alkaline phosphatase (ALP) For ALP qualitative determination, MSCs (1 × 104 cells/cm2 of initial density) were seeded onto different Ti surfaces in 24-well plates and then incubated in a cell incubator for 7 days. After being rinsed and fixed, the cells were stained with BCIP/NBT ALP color development kit. Representative images were obtained by a stereoscopic microscope (Leica, Germany). For ALP quantitative investigation, the cells grown on different Ti substrates were firstly incubated for 7 and 14 days. Then, the cells were lysed by 1% triton X-100. The cell lysate was obtained and centrifuged. After that, total intracellular protein and ALP activity were determined with BCA kit and p-nitrophenyl phosphate assaykit, respectively.
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Fig. 4. Cell compatibility evaluation of 5 kinds of Ti samples: (a) Live/Dead cell staining with FDA/PI for mesenchymal stem cells (MSCs) grown on the Ti, AHTi, AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs substrates, scale bar: 200 μm; (b) dead cell number (n = 10); (c) LDH activity (n = 5); (d) Cell viability (n = 5). ABS means absorbance. All the quantitative data were indicated as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
monometallic MPNs, two results could make a strong case that the four metal ions were successfully combined with TA to generate MPNs on the Ti plates: (1) There were significant differences on C and O signals between the AHTi-TA and MPNs coated Ti surfaces. Take the Sr-MPNs substrates as an example, compared with the AHTi-TA surfaces, the elemental content of C significantly increased to 62.10 at% after being soaked in the mixed solutions of TA and Sr2+ for 40 h (Fig. S9). Nevertheless, at 80 h after immersion, the content of C had almost no change (Fig. 1, i). The C1s spectra of AHTi-TA and Sr-MPNs surfaces could be fitted with 4 peaks assigned to C-C/C = C/C-H at 284.54 eV, C-O/C-OH at 286.17 eV, C = O at 288.57 eV, and O-C = O at 291.21 eV (Fig. S7 and S8a). By contrast, O1s spectra are more reflective of the coating process. The O1s spectrum detected from the AHTi-TA surfaces was fitted with three peaks assigned to HO-C at 529.60 eV, O-Ti at 531.50 eV, and O = C at 532.81 eV (Fig. S7) [18,25]. In the fitted O1s spectrum of Sr-MPNs surfaces, however, part of the HO-C peak shifted from 529.60 eV to 531.19 eV, mainly because of electron transfer from TA to Sr2+ [18]. (2) The characteristic peaks of metallic elements, including Sr3d, Cu2p, Zn2p, and Mn2p, were observed in the XPS survey spectra of their corresponding MPNs. After being soaked in the mixed solutions of TA and M2+ for 80 h, the contents of Sr2+, Cu2+, Zn2+, and Mn2+ on the modified Ti surfaces were 2.46, 2.71, 2.58, and 2.52 at%, respectively (Fig. 1i and Fig. S10). It was also important to note that, variation trend of Sr element content on the Sr-MPNs surfaces was similar to that of WCA as the soaking time increased from 20 to 80 h. The same reason was responsible for the result, that is, almost only TA was deposited uniformly for the last 40 h of the 80 h soaking process. As for Sr/Cu-MPNs substrates, the characteristic peaks of Sr and Cu appeared simultaneously in the XPS survey spectra (Fig. 1, h). However, the strontium-to-copper signal ratio (Sr/ Cu) of 0.781 was significantly lower than the concentration ratio of Sr2+ to Cu2+ (1 : 1) in the mixed solution of Sr2+, Cu2+, and TA (Fig. 1, i). So did the Zn/Cu/Mn-MPNs substrates (Fig. S12). We suspect this may be because different metal ions display different chelating abilities with TA [26]. Nevertheless, the content ratio of strontium and copper in
increased up to about 300 nm when the immersion time was increased to 80 h (Fig. 1d & f and Fig. S3). Interestingly, extremely similar results could be observed for the surface topography and thickness of multimetallic MPNs (Fig. 1c, c1, e & f and Figs. S4 & S5). The results also indicate that, for the multimetallic MPNs system of divalent metal ions, the interactions among different metal ions have little influence on the formation of MPNs. The change of surface wettability also confirmed the successful preparation of MPNs on Ti surfaces. Water contact angles (WCAs) of pure Ti and AHTi plates were similar to our previous studies (Fig. S1, c) [8,24]. After immersion in 5.88 mM TA solution for 80 h, the WCA of AHTi-TA substrates significantly went up to 12 ± 1.9° (Fig. S3, c). It was probably because the microporous structure of AHTi substrates was covered by TA (Fig. S2) and there were plenty of hydrophobic phenyl rings in TA molecules. Hydrophilic phenolic hydroxyl groups would be reduced once part of them complexed with metal ions, which directly led to a significant increase in the WCAs of MPNs coated Ti surfaces (Fig. 1g and Figs. S1 & S3–S5). It was worth noting that, however, the WCAs of MPNs coated Ti surfaces showed the characteristic of climbing up and then declining when the soaking time increased from 20 to 80 h (Fig. S3, c). In addition, there was no ionic effect on the MPNs wettability, and all the MPNs coated Ti surfaces (being soaked for 80 h) showed extremely similar wettability (Fig. 1g and Figs. S1 & S3–S5). The result was almost completely identical with the study of Yun et al [22]. We then explored the formation of MPNs on the Ti foils from a chemical point of view via XPS measurement. As shown in Fig. 1h and Fig. S6-S12, the characteristic peaks of carbon (C), argon (Ar), and oxygen (O) appeared in all the XPS survey spectra. In general, Ar signal was believed to have originated in the detection atmosphere [24]. On the surfaces of Ti and AHTi substrates, C signal was due to the hydrocarbon of environmental contamination [24]. The carbon-to-oxygen signal ratio (C/O) on the AHTi-TA and MPNs coated Ti surfaces was close to that of the theoretical value for TA (C/O = 1.65), indicating that TA or MPNs was successfully coated onto the AHTi surfaces. For 6
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Fig. 5. In vitro osteogenic differentiation: (a) ALP, Col I, and ECM mineralization staining of MSCs grown onto the Ti, AHTi, AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs surfaces after incubation for 7, 14, or 21 days (scale bar: 1 mm); quantitative evaluations of ALP activity (b), Col I secretion (c), and ECM mineralization (d). ABS means absorbance. All the quantitative data were indicated as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
also been observed on the surfaces of Sr/Cu-MPNs plates, indicating that there was nometal ion effecton the disassembly of multimetallic MPNs (Fig. 2, b & b1). Regardless of the conditions, Cu2+ exhibited extremely similar releasing behaviours to Sr2+ (Fig. 2, c & c1). We had also observed the microtopography of Sr-MPNs and Sr/Cu-MPNs surfaces after being soaked in PBS with different pH for 1 and 14 days. As shown in Fig. 2d and e, at 1 day after immersion, the degree of membrane dissociation varied significantly: In pH = 7.4 and 6.5 PBS, relatively intact MPNs could be observed, while only a thin layer remained in an acidic solution of pH = 4.5. At 14 days, there was stilla small area of continuous MPNs on the substrates soaked in pH = 7.4 PBS, whereas coatings treated by acidic solution had degraded completely. These results confirm the results of ion release behaviours well. All in all, the features described above of MPNs opportunely meet the requirements of different in vivo conditions for Ti-based implants: In normal bone tissue (pH = 7.4), the implants are asked to continuously stimulate osteogenesis-related cells for a long time to promote new bone formation; in weakly acidic bone resorption microenvironment
the MPNs could be adjusted by controlling the concentration ratio of Sr2+ to Cu2+ in the mixed solution of Sr2+, Cu2+, and TA (Fig. S11). In a word, we may regulate the amount and species of the introduced metal ions with ease and precision, which lays the foundation for obtaining multifunctional Ti-based implants. Fig. 2 displayed the release behaviours of Sr2+ and/or Cu2+ from Sr-MPNs and Sr/Cu-MPNs surfaces, which also reflected the pH-disassembly kinetics of the MPNs. Obviously, the stability of the investigated MPNs reduced gradually when the pH decreased from 7.4 to 4.5. At pH = 7.4, Sr2+ was released from Sr-MPNs substrates for up to 15 days (Fig. 2, a). During the first 3 days, the average rate of ion release reached a maximum of 1.1 μg/cm2/day (Fig. 2, a1). In pH = 6.5 PBS, the average release rate for the first three days increased dramatically to 1.8 μg/cm2/day (Fig. 2, a1), and strontium ions were released completely at the initial 7 days (Fig. 2, a). At a pH of 4.5, the MPNs became unstable and were rapidly disassembled for the first three days. Seventy-two hours later, it was difficult todetect Sr2+ in the recovered PBS (Fig. 2, a1). Almost identical release characteristics of Sr2+ had 7
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7 days, MSCs grown on the MPNs coated Ti substrates showed the highest cell viability when comparing with the other Ti plates (p < 0.01 or p < 0.001). Noteworthily, TA also promoted the proliferation of MSCs. Similar tendencies were observed at 14 days after incubation. The reason of TA or MPNs displaying a good biocompatibility may lie in the free radical scavenging capability of phenolic hydroxyl in TA molecules [31,32]. All the above results indicated the excellent biocompatibility of MPNs coated Ti substrates. As displayed in Fig. 5, after incubation for 4 or 7 days, the alkaline phosphatase (ALP) activity on the surfaces of MPNs coated Ti foils was significantly higher than that on the Ti, AHTi, and AHTi-TA substrates (p < 0.05 or p < 0.001), which was confirmed by the ALP staining (Fig. 5, a). In bone tissue, Col I is the predominant structural protein and is usually secreted in the later stages of osteogenic differentiation [8]. Herein, we could observe that whether at 2 or 3 weeks after culturing, MSCs grown on the Sr-MPNs and Sr/Cu-MPNs samples secreted more Col I than those cultured on the Ti, AHTi, and AHTi-TA substrates (p < 0.05, p < 0.01 or p < 0.001) (Fig. 5, a & c). Extremely similar results could be observed for the ECM mineralization of MSCs seeded on these 5 kinds of Ti surfaces (Fig. 5, a & d). Nevertheless, there was no significant effect of TA on the cell osteogenic differentiation of MSCs (Fig. 5). Taken together, the MPNs coated Ti substrates (i.e., Sr-MPNs and Sr/Cu-MPNs substrates) were confirmed topromote the expressions of ALP and Col I in MSCs, in turn accelerating their ECM mineralization.
(pH = 6.5), the accelerated ion release can rapidly inhibit osteoclast activity; in the infection sites (pH = 4.5), the faster ion release makes the bactericidal ions locally accumulate in high concentrations over a short period of time, playing a rapid bactericidal effect. Also, we had briefly evaluated the biological properties of MPNs coated Ti substrates (i.e., Sr-MPNs and Sr/Cu-MPNs substrates), including protein adhesion, cell adhesion, cytotoxicity, cell proliferation and osteogenic differentiation of MSCs. For protein adhesion investigation, BSA and FN were respectively used as anionic and cationic model proteins. Fig. 3a showed that although all samples were negatively charged, positively charged BSA could be effectively adsorbed on the surfaces of each substrate. Nevertheless, the amount of BSA adsorbed on the TA and MPNs coated Ti substrates was more than twice that on the Ti and AHTi surfaces. There was no significant difference of BSA adsorption among the AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs substrates. Similar results of FN adsorption were observed (Fig. 3, b). All of the above observation was also consistent with the work of Yang et al [27]. TA plays a crucial role in protein adsorption as same as dopamine coating of implants [28]. Phenyl rings and phenolic hydroxyls in TA molecules serve as the adhesion sites of hydrophobic and hydrophilic regions in protein molecules, respectively. It ensures minimal conformational changes in the adsorbed proteins and maintains their native structures, which facilitates the retention of their activity [29]. As shown in Fig. 3c, most of cells adhered to pure Ti surfaces displayed triangle-like morphology. Incomplete spreading state was more pronounced on the surfaces of AHTi substrates, even cells with reduced spreading and less actin filaments on the verge of apoptosis could be clearly observed (white arrow in Fig. 3c). Therefore, the cell area of MSCs grown on the AHTi surfaces was significantly smaller than that of cells on the pure Ti substrates (p < 0.001) (Fig. 3, d). Obviously, TA coatings greatly improved the living microenvironment of cells. The cells showed more pseudopodia and favourable spreading. On the surfaces of Sr-MPNs and Sr/Cu-MPNs substrates, MSCs exhibited better spreading and more cytoskeleton formation than those adhered to the others. Moreover, MPNs coatings significantly increased cell number of MSCs (p < 0.001) (Fig. 3, e). These phenomena maybe due to the positive stimulatory effect of released metal ions (Sr2+ and/or Cu2+) on the MSCs [8,11,24,30]. In addition, the average cell adhesion forces of MSCs adhered to the different Ti samples were detected to investigate the influences of TA or MPNs on the initial cell adhesion. At 12 h after seeding, the forces obtained from AHTi-TA, Sr-MPNs, and Sr/Cu-MPNs substrates were 291.76 ± 43.46, 287.87 ± 44.24, and 284.38 ± 40.44 pN, which were significantly higher than those measured from pure Ti and AHTi surfaces (p < 0.001) (Fig. 3, f). Twelve hours later, the average cell adhesion forces of MSCs grown on these 3 kinds of Ti surfaces were dramatically increased to 446.06 ± 43.25, 453.39 ± 83.78, and 461.46 ± 46.97 pN (Fig. 3, f). The results indicated that TA or MPNs could effectively enhance cell anchorage. However, no effect of metal ions on cell adhesion was observed. Live/Dead cell staining was then applied to observe the living (green fluorescent) and dead cells (red fluorescent; white dotted circle in Fig. 4a). Compared to other substrates, more dead cells were observed on the AHTi surfaces. TA, especially MPNs, significantly improved the biocompatibility of the substrates. It could be seen that the number of dead cells decreased dramatically from 854.2 ± 266.8 cells/cm2 of AHTi surfaces to 229.3 ± 205.8 cells/cm2 of Sr-MPNs surfaces and 120.4 ± 95.4 cells/cm2 of Sr/Cu-MPNs surfaces (p < 0.001) (Fig. 4, b). Furthermore, we used a lactate dehydrogenase (LDH) assay to quantitatively evaluate the cytotoxicity of different Ti surfaces via measuring LDH released from damaged cells. As shown in Fig. 4c, the highest LDH activity was detected on the AHTi substrates after incubation for 1 or 3 days, which was significantly reduced by TA or MPNs (p < 0.05, p < 0.01, or p < 0.001). The proliferation of MSCs cultured on Ti, AHTi, AHTi-TA, and MPNs coated Ti surfaces were then evaluated by the CCK-8 method (Fig. 4, d). After seeding for
4. Conclusions In summary, in this study, we had successfully prepared MPNs coatings on the Ti-based implants surfaces to achieve the pH-controlled release of common divalent metal ions (e.g., Sr2+, Cu2+, Zn2+, and Mn2+). When the final concentrations of TA and M2+ were constant (5.88 mM for TA and 9.25 mM for M2+), the thickness of MPNs and the amount of introduced metal ions in MPNs showed a strong immersion time dependence. The highlight of this study lies in the pH-responsive disassembly of MPNs. In PBS, the release rate of metal ions increased rapidly as the pH decreased from 7.4 to 4.5. No metal ion effect was observed on the formation of MPNs and the release behaviours of metal ions. The evaluation of MPNs’ biological properties confirmed that MPNs coated Ti surfaces displayed excellent biocompatibility and could promote the adhesion and proliferation of MSCs. What’s more, a continuous release of bioactive divalent metal ions (Sr2+ and/or Cu2+) significantly accelerated the osteogenic differentiation of MSCs grown on the Sr-MPNs and Sr/Cu-MPNs surfaces. Therefore, the surface modification method based on MPNs provides an extremely feasible solution for preparing Ti-based implants that can adapt to different in vivo microenvironments. CRediT authorship contribution statement Kui Xu: Conceptualization, Methodology, Data curation, Software, Writing - original draft, Writing - review & editing, Supervision. Mi Zhou: Data curation, Writing - original draft. Ming Li: Writing - original draft. Weizhen Chen: Data curation, Software. Yabin Zhu: Writing - review & editing, Supervision. Kaiyong Cai: Writing - review & editing. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgments The work was financially sponsored by K.C. Wong Magna Fund in Ningbo University and Zhejiang Key Laboratory of Pathophysiology (No. 201911). K. Xu really appreciated the financial support from the Talent Introduction Fund of Ningbo University (Nos. 421700020, 8
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421709530, and 421804880). In addition, we would like to gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 81802133), the Project of Scientific Innovation Team of Ningbo (No. 2015B11050), and the Ningbo Natural Science Foundation (No. 2018A610261).
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