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Enhanced Osseointegration of Porous Titanium Modified with Zeolitic Imidazolate Framework-8 Xin Zhang, Junyu Chen, Xiang Pei, Jian Wang, Qianbing Wan, Shaokang Jiang, Chao Huang, and Xibo Pei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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ACS Applied Materials & Interfaces

Enhanced Osseointegration of Porous Titanium Modified with Zeolitic Imidazolate Framework-8 Xin Zhang,#,†,‡,ǁ Junyu Chen,#,†,‡ Xiang Pei,§ Jian Wang,†,‡ Qianbing Wan,†,‡ Shaokang Jiang,‡,ǁ Chao Huang,*,ǁ Xibo Pei*,†,‡ †

State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, ‡Department of Prosthodontics, West China Hospital of Stomatology, § Postanesthesia Care Unit, West China Hospital of Stomatology, and ǁCollege of Chemistry, Sichuan University, Chengdu, P.R. China #

Co-first authorship

KEY WORDS Zeolitic imidazolate framework-8, Titanium implants, Surface modification, Osseointegration, In vitro, In vivo

ABSTRACT Nanoscale

zeolitic

imidazolate

framework-8

(ZIF-8)-modified

titanium

(ZIF-8@AHT) can enhance osteogenesis in vitro. In this study, we systematically and quantitatively examined the effects of ZIF-8@AHT on osteogenesis, and investigated its ability to form bone in vivo. First, we coated various quantities of nanoscale ZIF-8 crystals on alkali- and heat-treated titanium (AHT) by controlling the concentration of the synthesis solution. We then characterized the ZIF-8@AHT materials using scanning electron microscopy (SEM), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and determination of the sessile drop contact angle. To illustrate the combined effects of micro/nanotopography and ZIF-8@AHT composition on bone regeneration, we cultured MC3T3-E1 preosteoblast cells on various titanium substrates in vitro by setting pure titanium (Ti) and AHT as control groups. The ZIF-8@AHTs enhanced cell bioactivity compared with AHT and Ti, as evidenced by increased extracellular matrix (ECM) mineralization, collagen secretion and the upregulated expression of osteogenic genes (Alp, Col1, Opg, and Runx2) and 1 ACS Paragon Plus Environment


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osteogenesis-related proteins (ALP and OPG). ZIF-8@AHT-1/8 exhibited better osteogenic activity compared with the other ZIF-8@AHT groups investigated. We subsequently inserted Ti, AHT, and ZIF-8@AHT-1/8 implants into the healed first molars (M1s) of mice, and found that ZIF-8@AHT-1/8 also

promoted

osseointegration at the bone–implant interface. These results suggest that ZIF-8@AHT-1/8 has great potential for practical application in implant modification.

1. INTRODUCTION Generally, biomaterials are used to construct medical devices or implants to replace the diseased or lost biological structures.1-3 Since Bothe et al. first applied titanium devices to biomedical domains in a mouse model,4 titanium has been widely used in metallic implants because it has superior biocompatibility, excellent corrosion resistance, and a low modulus.5 However, titanium and its alloys usually have insufficient osseointegration for implant longevity owing to their suboptimal osteoconductivity, which can lead to implants failure.6 To improve the clinical success rate of titanium implants, two main surface modification strategies have been devised: topological structure design (TSD) and surface chemistry design (SCD).7, 8 In the case of TSD, several studies have showed that the use of micro- and submicro-structures that directly correspond to the dimensions of cells and resorption pits may result in enhanced osteoblast differentiation.9, 10 Nanostructures with feature sizes comparable to those of membrane receptors and proteins can increase cell proliferation,11 adhesion,12 and spreading.13 Moreover, it has been claimed that nanostructures cannot appreciably regulate osteoblast differentiation without microstructures,14 and the initial cellular responses to the microstructure alone are not ideal.15 In terms of SCD, many kinds of biochemicals such as hydroxyapatite, ions, drug molecules and even genes have been loaded into or coated on titanium surfaces for different applications.5,

16, 17

For example, vancomycin has been loaded into

Ag-implanted TiO2 nanotubes to improve their antibacterial activity,16 and the 2 ACS Paragon Plus Environment

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biodegradable microRNA miR-29b has been used as a surface coating to promote the osteogenic bioactivity of the implant.17 However, the control of surface chemistry, which affects the quality, composition and stability of the surface, remains challenging.18, 19 Zeolitic imidazolate frameworks (ZIFs), a subclass of metal organic frameworks (MOFs),20 may provide a better alternative for achieving controllable modification with stable cellular responses. ZIFs consist of tetrahedral metal ions (specifically Zn and Co) bridged by imidazolate ligands (Im), and have the advantages of both MOFs and zeolites,21 such as controllable synthesis and good chemical and thermal stability.22-26 These features make ZIFs attractive candidates for use in catalysis,27 sensing,28 adsorption29,

30

and separation.31 Moreover, their potential for use in

biomedical applications is also attracting increasing attention.32, 33 For instance, Sun et al. have reported a pH-sensitive drug delivery vehicle based on ZIFs.34 Shieh et al. embedded catalase molecules into ZIF-90 to protect catalase from inhibition by proteinase K.35 Christopher et al. reported the potential use of ZIFs in bioimaging.36 We recently reported that porous titanium modified with nanoscale ZIF-8 coating enhances osteogenic and antibacterial activity compared with pure or porous titanium.37 Compared with pure or porous titanium, nanoscale ZIF-8-coated titanium increases ECM mineralization, promotes alkaline phosphatase (ALP) activity, upregulates the expression of osteogenesis-related genes (Alp and Runx2) in MG63 cells, and inhibites the growth of Streptococcus mutans. These results indicate that nanoscale ZIF-8 coatings are potentially useful for the surface modification of implants.37 However, there is still little information about the use of ZIFs coatings in bone tissue engineering. Specifically, a detailed grouping is considered necessary to quantitatively determine how the density and particle size of the coating, and the amount of released zinc ions, affect osteogenic activity. Moreover, the impact of ZIF-8 coating on the growth of collagen fiber is also unknown. This warrants further study because collagen fiber can direct the mineralization of hydroxyapatite to form mature bone.38 3 ACS Paragon Plus Environment


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Furthermore, the ability of ZIF-8@AHT to form bone in vivo has not been fully determined until now. Consequently, in the current study we fabricated nanoscale ZIF-8 coatings with controllable particle sizes and densities on micro-roughened titanium plates, and characterized their structure and morphology. We carried out a detailed investigation of the combined effects of the topological structure and surface chemistry of ZIF-8@AHT. Furthermore, we used MC3T3-E1 preosteoblast cells to evaluate the effect of ZIF-8 coatings on osteogenic differentiation in vitro, and used a mouse model to examine the osseointegration of ZIF-8@AHT in vivo. A systematic evaluation of the reciprocity at the interface between cells/tissues and ZIF-8 coatings that involves in vitro cell behavior and in vivo osteogenesis assessment will provide a better understanding of the mechanisms by which ZIF-8 coatings induce osseointegration.

2. MATERIALS AND METHODS 2.1. Materials. Commercial pure Ti plates (10 × 10 × 0.5 mm, Guantai co., Ltd., Hebei, China) were used in the in vitro experiments. Zinc nitrate hexahydrate, 2-methylimidazole (MeIm) and methanol were purchased from the Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All the chemical reagents mentioned above were of analytical grade and were used as received without further purification. MC3T3-E1 cells were supplied by the State Key Laboratory of Oral Diseases (Sichuan University, China). Fetal

bovine

serum

(FBS),

α-minimum

essential

medium

(α-MEM),

penicillin/streptomycin (PS) and paraformaldehyde (PFA) were obtained from HyClone (Logan, USA). Fluorescein isothiocyanate-phalloidin (FITC-phalloidin) was acquired from Yeasen Biotechnology Co., Ltd. (Shanghai, China). The ALP activity kit was from Jiancheng Co. (Nanjing, China). A bicinchoninic acid (BCA) protein assay kit and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Picric acid was from Xiya 4 ACS Paragon Plus Environment

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Reagent Co., Ltd. (Chengdu, China). The enzyme-linked immunosorbent assay kit (ELISA) was supplied by the Cloud-Clone Corp. (Wuhan, China). The RNAzol reagent, PrimeScript™ RT reagent kit, and SYBR Premix Ex™ Taq II kit were acquired from TaKaRa (Dalian, China). A 0.1% Sirius Red solution, isoflurane, sodium pentobarbital, and buprenorphine were obtained from Sigma-Aldrich (St. Louis, USA).

2.2. Sample Preparation and Characterization. Both Ti and AHT were prepared according to the procedures described in the literature.39 We used a simple and environmentally friendly hydrothermal strategy that did not involve organic solvents to coat ZIF-8 onto AHT. ZIF-8 coatings of various densities were obtained by controlling the concentration of the synthesis solution. More specifically, zinc nitrate hexahydrate (0.11 g) and MeIm (2.27 g) were dissolved in 40 mL deionized (DI) water, and stirred gently for 20 min. The obtained milky solution was diluted to concentrations of 1/2, 1/4, 1/8 and 1/16. The solution was then transferred to Teflon-lined autoclaves in which the AHT was placed horizontally, and heated at 37 ℃ for 6 h. The ZIF-8@AHT samples were obtained by thoroughly rinsing with DI water (10 mL × 3) and drying at 37 ℃ for 24 h. The samples were denoted as ZIF-8@AHT-1, ZIF-8@AHT-1/2, ZIF-8@AHT-1/4, ZIF-8@AHT-1/8 and ZIF-8@AHT-1/16, where “1”, “1/2”, “1/4”, “1/8” and “1/16” denote the concentrations of the synthesis solutions. The precipitates from the mother solution were harvested by centrifugation (5000 rpm, 10 min) and washed with DI water (10 mL × 3). It should be noted that in the case of ZIF-8@AHT-1/16, further 5 mL of methanol was added and the rotational speed was maintained at 9000 rpm for 10 min. Finally, the obtained precipitates were dried at 37 ℃ for 24 h. The morphology of the samples was analyzed with a SEM instrument (KYKY Technology Development Ltd.), which was operated at 10 kV. Sessile drop contact angles of DI water were used to determine the wettability of the various air-dried samples using a DSA25 contact angle goniometer (KRÜSS GmbH, Germany) 5 ACS Paragon Plus Environment


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equipped with a digital camera and image analysis software. The volume of the drops was 5 µL and the time between drop measurements was 10–20 s. XRD patterns of the precipitates were measured using a X’Pert pro MPD diffractometer (Philips, Japan) with Cu Kα radiation (15 mA and 40 kV) at a scan rate of 1° min-1 over a wide range of angles (2θ = 5–50°). FT-IR spectra of the precipitate samples were recorded on a Thermo Fisher Scientific FT-IR spectrometer (Nicolet 6700, USA) in the range 400– 4000 cm-1 using a potassium bromide disk. The concentration of zinc ions released from the ZIF-8@AHT samples was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES; IRIS Advantage, Thermo Scientific). The ZIF-8@AHT specimens were soaked in 3 mL of phosphate buffered saline (PBS) for 6 h, 12 h, 1 day, 2 days, and 4 days at 37 ℃ without agitation, and the leachates were collected at each time-point.

2.3. In Vitro Studies. 2.3.1. Cell Culture. The MC3T3-E1 cells were cultured in 75-cm2 flasks (Coring, USA) containing 8 mL of α-MEM supplemented with 10% FBS and 1% PS in a humidified atmosphere of 5% CO2 at 37 ℃. The culture medium was refreshed every 2 days, and the cells were subcultured every 6 days.

2.3.2 .Cell Proliferation. The MC3T3-E1 cells were seeded on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) at a cell density of ~2 × 104 cells/mL. Cell proliferation was investigated by CCK-8 assay after culturing for either 1 or 4 days. At the predetermined time-point, the cells were rinsed twice with PBS. Fresh culture medium (300 µL) and CCK-8 solution (30 µL) were added to each well, followed by incubation at 37 ℃ for 2 h. Subsequently, 100 µL of the supernatant was transferred to a 96-well plate, and the absorbance at 450 nm was determined using a microplate reader (Thermo, USA). All experiments were repeated three times. 6 ACS Paragon Plus Environment

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2.3.3. Cell Morphology. The MC3T3-E1 cells were seeded on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) for 2 days at a cell density of ~2 × 104 cells/mL. The samples were washed twice with PBS and fixed with 2.5% glutaraldehyde at 37 ℃ for 4 h. The samples were dehydrated using a graded ethanol series (30%, 50%, 75%, 90%, 95% and 100%; 15 min each), then dried overnight in a vacuum desiccator. Finally, the specimens were coated with gold and examined by SEM at 5 kV.

2.3.4. Initial Cell Adhesion. The MC3T3-E1 cells were seeded on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) at a cell density of ~2 × 104 cells/mL for 1, 4 and 24 h. At each time-point, the cells were rinsed twice with PBS and fixed with 4% PFA at room temperature (RT) for 10 min. The cells were permeabilized with 0.1% Triton X-100 for 2 min, and washed thrice with PBS. The samples were then stained with FITC-phalloidin following the manufacturer’s instructions, and with DAPI for a further 5 min in the dark. The stained cytoskeletal F-actin (green) and cell nuclei (blue) were examined using a fluorescence microscope (Olympus, Japan).

2.3.5. Alkaline Phosphatase Activity Assay. An osteogenic medium supplemented with 10 mM β-glycerol phosphate and 0.2 mM ascorbic acid was used for the following osteogenic-related studies. Osteogenic activities were studied in vitro after 7 days of cell culture in the medium. The osteogenic medium was refreshed every 2 days. The MC3T3-E1 cells were seeded on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) at a cell density of ~2 × 104 cells/mL. After culturing for either 7 or 14 days, ALP activity was evaluated using an ALP activity kit (Jiancheng Co., Nanjing, China) according to the manufacturer’s instructions. Briefly, the cell lysates were obtained by applying repeated freeze-thaw cycles, followed by incubation with substrate solution 7 ACS Paragon Plus Environment


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at 37 ℃ for 1 h. The color development reagent was then added to each well, and production was immediately measured at 405 nm on a microplate reader. The ALP activity was normalized to the total protein content determined by a BCA protein assay kit. The experiments were conducted in triplicate.

2.3.6. Extracellular Matrix Mineralization. ECM mineralization was determined by Alizarin Red staining. The MC3T3-E1 cells were seeded on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) for either 7 or 14 days with a cell density of ~2 × 104 cells/mL. The samples were washed twice with PBS and fixed with 75% ethanol at RT for 1 h. The MC3T3-E1 cells were then stained with 40 mM Alizarin Red in Tris-HCl solution (pH=4.2) for 15 min at RT. The stained samples were washed thoroughly with DI water to remove excess dye, and the substrates were examined under a stereomicroscope (Olympus, Japan). For the quantitative analysis, cetylpyridinium chloride (10%) was added to each well to dissolve the stain for the quantitative analysis. After incubating for 15 min at RT, the stain was eluted in 10% cetylpyridinum chloride in 10 mM sodium phosphate, and the absorbance at 542 nm was measured using a microplate reader. The experiments were repeated three times.

2.3.7. Collagen Secretion. The collagen secretion of the cells was determined by Sirius Red staining. The MC3T3-E1 cells were cultured on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) at a cell density of ~2 × 104 cells/mL. After culturing for either 7 or 14 days, the samples were washed twice with PBS and fixed in 4% PFA for 30 min at RT. The cells were then rinsed twice with PBS and stained in a 0.1% Sirius Red solution in saturated picric acid for 18 h at RT. The cells were then rinsed thoroughly with 0.1 M acetic acid until the rinse solution was colorless, and the morphologies of the surfaces were examined under a stereomicroscope (OLYMPUS, Japan). With respect to quantitative analysis, 0.5 mL of the destain solution (0.2 M NaOH: methanol = 1:1) was added to 8 ACS Paragon Plus Environment

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each well to dissolve the stain. The absorbance of the solution at 492 nm was determined using a microplate reader. The experiments were carried out in triplicate.

2.3.8. Osteoprotegerin Secretion. We investigated the concentration of osteoprotegerin (OPG) in the culture medium using an ELISA kit according to the manufacturer’s instructions. Titanium surfaces (Ti, AHT, ZIF-8@AHTs) with MC3T3-E1 cells at a density of ~2 × 104 cells/mL were cultured for 14 days. Briefly, the supernatant of the culture medium was collected via centrifugation (10000 rpm, 30 min) for subsequent reagent preparation, sample preparation, and assay procedures. The optical density of each well at 450 nm was measured using a microplate reader, and the concentration of OPG was determined by a linear interpolation of the standard curve. The experiments were conducted in triplicate.

2.3.9. Real-time Polymerase Chain Reaction (RT-PCR) Analysis. We investigated the expression of osteogenic-related genes in the MC3T3-E1 cells using RT-PCR. The MC3T3-E1 cells were cultured on the titanium surfaces (Ti, AHT, ZIF-8@AHTs) at a cell density of ~2 × 104 cells/mL for either 7 or 14 days. At each time-point, the cells were washed twice with PBS, and the total RNA was extracted using 1 ml of RNAzol reagent. The RNA concentrations were determined by spectrophotometry. The total RNA was reverse-transcribed into complementary DNA (cDNA) using a PrimeScript™ RT reagent kit. We carried out RT-PCR on Alp, Opg, collagen type-1 (Col1) and runt-related transcription factor 2 (Runx2) using SYBR Premix Ex™ Taq II. The relative expression levels of the target genes were normalized based on the expression levels of the reference gene β-actin. Each analysis was performed three times, and the primer sequences are listed in Supporting Information Tbl. S1.

2.4. In Vivo Animal Studies. 9 ACS Paragon Plus Environment


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2.4.1. Animals. Every procedure was approved by the Animal Care Committee of the West China Hospital of Stomatology, in accordance with the ARRIVE guidelines. Efforts were taken to ensure that the guiding principles of the three R’s were followed.40 Wherever possible, mathematical modeling and quantitative in vitro assays were used instead of animals. Eighteen wild type and ACTb-EGFP skeletally mature female mice at the age of 2– 3 months old were used in the surgery. They were housed in a climate-controlled environment (25 ℃ and 50% humidity), with 12 h light/dark cycles. Food and water were provided ad libitum. To ensure the guiding principles of the three R’s, the ZIF-8@AHT-1/8 was selected as the experimental group for in vivo studies because it demonstrated the best osteogenic activity in vitro. The mice were randomly assigned to three groups corresponding to Ti, AHT, and ZIF-8@AHT-1/8.

2.4.2. Surgical Procedures. The mice were anesthetized via inhalation anesthesia (2% isoflurane), followed by an intraperitoneal injection of sodium pentobarbital (1%, 50 mg/kg), and a sub-cutaneous injection of buprenorphine (0.1 mg/kg). The maxillary M1s were extracted using small forceps, and the first molar (M1) sites were allowed to heal for 3 weeks. After creating an osteotomy in each healed M1 site using a dental engine (1000 rpm) and a drill bit (0.52mm Drill bit City, Chicago, USA), implants (0.54 mm, Fairfax Dental Ltd., London, UK) (Ti, AHT and ZIF-8@AHT-1/8) were inserted into the osteotomy. The Ti implant was used without further treatment whereas the AHT and ZIF-8@AHT-1/8 implants were prepared according to the same procedures used for the AHT and ZIF-8@AHT-1/8 plates. All implants were positioned at the height of the gingiva. After the operation, buprenorphine (0.1 mg/kg) was injected subcutaneously for 3 consecutive days to relieve pain, and the mice were provided with a soft food diet. There was no evidence of infection or prolonged inflammation at any of the surgical sites. Subsequently, the mice were sacrificed on 10 ACS Paragon Plus Environment

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post-implantation day 7 (PID 7) and the tissues were prepared.

2.4.3. Histology, Histomorphometry, and Immunohistochemistry. Alveolar bone was harvested under ribonuclease-free conditions and washed with 1 × PBS at 4 ℃. After fixing in 4% PFA overnight, the tissues were decalcified in 19% ethylenediaminetetraacetic acid (EDTA) for 21 days at 4 ℃, and prepared for paraffin embedding. Paraffin embedding was carried out following standard protocols, and the samples were cut into 8 µm-thick sections. See Supporting Information for details.

2.4.4. Histomorphometric Analyses. The histomorphometric analyses were conducted using ImageJ software (version 1.49v). At least six histologic sections were used to detect and quantify peri-implant tissues for every test. The photography was carried out by a single investigator. We calculated the ALP pixels (AP) and total pixels (TP) within the defined region of interest (ROI), and obtained a percentage representing ALP secretion by dividing TP with AP. We used a similar method to quantify tartrate-acid resistant phosphatase (TRAP) secretion. The RUNX2-positive (RUNX2+ve) and osterix-positive (osterix+ve) cells were counted directly using ImageJ software.

2.5. Statistical Analyses. The data are expressed as the mean ± standard deviation of independent replicates. The results of the in vitro and in vivo experiments were statistically analyzed using IBM SPSS version 19.0 software. Significant differences between different groups were

assessed

using

one-way

analysis

of

variance

(ANOVA)

with

the

Student-Newman-Keuls test for multiple comparisons. P ≤ 0.05 was considered statistically significant.

3. RESULTS 11 ACS Paragon Plus Environment


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3.1. Characterization of Samples. The morphology of the Ti, AHT, and ZIF-8@AHT samples was examined using SEM. The obtained micrographs were processed and analyzed with ImageJ software to determine the diameter of AHT pores and ZIF-8 crystals. As shown in the images, some minor scratches caused by the polishing treatment were clearly visible on the Ti surface (Fig. 1A), whereas the AHT had a rough surface with pores of approximately 1 µm in diameter (Fig. 1B). ZIF-8 crystals were randomly distributed in the pore spaces of AHT and the number of ZIF-8 crystals decreased gradually from ZIF-8@AHT-1 to ZIF-8@AHT-1/16 (Figs. 1C-G). The crystals in ZIF-8@AHT-1, ZIF-8@AHT-1/2, ZIF-8@AHT-1/4, and ZIF-8@AHT-1/8 were rhombic dodecahedral in shape and approximately 300 nm in size, whereas the crystals in ZIF-8@AHT-1/16 were spherical in shape and approximately 200 nm in size (Figs. 1C-G). We measured the static water contact angles to evaluate the surface wettability of the modified surfaces. As displayed in Fig. 1H, the contact angle for Ti was approximately 100° whereas that for AHT (Supporting Information Video S1) was very small (< 3°). Moreover, the contact angle decreased gradually from ZIF-8@AHT-1 to ZIF-8@AHT-1/16, which was in accordance with the decreasing trend in the number of crystals.

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Figure 1. SEM images of different substrates: (A) Ti, (B) AHT, (C) ZIF-8@AHT-1, (D)

ZIF-8@AHT-1/2,

(E)

ZIF-8@AHT-1/4,

(F)

ZIF-8@AHT-1/8,

(G)ZIF-8@AHT-1/16; (H) qualitative results of contact angle. n=6 per group. * p < 0.05; # p < 0.05 compared with other groups.

To investigate the phase purity of the as-prepared ZIF-8 coatings, the ZIF-8 crystals in the mother solution were collected and analyzed using FT-IR and XRD spectroscopy. As shown in the FT-IR spectra (Fig. 2A), the peak positions and their assignments agreed well with those reported in the literatures,31, 41 implying that the crystals were of pure phase. A comparison of the experimental and simulated XRD patterns is shown in Fig. 2B. The XRD patterns of the as-prepared ZIF-8 crystals agreed well with the simulated one, indicating the phase purity of the ZIF-8 crystals, which was consistent with the FT-IR analysis result. Therefore, the coatings prepared on AHT were ZIF-8 crystals without any other impurities.



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Figure 2. (A) FT-IR spectra of as-prepared ZIF-8 crystals; (B) XRD patterns of the simulated and as-prepared ZIF-8 crystals.

3.2. Biocompatibility of ZIF-8@AHT. The quantity of zinc ions released from the ZIF-8@AHT samples was determined by ICP-AES. Zinc ions were slowly released from the ZIF-8@AHTs without a burst-release effect (Fig. 3A). There was a small, acute release of zinc ions in the first 12 h, and 1.08, 0.76, 0.72, 0.56 and 0.24 µg/mL of zinc ions were released from ZIF-8@AHT-1,

ZIF-8@AHT-1/2,

ZIF-8@AHT-1/4,

ZIF-8@AHT-1/8

and

ZIF-8@AHT-1/16 at 12 h, respectively. After 1 day, the release rate decreased slowly. Four days after treatment with the buffer solution, the concentrations of the released zinc ions were only 1.69, 1.46, 1.23, 0.96 and 0.39 µg/mL, respectively. Overall, the amount of zinc generated from ZIF-8@AHTs decreased step-by-step from ZIF-8@AHT-1 to ZIF-8@AHT-1/16 at each time-point. We used a CCK-8 assay to measure the proliferation of MC3T3-E1 cells to investigate the biocompatibility of the ZIF-8@AHTs. As shown in Fig. 3B, no significant difference in cell proliferation was found between the modified groups and the control groups. Secondly, we investigated initial cell adhesion on the titanium surfaces by staining with FITC and DAPI, which reveal F-actin and nuclei, respectively. Specifically, the cells on all samples exhibited a similar spherical morphology in the first hour. At 4 h, the expression levels of F-actin and extensions of 14 ACS Paragon Plus Environment

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filopodia increased markedly (Supporting Information Fig. S1). After culturing for 24 h, the cells on the Ti surface exhibited a round morphology with numbers of short filopodia, whereas active polygonal lamellipodium protrusions were observed in the AHT and ZIF-8@AHT samples (Fig. 3C). We further confirmed this result by cell morphology analysis using SEM (Fig. 4).

Figure 3. (A) Zinc ion release kinetics of the ZIF-8@AHTs in 10% FBS-containing α-MEM; (B) Cell proliferation examined with a CCK-8 assay after MC3T3-E1 cells were cultured for either 1 or 4 days; (C) Fluorescence microscopy images of MC3T3-E1 cells cultured on the titanium surfaces for 24 h (white arrows: polygonal lamellipodium protrusions); F-actin was stained with FITC (green) and the nucleus with DAPI (blue). * p < 0.05; # p < 0.05 compared with other groups.



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Figure 4. SEM morphology of MC3T3-E1 cells cultured on the titanium surfaces for 2 days (white arrows: polygonal lamellipodium protrusions; black arrows: the round MC3T3-E1 cells with numbers of short filopodia).

3.3. In vitro Osteogenic-Related Assay. We investigated the differentiation of osteoblasts cultured on the samples by measuring the ECM mineralization and collagen secretion of the MC3T3-E1 cells using Alizarin Red and Sirius Red staining, respectively. As shown in Figs. 5A-B, the mineralization of the cells on the ZIF-8@AHTs was significantly greater (p < 0.05) than that on the AHT and Ti surfaces after culturing for 7 days. This trend became more evident after the cells had been cultured for 14 days. Based on the optical images (Fig. 5A), the MC3T3-E1 cells on all of the five ZIF-8@AHT groups displayed much denser mineral nodules and a higher degree of mineralization on Day 14 than those on the AHT and Ti surfaces. Among the five ZIF-8@AHT groups, ZIF-8@AHT-1/2,

ZIF-8@AHT-1/8,

and

ZIF-8@AHT-1/16

exhibited

greater

mineralization compared with ZIF-8@AHT-1 and ZIF-8@AHT-1/4 (p < 0.05) (Fig. 5B). Furthermore, collagen production quantified by Sirius Red staining showed that more collagen was secreted on ZIF-8@AHTs and AHT than on Ti at the early stage (Day 7). After 14 days, apparently denser collagen was deposited on the ZIF-8@AHTs and AHT than on the Ti (Fig. 5D). Notably, collagen secretion by 16 ACS Paragon Plus Environment

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cultured cells was highest on ZIF-8@AHT-1/8 and ZIF-8@AHT-1/16. These results were further evidenced by the quantitative analysis (Fig. 5C).

Figure 5. (A) Matrix mineralization of cells cultured on different titanium surfaces on Days 7 and 14; (B) Colorimetric quantitative results of ECM mineralization; (C) Colorimetric qualitative results of collagen secretion; (D) Collagen secretion of cells cultured on various surfaces on Days 7 and 14. * p < 0.05; # p < 0.05 compared with other groups.

To further evaluate the osteogenic activity at the molecular level, we investigated ALP activity, OPG secretion, and the expression of osteogenic-related genes on different titanium surfaces. The MC3T3-E1 cells cultured on the ZIF-8@AHTs had 17 ACS Paragon Plus Environment


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significantly higher ALP activity than the cells on AHT and Ti on Days 7 and 14 (Fig. 6A). ZIF-8@AHT-1/8 in particular displayed significantly higher ALP activity on Day 14 than the other samples (p < 0.05). The OPG secretion data for the various samples are provided in Fig. 6B. On Day 14, ZIF-8@AHT-1/4, ZIF-8@AHT-1/8, and ZIF-8@AHT-1/16 exhibited significantly higher OPG secretion. In this study, we used RT-PCR to determine the expression levels of Alp, Col1, Opg, and Runx2 mRNA after culturing for either 7 or 14 days (Fig. 6C). At the early stage (Day 7), there was much higher Alp, Col1, and Runx2 expression in the cells cultured on some of the ZIF-8@AHTs than in those on the Ti surface (p < 0.05). After culturing for 14 days, the cells on ZIF-8@AHT-1/8 and ZIF-8@AHT-1/16 exhibited notably higher expression levels of Alp compared with the other groups (p < 0.05). Cells cultured on ZIF-8@AHT-1/8 displayed the highest Col1 and Runx2 expression of

all

the

groups;

Moreover,

Opg

expression

in

ZIF-8@AHT-1/4

and

ZIF-8@AHT-1/8 was markedly higher than in the other groups (p < 0.05).

Figure 6. (A) Quantitative ALP activity of cells cultured on various surfaces on Days 7 and 14; (B) OPG expression detected by ELISA after MC3T3-E1 cells were cultured on various surfaces for 14 days; (C) Expression of osteogenic-related genes in MC3T3-E1 cells cultured on various surfaces measured by quantitative RT-PCR. * p < 0.05; # p < 0.05 compared with other groups. 18 ACS Paragon Plus Environment

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3.4. In Vivo Osseointegration Studies. We used an M1 extraction mouse model to investigate the combined effects of micro/nanotopography and coating composition on osseointegration in vivo. As shown in Fig. 7A, the implants (Ti, AHT, or ZIF-8@AHT-1/8) were first inserted into the healed M1 sites of the mice. After 7 days, the mice were sacrificed and the samples were harvested for further study. We analyzed samples taken from the sagittal and transverse directions (Fig. 7B), and obtained the threads of bone–implant interface in the images. Pentachrome staining revealed that more mature bone collagen (yellow) formed around ZIF-8@AHT-1/8 than around AHT and Ti (Fig. 7C). Aniline blue staining revealed that more mineralized bone matrix was formed around the ZIF-8@AHT-1/8 and AHT samples than around the Ti (Fig. 7D). Although the difference in new bone volume between AHT and ZIF-8@AHT-1/8 shown in Fig. 7D was not decisive, a soft tissue gap was found at the bone–implant interface in the AHT group, whereas most of the newly formed bone contacted the implant surface directly without infiltration of soft tissue in the ZIF-8@AHT-1/8 group.



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Figure 7. (A) Schematic of the surgery; (B) Sagittal and transverse direction of the implants placed into osteotomies; (C) Pentachrome staining, and (D) Aniline blue staining of Ti, AHT and ZIF-8@AHT-1/8. Scale bar representing 100 µm; n=6 per group.

The results obtained from pentachrome staining were confirmed by picrosirius red (PR) staining (Fig. 8A), which also detected type I collagen (bright red area) and type III collagen (green area) formed at the bone–implant interface. As shown in Fig. 8A, more collagen was detected on ZIF-8@AHT-1/8 than on AHT or Ti at the early stage (Day 7). This trend became more noticeable at the bone–implant interface. ALP is a cell surface glycoprotein that facilitates the mineralization of collagenous matrices,42 whereas TRAP is an osteoclast marker enzyme.43 We investigated osteogenic capability in vivo to further evaluate ALP expression and TRAP activity. 20 ACS Paragon Plus Environment

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We detected significantly higher ALP expression in the ZIF-8@AHT-1/8 and AHT groups than in the Ti group (p < 0.05) (Figs. 8B-C). ZIF-8@AHT-1/8 showed minimal TRAP activity, whereas we detected significantly higher TRAP activity in the Ti group (p < 0.05) (Figs. 8D-E).

Figure 8. (A) Picrosirius red staining, and (B) ALP staining of Ti, AHT and ZIF-8@AHT-1/8; (C) Colorimetric quantitative results of ALP staining; (D) TRAP staining of Ti, AHT and ZIF-8@AHT-1/8; (E) Colorimetric quantitative results of TRAP staining. Scale bar representing 100 µm; n=6 per group. * p < 0.05.

Osterix and RUNX2 are important early and mature osteoblastic marker enzymes, and early indicators of osseointegration.44,

45

We carried out immunochemistry

staining to determine the expression levels of osterix and RUNX2. After implantation for 7 days, the number of osterix+ve and RUNX2+ve cells appeared to be greater in the 21 ACS Paragon Plus Environment


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ZIF-8@AHT-1/8 group than in AHT and Ti groups (Figs. 9A-D) (p < 0.05). AHT exhibited significantly higher osterix and RUNX2 secretion than Ti (p < 0.05).

Figure 9. (A) Osterix staining of Ti, AHT and ZIF-8@AHT-1/8; (B) Colorimetric quantitative results of osterix staining; (C) RUNX2 staining of Ti, AHT and ZIF-8@AHT-1/8; (D) Colorimetric quantitative results of RUNX2 staining. Scale bar representing 100 µm; n=6 per group. * p < 0.05.

4. DISCUSSION In a previous study, we qualitatively proved that a nanoscale ZIF-8@AHT coating had the potential as an implant modification in vitro.37 However, the quantitative effect of ZIF-8 coatings on the growth of bone and collagen fiber remains unclear. Furthermore, a comprehensive evaluation of osseointegration following ZIF-8@AHT implantation in vivo would be of great significance. Therefore, in this study we coated various quantities of nanoscale ZIF-8 crystals onto AHT surfaces, and found that ZIF-8@AHT-1/8 exhibited the best osteogenic activity both in vitro and in vivo. As illustrated by the in vitro tests, cells on AHT and ZIF-8@AHTs exhibited active polygonal lamellipodium protrusions, whereas a round morphology with numbers of short filopodia was observed in the Ti group (Figs. 3-4). This result can be explained 22 ACS Paragon Plus Environment

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by the rough surfaces and superior wettability of AHT and ZIF-8@AHTs. Briefly, it has been reported that titanium implants with rough surfaces can enhance osteoblast adhesion and extension compared to those with smooth surfaces.46 Moreover, implants with higher surface hydrophilicity can also facilitate the attachment of osteoblasts.47 In further, according to the in vitro osteogenic-related assays, we found that cells on ZIF-8@AHT-1/8 showed the highest ECM mineralization and collagen secretion ability, ALP activity, and expression of osteogenesis-related genes (Alp, Col1, Opg, and Runx2). These results will be discussed in detail in the following sections. A biomimetic approach to tissue engineering has attracted a great deal of attention recently.14,

48, 49

For example, Gittens et al. have reported that a combination of

micro/submicro-structures with a high density of nanostructures promote osteocalcin (OCN) and OPG expression in MG63 cells.14 Similarly, Kubo et al. have reported that randomly distributed nanofeatures of approximately 300 nm can greatly enhance ALP activity, Col1 and Ocn expression, and the total calcium deposition of bone marrow cells.49 ZIF-8@AHT, which has nanostructures (ZIF-8 crystals) of a similar size to membrane receptors and proteins as well as micro/submicro-structures (on the AHT surface) corresponding to the size of resorption pits and cell dimensions, mimics the morphology of biomineralization, and may therefore represent a potential approach to tissue engineering. In fact, biomimetic ZIF-8@AHT with crystals of approximately 300 nm distributed in a random manner markedly promotes the differentiation of MC3T3-E1 cells in vitro. Surface wettability can also affect implant osseointegration.50 For example, Ko et al. have reported that hydrophilic surfaces can strengthen the binding of adhesion proteins to the surfaces of osteogenic cells, and facilitate their growth.51,

52

Furthermore, greater implant wettability can accelerate healing and early osseointegration.47 We expected that as the wettability gradually increased from ZIF-8@AHT-1 to ZIF-8@AHT-1/16 (Fig. 1H), there would be a similar increase in osteogenic activity. Actually, the osteogenic activity from ZIF-8@AHT-1 to 23 ACS Paragon Plus Environment


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ZIF-8@AHT-1/8 was mostly in agreement with the tendency of wettability, whereas there

was

a

slight

decrease

for

ZIF-8@AHT-1/16

in

comparison

with

ZIF-8@AHT-1/8, indicating that osteogenic activity is simultaneously modulated by other factors. Briefly, it is well known that zinc is an essential ingredient of many transcription factors, and an appropriate amount of zinc can promote osteoblast differentiation.53

For instance, Seo et al. reported that the ALP secretion of

MC3T3-E1 cells was promoted by the addition of zinc; ALP secretion increased with the increase in zinc ion content after culturing for 10 days, but no obvious difference was observed when the concentration was approximately 0.96–1.6 µg/mL.53 In this study, the highest zinc concentrations for ZIF-8@AHT-1, ZIF-8@AHT-1/2, ZIF-8@AHT-1/4, and ZIF-8@AHT-1/8 was 1.69, 1.46, 1.23, and 0.96 µg/mL, respectively (all approximately in the 0.96–1.6 µg/mL range). For ZIF-8@AHT-1/16, however, the released zinc ions were significantly lower (0.39 µg/mL) than in the other four groups, which probably resulted in a decrease in the osteogenic activity. On the other hand, the crystal morphology and size in ZIF-8@AHT-1/16 differed slightly from those in the other four groups (Figs. 1C-G), which can be explained by the XRD analysis results (Fig. 2B). Specifically, the relative intensity of ZIF-8@AHT-1/16 at the (110) reflection was much lower than that in the simulated pattern, whereas the (112) reflection showed the most prominent peak. The change in the XRD pattern and shape implied that the crystals preferred to orient in the (112) planes, and the crystallinity might have changed.54 Moreover, crystallinity has been proved to affect the proliferation and differentiation of osteogenic cells.55 Therefore, the combination of low zinc concentration and poor crystallinity may have resulted in a lower osteogenic

activity

for

ZIF-8@AHT-1/16

in

comparison

with

that

for

ZIF-8@AHT-1/8. We selected ZIF-8@AHT-1/8 for further in vivo studies because it demonstrated the best osteogenic activity in vitro. Briefly, we inserted Ti, AHT, and ZIF-8@AHT-1/8 implants into the healed M1 mouse extraction sites, and carried out in vivo osseointegration studies. As expected, osseointegration was better in the 24 ACS Paragon Plus Environment

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ZIF-8@AHT-1/8 implants than in the Ti or AHT implants, with newly formed bone directly contacting the implant without any infiltration of soft tissue between the implant and the bone (Fig. 7D). Clinically, the success of implants depends largely on early osteogenesis,56 which is determined by the ability of osteoblastic cells to form new bone, and is regulated by related genes and proteins.57 Alp, Col1, and Runx2 genes are early-stage markers of osteoblast differentiation,44, 58-61 and Opg is crucial for protecting osteogensis and inhibiting the activity of osteoclasts.62 Based on the RT-PCR analysis in vitro (Fig. 6C), the cells cultured on the ZIF-8@AHTs exhibited significantly higher expression levels of these genes compared with those on AHT and Ti. Moreover, in contrast to AHT and Ti, the secretion of ALP protein, which is capable of promoting the mineralization of collagenous matrices,42 was enhanced both in vitro and in vivo (Figs. 6A, 8B-C). Moreover, both osterix protein and RUNX2 protein can facilitate the differentiation of preosteoblasts and multipotent mesenchymal cells into fully functioning osteoblasts, thereby promoting osteogenesis.54,

60-63

As the in vivo

immunochemistry staining shows (Figs. 9A-D), the number of osterix+ve and RUNX2+ve cells appeared to be greater in ZIF-8@AHT than in AHT and Ti, confirming the increased osteogenic activity of these ZIF-8 modified groups. With regard to osteoclast activity, OPG is able to inhibit the differentiation and function of osteoclasts, whereas TRAP can permit the migration of osteoclasts across the bone surface to new resorptive sites and thus facilitate bone resorption.64-66 As shown in Fig. 6B and Figs. 8D-E, the increased secretion of OPG protein in vitro and the decreased secretion of TRAP protein in vivo in the presence of ZIF-8 coatings implies that the coating inhibited osteoclast activity. These findings suggest that by simultaneously protecting bone regeneration and suppressing bone resorption, ZIF-8 coatings can promote osteogenesis both in vitro and in vivo. It is well known that peri-implantitis can lead to loss of supporting bone around implants.67 Accordingly, it would be very interesting if the coated implants exhibited superior osteogenic and antibacterial activity. Specifically, some antibacterial drugs 25 ACS Paragon Plus Environment


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can probably be loaded into ZIF-8 crystals, an ideal vehicle for drug delivery.34 Moreover, although the mouse is one of the most commonly utilized animal models for medical research,68 further studies on larger animals are necessary for simulating osseointegration following implantation under more complicated conditions. Moreover, the ALP, RUNX2, COL1, OPG, and osterix signaling cascades can be activated by the Wnt pathway, thereby controlling bone regeneration.69,

70

In the

present study, the increased expression of ALP, RUNX2, OPG, osterix as well as Alp, Col1, Opg, and Runx2 indicated that the Wnt signaling pathway may have been activated by ZIF-8@AHT-1/8; this requires further investigation.

4. CONCLUSION In this work, we reported an environmentally friendly method for coating nanoscale ZIF-8 onto AHT surfaces, thereby significantly enhancing the osteogenic activity of the modified titanium implants. According to the in vitro osteogenic-related assay, the ZIF-8@AHTs not only improved ECM mineralization and collagen secretion, and up-regulated the expression of osteogenic genes (Alp, Col1, Opg, and Runx2), but also promoted the secretion of osteogenesis-related proteins (ALP and OPG) in MC3T3-E1 cells. Among the five ZIF-8@AHT groups, ZIF-8@AHT-1/8 exhibited the best osteogenic activity. Furthermore, the in vivo results showed that osseointegration was better in the ZIF-8@AHT-1/8 implants than in the Ti and AHT implants, with newly formed bone directly binding to the implant without the intervention of fibrous soft tissue. Overall, ZIF-8@AHT-1/8 provides an alternative for clinically improving the osseointegration of titanium implants.

ASSOCIATED CONTENT Supporting Information Sequence of primers used for the real-time PCR analysis; histology, histomorphometry, and immunohistochemistry staining process of alveolar bone; static water contact angle of AHT; expressions of F-actin and extensions of filopodia 26 ACS Paragon Plus Environment

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after culturing for 1 h, 4 h, and 24 h. (PDF)

AUTHOR INFORMATION Corresponding Authors *Xibo Pei: E-mail: [email protected]. *Chao Huang: E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (21501123, 81601613), Science & Technology Support Program of Sichuan Province (2016FZ0085, 2014SZ0201). The authors are grateful to Mr. Weifeng Zhao for helpful manuscript revisions. We would like to acknowledge the comprehensive training platform of ‘specialized laboratory, College of Chemistry, Sichuan University’ for ICP, FT-IR and XRD analyses.

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68. Alghamdi, H. S.; Bosco, R.; Both, S. K.; Iafisco, M.; Leeuwenburgh, S. C.; Jansen, J. A.; van den Beucken, J. J., Synergistic Effects of Bisphosphonate and Calcium Phosphate Nanoparticles on Peri-Implant Bone Responses in Osteoporotic Rats. Biomaterials 2014, 35, 5482–5490. 69. Brun, J.; Fromigue, O.; Dieudonne, F. X.; Marty, C.; Chen, J.; Dahan, J.; Wei, Y.; Marie, P. J., The LIM-Only Protein FHL2 Controls Mesenchymal Cell Osteogenic Differentiation and Bone Formation through Wnt5a and Wnt10b. Bone 2013, 53, 6–12. 70. Ping, Z.; Hu, X.; Wang, L.; Shi, J.; Tao, Y.; Wu, X.; Hou, Z.; Guo, X.; Zhang, W.; Yang, H.; Xu, Y.; Wang, Z.; Geng, D., Melatonin Attenuates Titanium Particle-Induced Osteolysis via Activation of Wnt/beta-catenin Signaling Pathway. Acta Biomater. 2017, 51, 513–525.

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SEM images of different substrates: (A) Ti, (B) AHT, (C) ZIF-8@AHT-1, (D) ZIF-8@AHT-1/2, (E) ZIF8@AHT-1/4, (F) ZIF-8@AHT-1/8, (G)ZIF-8@AHT-1/16; (H) qualitative results of cantact angle. n=6 per group. * p < 0.05; # p < 0.05 compared with other groups. 199x141mm (300 x 300 DPI)

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(A) FT-IR spectra of as-prepared ZIF-8 crystals; (B) PXRD patterns of the simulated and as-prepared ZIF-8 crystals. 199x88mm (300 x 300 DPI)


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Figure 3. (A) Zinc ion release kinetics of the ZIF-8@AHTs in 10% FBS-containing α-MEM; (B) Cell proliferation examined with a CCK-8 assay after MC3T3-E1 cells were cultured for either 1 or 4 days; (C) Fluorescence microscopy images of MC3T3-E1 cells cultured on the titanium surfaces for 24 h (white arrows: polygonal lamellipodium protrusions); F-actin was stained with FITC (green) and the nucleus with DAPI (blue). * p < 0.05; # p < 0.05 compared with other groups 150x113mm (300 x 300 DPI)



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Figure 4. SEM morphology of MC3T3-E1 cells cultured on the titanium surfaces for 2 days (white arrows: polygonal lamellipodium protrusions; black arrows: the round MC3T3-E1 cells with numbers of short filopodia) 180x87mm (300 x 300 DPI)


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(A) Matrix mineralization of cells cultured on different titanium surfaces on Days 7 and 14; (B) Colorimetric quantitative results of ECM mineralization; (C) Colorimetric qualitative results of collagen secretion; (D) Collagen secretion of cells cultured on various surfaces on Days 7 and 14. * p < 0.05; # p < 0.05 compared with other groups. 199x200mm (300 x 300 DPI)



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(A) Quantitative ALP activity of cells cultured on various surfaces on Days 7 and 14; (B) Osteocalcin protein expression detected by ELISA after MC3T3-E1 cells were cultured on various surfaces for 14 days; (C) Expression of osteogenic-related genes in MC3T3-E1 cells cultured on various surfaces measured by quantitative RT-PCR. * p < 0.05; # p < 0.05 compared with other groups. 199x108mm (300 x 300 DPI)


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(A) Schematic of the surgery; (B) Sagittal and transverse direction of the implants placed into osteotomies; (C) Pentachrome staining, and (D) Aniline blue staining of Ti, AHT and ZIF-8@AHT-1/8. Scale bar representing 100 µm; n=6 per group. 189x166mm (300 x 300 DPI)



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(A) Picrosirius red staining, and (B) ALP staining of Ti, AHT and ZIF-8@AHT-1/8; (C) Colorimetric quantitative results of ALP staining; (D) TRAP staining of Ti, AHT and ZIF-8@AHT-1/8; (E) Colorimetric quantitative results of TRAP staining. Scale bar representing 100 µm; n=6 per group. * p < 0.05. 174x157mm (300 x 300 DPI)


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(A) Osterix staining of Ti, AHT and ZIF-8@AHT-1/8; (B) Colorimetric quantitative results of osterix staining; (C) RUNX2 staining of Ti, AHT and ZIF-8@AHT-1/8; (D) Colorimetric quantitative results of RUNX2 staining. Scale bar representing 100 µm; n=6 per group. * p < 0.05. 177x110mm (300 x 300 DPI)



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contents graphic 83x35mm (300 x 300 DPI)


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Enhanced Osseointegration of Porous Titanium ... - ACS Publications

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