Double layers of gold nanoparticles immobilized titanium implants improve the osseointegration in rabbit models

Journal Pre-proof Double layers of gold nanoparticles immobilized titanium implants improve the osseointegration in rabbit models

Wan-Kyu Ko, Seong Jun Kim, Dong Nyoung Heo, In-Bo Han, Sewha Kim, Il Keun Kwon, Seil Sohn PII:

S1549-9634(19)30213-8

DOI:

https://doi.org/10.1016/j.nano.2019.102129

Reference:

NANO 102129

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Revised date:

13 November 2019

Please cite this article as: W.-K. Ko, S.J. Kim, D.N. Heo, et al., Double layers of gold nanoparticles immobilized titanium implants improve the osseointegration in rabbit models, Nanomedicine: Nanotechnology, Biology, and Medicine(2019), https://doi.org/ 10.1016/j.nano.2019.102129

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© 2019 Published by Elsevier.

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Double layers of gold nanoparticles immobilized titanium implants improve the osseointegration in rabbit models Wan-Kyu Ko, MS

1,2,†

, Seong Jun Kim, BS

1,2,†

, Dong Nyoung Heo, PhD 3, In-Bo Han, MD, PhD 1,

Sewha Kim, MD, PhD 4, Il Keun Kwon, PhD 3, Seil Sohn, MD, PhD 1,*

1

Department of Neurosurgery, CHA Bundang Medical Center, CHA University, 59, Yatap-ro,

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Bundang-gu, Seongnam-si, Gyeonggi-do, 13496, Republic of Korea 2

Department of Biomedical Science, CHA University, Bundang-gu, Seongnam-si, Gyeonggi-do,

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Republic of Korea 3

Department of Dental Materials, School of Dentistry, Kyung Hee University, Dongdaemun-gu,

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Seoul, 02447, Republic of Korea 4

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Department of Pathology, CHA Bundang Medical Center, CHA University, 59, Yatap-ro, Bundang-

Corresponding author:

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Seil Sohn, MD, PhD

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*

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gu, Seongnam-si, Gyeonggi-do, 13496, Republic of Korea

Department of Neurosurgery,

CHA University College of Medicine 59, Yatap-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13496, Korea Phone: 82-31-881-7966 Fax: 82-2-780-5269 E-mail: [email protected]

†

Wan-Kyu Ko and Seong Jun Kim contributed equally as the first authors

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Conflict of interest: The authors declare no competing financial interest. Funding: This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number: NRF2017R1D1A1B03032980) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare,

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Republic of Korea (grant number: HI16C1559).

Word count for Abstract: 148

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Word count for manuscript: 4994

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Number of References: 41

Number of tables: 0

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Number of figures: 8

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Number of Supplementary online-only files, if any: 1

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Abstract Osseointegration is important in osteopenia and osteoporosis patients due to their low bone densities. Gold nanoparticles (GNPs) are greatly beneficial materials as osteogenic agents. The aim of this study is to investigate osseointegration between bones and double layers of GNP-immobilized titanium (Ti) implants. The physicochemical properties of the Ti surface were evaluated by scanning electron microscopy, by atomic force microscopy, by means of the contact angle using water drops, and by x-

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ray photoelectron spectroscopy. Osteogenic differentiation of human bone-marrow-derived

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mesenchymal stem cells was analyzed and showed the higher values in double layers of GNP (GNP2) groups. In addition, we performed an in vivo study using hydroxyapatite (HA) and GNP2 spine

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pedicle screws in ovariectomized (OVX) and SHAM rabbits. Osseointegration parameters also showed higher values in GNP2 than in HA groups. These findings suggest that implants with double

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layers of GNPs can be a useful alternative in osteoporotic patients.

Keywords: Gold nanoparticle; Osseointegration; Hydroxyapatite; Mesenchymal stem cell; Titanium implant

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Background A successful implant anchorage during orthopedic surgery depends on strong osseointegration between bones and titanium (Ti) implants. Osseointegration is the important prerequisite between bones and anchored implants in orthopedic surgeries. 1 Osseointegration refers to the direct contact between living bone and an implant. In other words, osseointegration is the direct anchorage between bones and implants without fibrous tissue at the bone–implant interface.2 To improve the osseointegration between bones and Ti implants, polymethylmethacrylate (PMMA)

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cement has been injected into bones prior to screw insertion in spine surgery cases.3 Although PMMA can augment the bone–implant fixation, it has severe side effects such as cement leakage, an increased risk of pedicle fracture, neural injuries, and vascular injuries.3

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The calcium phosphate hydroxyapatite (HA) is the key component of human bone. Therefore, HA coating of the surfaces of Ti implants has been studied to enhance the osseointegration of implants.4

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HA promotes the stability of the bone–Ti interface without the side effects of PMMA.4 However, HA coating requires a very high sintering temperature, complex coating processes, and industrial coating

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equipment.5 In addition, implanted screws coated with HA can separate during the osseointegration process, as HA has poor adhesion with screws.5, 6

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Gold nanoparticles (GNPs) are highly beneficial materials as osteogenic agents. The GNPs induce the osteogenic differentiation of osteo-progenitor cells such as mesenchymal stem cells (MSCs) without

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cell cytotoxicity.7, 8 GNPs also induce the activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway.7 The p38 MAPK pathway causes the up-regulation of osteogenic genes

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such as runt-related transcription factor 2 (Runx2).7 Runx2 is an essential transcription factor for osteoblast differentiation.9 Increased levels of Runx2 can improve bone formation for osseointegration between bones and implants. 10 Therefore, GNPs have been applied to implant surfaces to improve the osseointegration between bones and implants. 11-13 In addition, GNPs improve osteogenesis in calvarial defect models.14 Through gold–sulfur (Au–S) bonding, GNPs are easily conjugated with the surfaces of Ti which are modified by (3-mercaptopropyl) trimethoxysilane (SH(CH2)3Si(OCH3)3, 3-MTPMS).15 Ti implants have limited area of the surfaces. To overcome the limited area, we immobilized the surfaces of Ti samples with double layers of GNPs through disulfur–Au bonding (Au–S–S–Au).16 We analyzed the bone–implant interface to evaluate osseointegration. We measured the parameters of the bone-to-implant contact (BIC), the bone surface (BS)/tissue volume (TV), and the bone volume (BV)/TV in the bone–implant interface using ovariectomized (OVX) or SHAM rabbit models.

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In this study, we aimed to evaluate the osseointegration between bones and double layers of GNPimmobilized Ti implants. We also added an HA-coated group as a positive control in this study.

Methods Chemicals and materials Chloroauric acid (HAuCl4), trisodium citrate, 3-MTPMS, anhydrous toluene (99.8%), n-hexane, and 1,6-hexanedithiol (SHCH2(CH2)4CH2SH) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anhydrous ethyl alcohol (99.9%) was obtained from Samchun (Seoul, Korea). Deionized water (DW,

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18.2 MΩ) was purified using an EXL-3 water purification system (Vivagen, Korea). Pristine TiO2 disks of ASTM F67 grade 4 and TiO 2 pedicle screws were obtained from Medyssey (Seoul, Korea). Human bone-marrow-derived MSCs (passage 4) were purchased from Invitrogen (Carlsbad, CA,

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USA) and cultured in the growth medium (GM, MesenPRO RSTM basal medium mixed with the

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MesenPRO RSTM growth supplement (Thermo Fisher Scientific, Rockford, IL, USA)). Osteogenic medium (OM) consisting of Dulbecco’s modified eagle medium (DMEM, Invitrogen) supplemented

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with 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY), 1% penicillin–streptomycin (PS, GIBCO), 10mM–β–glycerol phosphate (Sigma), 300 µM of ascorbic acid (Sigma), and 0.1 µM of dexamethasone (Sigma) was used for the osteogenic differentiation of MSCs. The 100-mm and 24-

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NJ).

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well cell culture plates were both purchased from Falcon Becton Dickinson (Falcon, Lincoln Park,

Preparation and characterization of the GNPs After HAuCl4 of a 500 µM solution in DW (800 mL) was refluxed, 2% trisodium citrate (Sigma, 15 mL) was quickly added. After 15 min, the GNPs were determined to have been prepared when the color of the solution changed to dark red. Ultraviolet/visible (UV/Vis) absorption values according to the GNP concentration were demonstrated in the form of a calibration curve using a spectrophotometer (UV-1650PC, Shimadzu, Japan). Transmission electron microscopy (TEM) images of the GNPs were obtained from an H-7100 device (Hitachi, Japan). The GNP calibration curve was obtained as follows: 580 µM of GNPs was diluted with DW to concentrations of 290, 145, and 72.5 µM. The absorbance values were measured with a UV/Vis spectrophotometer.

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Preparation of Ti samples The pristine Ti disks had a diameter of 8 mm and thickness of 1 mm. The Ti pedicle screws had a diameter of 3.0 mm, a length of 9.0 mm, and a thread pitch of 1.5 mm. The Ti samples were ultrasonically cleaned three times in n-hexane, acetone, ethanol and DW for 20 min each and then dried under a N2 stream. The cleaned Ti samples were integrated with HA or GNPs. The HA in the TiHA disks and the HA screws were physically coated by a Dentis device (Dentis Implant Co., Daegu, Korea) using a blasting method17 on the surfaces of the TiO2 samples. The synthesized GNP solution was reacted with disks or screws to immobilize the GNPs as follows: In the first procedure, the cleaned Ti was immersed in a 2.5 N NaOH solution to form active OH groups on the surface at 60 ºC

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for 24 h.18 It was then rinsed thoroughly with DW and ultrasonically cleaned with DW (labeled as TiOH). In the second procedure, TiOH disks were immersed in a 2% 3-MTPMS toluene solution to

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form SH end groups on the TiOH surface at 60 ºC for 24 h.15 The disks were thoroughly washed with n-toluene, ethanol, DW, and dried under a N2 stream (labeled as TiSH). The TiSH samples were

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immersed in a GNP solution to immobilize the first layer of the GNPs at 37 ºC for 24 h,19 after which they were rinsed thoroughly with DW and ultrasonically cleaned with fresh DW (labeled as TiGNP1

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disks or GNP1 screws). The TiGNP1 disks and GNP1 screws were immersed in a 10 mM 1,6hexanedithiol ethanol solution at 60 ºC for 24 h to form SH end groups again on the surfaces.16 Subsequently, they were thoroughly washed with ethanol, DW, and dried under a N2 stream. The

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samples were immersed in a fresh GNP solution at 37 ºC for 24 h, rinsed thoroughly with DW, and

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ultrasonically cleaned. Finally, the double layers of GNPs were immobilized on the Ti surfaces

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(labeled as TiGNP2 disks or GNP2 screws).

Quantification of immobilized GNPs on disks and screws The immobilized GNPs on the Ti samples were calculated using an indirect method.20 Briefly, six Ti samples were immersed in 580 µM of GNPs (10 mL). The non-immobilized GNP solution after the immobilization processes was compared to the GNP calibration curve to quantify the immobilized GNPs.

Surface characterization of the surfaces of Ti disks SEM, AFM, XPS, and energy dispersive x-ray spectroscopy (EDS) evaluation of the surface of Ti disks were described in supplementary methods.

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Cell culture and cell seeding on Ti surfaces for the in vitro test Passage 4 of the MSCs was proliferated on 100-mm cell culture plates with GM at 37 ºC in an atmosphere humidified with 5% CO2. The medium for the cells was replaced every three days and sub-cultured when the cells were fluent to a level of 90%. Ti disks of all groups were sterilized by immersion in 70% ethanol for 10 min, after which they were dried under a UV lamp on a clean bench.

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Each Ti disk was then placed in each well of a 24-well plate.

Cell cytotoxicity test

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Passage 6 of the MSCs was carefully seeded at 5 × 104 cells in 80 µL on 24-well culture plates for TiO2, TiHA, TiGNP1, and TiGNP2. They were allowed to adhere for two hours, after which 1 ml of

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GM was gently added to each well (n = 4 per group). Cytotoxicity was confirmed with live/dead assay kits (Invitrogen) after culturing for 24 or 48 h, respectively. Subsequently, the cell-seeded Ti disks

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were washed with Dulbecco's phosphate-buffered saline (DPBS, GIBCO, Grand Island, NY). Solutions of 2 µM calcein AM and 4 µM EthD-1 were added to each well. After five minutes, the

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stained cells were observed using an inverted fluorescence microscope (Olympus, IX71, Japan). Under the same conditions, the cell cytotoxicity on the surfaces of the Ti disks was quantitatively

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evaluated using a cell counting kit (CCK-8, Dojindo Molecular Technologies Inc., Japan) after culturing for 24 or 48 h, respectively (n = 4 per group). At each predetermined time point, the GM

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was replaced with fresh GM containing CCK-8 (500 μL of 0.1 mL/ml). After incubation for 2 h, the absorbance was measured at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). The absorbance of each culture plate group at 24 or 48 h was fixed at 100% and the absorbance levels of the other groups were calculated relative to that level.

Osteogenic differentiation of MSCs Passage 6 of the MSCs was carefully seeded at 5 × 104 cells to a level of 80 µL on 24-well culture plates for TiO2, TiHA, TiGNP1, and TiGNP2. Afterwards, these samples were allowed to adhere for two hours, and 1 ml of OM was then gently added to each well (n = 4 per group). MSCs cultured with GM in 24-well plates were added as a negative control under identical conditions. All groups were incubated for 3, 7, or 14 days in each case.

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Alkaline phosphatase (ALP) activity and quantitative real-time polymerase chain reaction (qRT-PCR) ALP activity was described in supplementary methods. The expression of collagen type 1 (COL1), Runx2 and osteopontin (OPN) was assessed by qRT-PCR (supplementary methods).

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Animals

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Four New Zealand female white rabbits weighing between 4.0 and 4.2 kg were purchased from Orient Bio Inc. (Seongnam, Korea). They were housed individually in cages under the following conditions:

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room temperature (20–23 °C), 55% humidity, 12 h circadian light rhythm, and free access to water/food. All surgical interventions and postoperative animal care procedures were provided by the

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Institutional Animal Care and Use Committee (IACUC) of CHA University (IACUC180016).

Surgeries for in vivo modeling

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The anesthetizing drug (Zoletil® (50 mg/kg, Virbac Laboratories, France)/Rompun® (10 mg/kg,

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Bayer, Korea)) solution was intraperitoneally administered to the animals. Anesthesia was established within 15–20 minutes and the anesthesia was maintained with isoflurane (3%) in oxygen (2 L/minute)

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via a facemask.21 Two rabbits were bilaterally OVX for the OVX models22 and the other two rabbits were used for the SHAM models.

Implantation of GNP2 and HA screws Twenty-four Ti pedicle screws were used for the in vivo study. Twelve screws were HA screws and the other twelve were GNP2 screws. Six screws (HA = 3, GNP2 = 3) were alternately implanted in each rabbit. The entire back of each rabbit was thoroughly shaved. After the back was prepared with povidone iodine, a sterile drape was used to cover the entire animal. A midline skin incision was made followed by periosteal dissection to expose the vertebrae. The left-sided spino-laminar junction was exposed to implant the GNP2 screw. Subsequently, a defect with a diameter of approximately 1.5 mm was created at the spino-laminar junction with a diamond electrical drill. The GNP2 screw was

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implanted into the bones toward the direction of the opposite lamina. After three GNP2 screws were implanted, right-sided periosteal dissection was done and three HA screws were implanted via the same method. All surgeries were performed by one spine surgeon (Sohn. S). After the surgeries, the animals were examined daily for activity, eating, and wound healing. Three months after the implanting operation, the rabbits were sacrificed using an overdose of KCl intravenously. Two rabbits (OVX: 1, SHAM: 1) were used for micro computed tomography (µCT) imagery and the other two rabbits (OVX: 1, SHAM: 1) were used for the histological analyses.

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Three-dimensional (3D) measurements of bone parameters using µCT Shortly after sacrificing the rabbits, vertebral blocks (OVX: 1, SHAM: 1) including six screws (HA:

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3, GNP2: 3) were safely separated from the whole spine using a bone cutter. The blocks were converted into digital imaging and communication in medicine (DICOM) files using µCT equipment

reconstructed

using

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(Quantum FX, Perkin Elmer, Waltham, MA, USA). All of the screws in the vertebral blocks were computer

tomographic

volume

(CTvol)

software

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(https://www.bruker.com/service/support-upgrades/software-downloads/micro-ct/measurements-andvisualization.html, version: 2.3.2.0, Bruker Corporation, Billerica, MA, USA). BV/TV and BS/TV in the designated volume of interests (VOIs) were quantified by volumetric analyzing using CT(CTAn)

software

(https://www.bruker.com/service/support-upgrades/software-

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Analyzer

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downloads/micro-ct/measurements-and-visualization.html, version: 1.17.7.2+, Bruker). The VOIs included screws with the surrounding bones and tissues and were 3 × 3 × 3 mm3 in size. VOIs were

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randomly designated for each screw. BV (mm3)/TV (mm3) in each VOI was demonstrated as a percentage and the BS (mm2)/TV (mm3) in each VOI was shown as 1/mm (n = 3 per group).

Histological preparation and two-dimensional (2D) measurements of the screws Shortly after sacrificing the animals, vertebral blocks (OVX: 1, SHAM: 1) including six screws (HA: 3, GNP2: 3) were also safely separated from the whole spine. The specimen blocks including each screw were fixed in 10% neutral buffered formalin (Sigma) for five days and were dehydrated with alcohol. The specimens including the screws were then embedded in resin (Technovit 7200 VLC, Kulzer, Germany). To display the screw and the surrounding tissues from the blocks, the blocks were grounded and sectioned parallel to the long axis of the screw using a water-cooled band saw.23, 24 The sections were prepared with EXAKT grinding equipment (EXAKT 300, Norderstedt, Germany). The

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final thickness of the sections was 70 µm and the sections were stained with Masson-Goldner trichrome staining kits (Roth, Karlsruhe, Germany) according to the manufacturer's instructions. The stained images were evaluated using a light microscope (Olympus IX71). Two images at 40 × magnification were randomly designated. A region of interest (ROI, 400 × 800 µm) was also randomly designated within the 40 × magnification images. To depict the ROI images in detail, 100 × magnification was used. BIC and BV in the ROI were quantified as follows (n = 6 per group): The 800 µm length of the entire long axis of screw within the ROI was set as 100% and the bone line in contact with the screw surfaces was calculated. The 400 × 800 µm area of the ROI was set as 100% and

the

bone

area

stained

with

green

was

calculated

using

the

ImageJ

software

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(http://rsb.info.nih.gov/ij/index.html, ImageJ; National Institutes of Health (NIH), Bethesda, MD,

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USA).

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Statistical analyses

All values were presented as the mean ± standard deviation (SD). Two group comparisons were

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conducted with Student’s t-tests. Multiple comparisons among the groups were performed with a oneway analysis of variance (ANOVA). Tukey’s multiple-comparison test was used as a post hoc analysis method. Differences with p values for which *p < 0.05 and **p < 0.01 were considered

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Results

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statistically significant.

Characterization of GNPs

Spherical GNPs thirty nanometer (nm) in size are effective when used for the osteogenic differentiation of osteoblasts.25 We synthesized 30 nm GNPs using trisodium citrate with a carboxylate (COO¯) reduction method.20 The GNPs were immobilized on the surfaces of Ti disks and Ti screws (Figure 1). TEM images of the spherical GNPs are shown in Figure 2, A. The GNPs have different maximum wavelengths ranging from 400 to 800 nm in accordance with their size. The GNPs also have different maximum absorbance capabilities according to the concentration of the GNPs. 25 As the size of the GNPs was increased, the maximum wavelengths increased, and as the concentration of the GNPs was increased, the maximum absorbance of the GNPs increased. 20 The maximum absorbance of the synthesized GNPs was 522 nm (Figure 2, B), which demonstrated that the average

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size of the GNPs was 30 nm.25 Figure 2, C shows that the maximum absorbance values decreased proportionally in accordance with the concentration of the GNPs.

Surface analysis of Ti disks We successfully immobilized the first GNP layer onto the surfaces of the Ti samples using 3-MTPMS (Figure 1).15 The Ti samples were labeled as TiGNP1 for the disk and GNP1 for the screw. After treatment with 1,6-hexanedithiol on the first layer of the GNPs, 19 another GNP layer was combined

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with the thiol end group (SH) by S–Au bonding.16 Finally, double layers of GNPs were immobilized

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on the Ti disks and Ti screws (TiGNP2 disk, GNP2 screw) (Figure S1).

Figure S2 shows that the concentration of immobilized GNPs per TiGNP1 disk was 7.2 µg and that

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6.7 µg of GNPs was additionally conjugated per TiGNP2 disk. Figure S3 indicated that the

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immobilized GNP concentration at the GNP1 screw was 29.9 µg and that 23.9 µg was additionally conjugated at the GNP2 screw.

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The properties of each Ti surface from the pristine TiO2 to the TiGNP2 disks were determined by SEM, by AFM, and by assessing the contact angle using water drops (Figures 3 and 4). The SEM and

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AFM outcomes of the pristine TiO2 disk showed a uniformly flat surface (Figures 3 and 4, A). However, the TiOH disks demonstrated a fairly rough surface (Figure 4, A). The SEM images of

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TiSH were similar to those of the TiOH surface (Figure 3). The AFM images of TiSH showed a smooth surface (Figure 4, A). After the GNP reaction, there was a change in the surface morphology.

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As shown in Figure 3, the GNPs were deposited onto the TiGNP1 and TiGNP2 disks. Specifically, TiGNP2 showed that the surfaces were fully immobilized with GNPs (Figures 3 and 4, A). TiHA indicated rugged surfaces, as indicated in Figures 3 and 4, A. Figure 4, B shows that the TiSH surface was highly hydrophobic (90.98 º ± 7.62) due to the formation of thiol group.26 As illustrated in Figure 4, B, TiHA showed a slightly increased contact angle (50.02 º ± 3.83) relative to those of the TiGNP groups (TiGNP1: 47.27 º ± 2.87, TiGNP2: 42.58 º ± 3.35). The decreased contact angle was caused by the hydrophilic citrate layer on the TiGNP surface.27 The XPS spectra of TiO2 and TiOH showed peaks indicative of O 1s, Ti 2p, and C 1s (Figure S4 and Table S1). TiSH treated with 3-MTPMS showed 2p and Si 2p peaks (Figure S4), which demonstrated that the end group of the TiSH surface was immobilized with SH. TiGNP groups showed an Au 4f peak (Figure S4 and Table S1). The percentages of Au were determined to be 1.85% (TiGNP1) and 3.55% (TiGNP2). TiHA had Ca 2p (13.07%) and P 2p (21.75%) peaks (Figure S4 and Table S1).

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Figures 2–4 and S1–4 indicate that double layers of GNPs were successfully immobilized on the surface of Ti.

Increasing proliferation of MSCs on TiGNP2 disks To investigate whether Ti disks have cytotoxicity, we undertook live/dead staining and cell counting kit (CCK) assays for 48 h using human bone-marrow-derived mesenchymal stem cells (MSCs, Figure 5). As shown in Figure 5, A, the MSCs in all groups were stained with green (alive) or stained with

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red (dead).28 The number of living cells was visually decreased on the TiHA group compared to those

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of the plate group shown in Figure 5, A. On the other hand, the TiGNP groups showed increased numbers of cells compared to the plate group (Figure 5, A).

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The TiO2 and TiGNP groups demonstrated increased cell proliferation percentages as compared with the plate groups (Figure 5, B). In contrast, the TiHA group showed decreased cell proliferation

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percentages as compared to the plate groups (24 h: 92.76% ± 2.41, 48 h: 86.37% ± 1.29). As shown in

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Figure 5, B, the TiGNP2 group showed the highest cell proliferation rate (24 h: 114.66% ± 3.79, 48 h: 111.94% ± 3.05) among all of the groups. The increased proliferation of cells by GNPs is in accordance with a recently published report.29 They indicated that GNPs enhanced the proliferation of

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cells.29

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Improved osteogenic differentiation of MSCs on TiGNP2 disks We performed ALP activity and qRT-PCR analyses to evaluate the osteogenic differentiation of MSCs on disk groups. The cells were cultured with a GM or an OM for 3, 7, or 14 d. The typical enzyme, ALP, is expressed during the osteogenic differentiation of MSCs.30 As shown in Figure S5, ALP levels in all of the disk groups constantly increased for 14 d. The ALP levels of all groups demonstrated a significant difference at 14 d. (**p < 0.01, Figure S5); these levels were increased in the order of plate (GM), plate (OM), TiO2 (OM), TiHA (OM), TiGNP1 (OM), and TiGNP2 (OM) groups for 14 d (Figure S5). This result indicates that GNPs improved the osteogenic differentiation of MSCs more as compared to that by HA. Typical genes related to osteogenic differentiation, in this case COL1, Runx2, and OPN were investigated by qRT-PCR (Figure 6). COL1 is the most abundant protein in the bone matrix and usually is formed during the proliferation of osteoblast cells.31 COL1 mRNA in all groups was

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recorded at the maximum level at 3 d and steadily decreased up to 14 d (Figure 6, A). Moreover, the highest level of COL1 was observed at each time point in the TiGNP2 (OM) group (Figure 6, A). Runx2 is the key osteogenic transcription factor,31 and the mRNA expression levels showed constant increases in the order of the plate (GM), plate (OM), TiO2 (OM), TiHA (OM), TiGNP1 (OM), and TiGNP2 (OM) groups at each time point (Figure 6, B). OPN levels also demonstrated a similar increasing tendency for 14 d (Figure 6, C). OPN induces calcium deposition in the extracellular matrix during its mineralization stage,31 and it recorded the highest level in the TiGNP2 groups for 14 d (Figure 6, C). These data demonstrate that GNPs improved the osteogenic differentiation of MSCs (Figures S5 and

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6). Although TiHA enhanced the differentiation of MSCs, double layers of GNPs on TiGNP2 disks constantly showed the highest osteogenic differentiation values (Figures S5 and 6).

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After the confirmation of enhanced osteogenic differentiation in the TiHA and TiGNP groups, we performed an in vivo study using Ti pedicle screws in spine surgery. The pedicle screws were

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immobilized with double layers of GNPs (GNP2 screws) and HA (HA screws). These screws were

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then implanted into the vertebra in OVX and SHAM rabbit groups (Figure S6, A and B).

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processes.

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GNPs were rarely disconnected from the surfaces of the screws during the implanting

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We investigated whether GNPs would be disconnected from the surfaces of the screws during the implantation processes. SEM images and EDS results of the screws prior to implantation and SEM images and EDS results of the same screws when immediately unscrewed were compared (Figure S6, C and D). As shown in the EDS results (Figure S6, D), the weight percentages of the Au element were slightly decreased from 1.85 (before implantation) to 1.75 (after implantation). C and D of Figure S6 demonstrate that the stably immobilized double layers of GNPs did not become disconnected during the implantation processes.

Improved osseointegration of GNP2 screws in both OVX and SHAM rabbit models We designated the VOI, including bones and tissues (Figures 7, A–C). Afterwards, we quantified the osseointegration between the bones and screws in 3D space (Figure 7, D and E). The BV/TV in the

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OVX models was significantly increased in the GNP2 group as compared to the HA group (HA group vs. GNP2 group; 19.3% ± 1.6 vs. 23.1% ± 1.1, *p < 0.05, Figure 7, D). The GNP2 group also showed a significant increase in the SHAM model (HA group vs. GNP2 group; 29.3% ± 1.5 vs. 39.3% ± 3.7, *p < 0.05, Figure 7, D). The BS/TV values were significantly increased in the GNP2 groups of both the OVX (HA group vs. GNP2 group; 6.4/mm ± 0.3 vs. 7.8/mm ± 0.3, *p < 0.01, Figure 7, E) and the SHAM models (HA group vs. GNP2 group; 8.5/mm ± 0.2 vs. 9.8/mm ± 0.6, *p < 0.05, Figure 7, E). We present histological images of the HA and GNP2 screws in Figure 8. The parameters within the ROIs were quantified in the OVX and SHAM models (Figure S7). The percentage of BIC in the OVX

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models was significantly increased in the GNP2 group compared to that in the HA group (HA group

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vs. GNP2 group; 33.5% ± 9.5 vs. 54.4% ± 4.4, *p < 0.01, Figure S7, A). In the SHAM model, the percentage of BIC was also significantly increased in the GNP2 group compared to that in the HA

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group (HA group vs. GNP2 group; 56.3% ± 6.0 vs. 69.7% ± 8.7, *p < 0.05, Figure S7, A). The BV levels within the ROI were significantly increased in the GNP2 groups for both the OVX (HA group

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vs. GNP2 group; 52.0% ± 4.9 vs. 59.3% ± 3.9, *p < 0.05, Figure S7, B) and SHAM models (HA group vs. GNP2 group; 69.5% ± 3.1 vs. 81.1% ± 3.0, *p < 0.01, Figure S7, B). Taken together, the

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histological parameters in 2D and in 3D showed higher values in the GNP2 groups than in the HA

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Discussion

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groups.

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The number of osteopenia and osteoporosis patients has increased steadily due to the worldwide ageing of populations.32 Specifically, osseointegration is important in osteopenia and osteoporosis patients due to their low bone densities.33 To investigate whether the GNP2 screws improved osseointegration in OVX models, we performed bilateral OVX surgery on female adult rabbits. OVX surgeries may not be sufficient as the osteoporosis models, 34, 35 but we found that the bone parameters of the HA and GNP2 groups were constantly lower in the OVX models than in the SHAM models (Figures 7, 8 and S7). These decreased parameters in the OVX models indicate that bilateral OVX resulted in osteopenia at least. Masson-Goldner trichrome staining is widely used for histological evaluations of osseointegration between bones and implants.36 Specifically, staining results in green only for bones, making this method quite useful when investigating osseointegration between bones and implants. 36 The BIC and the BV in the ROI are the representative parameters to evaluate osseointegration. In this study, all

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osseointegration parameters for both the HA and GNPs groups were constantly higher in the SHAM models than in the OVX models (Figures 7, D, E and S7). To strengthen the osseointegration of implants, immobilization methods suitable for osteoinductive growth proteins including bone morphogenetic protein-2 (BMP-2), transforming growth factors (TGF-β), and peptides on the implant surfaces have been developed.37 Although these proteins are effective for the osseointegration of implants,38 osteoinductive proteins have a variety of disadvantages, such as low stability, a high cost, stringent storage requirements, and complicated immobilization processes.39 On the other hand, Ti samples require only just two steps for the

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immobilization of GNPs (Figure 1). In addition, the GNPs were strongly immobilized onto the

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surfaces of Ti, not only on the 2D flat surfaces but also on the 3D structure through Au–S bonding.40 To disconnect the Au–S bonds, a considerable amount of energy is required as compared to other

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bonds such as hydrogen or ionic bonds.41 The key osteogenic transcription factor, Runx2, was also increased by GNPs immobilized on Ti disks (Figure 6, B). In addition, the GNP2 screws indicated the

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stable immobilization of GNPs on the screw surfaces after implantation into the bones (Figure S6, C and D). The stably immobilized GNPs after implantation induced osseointegration between the bones

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and screws (Figures 7, 8, and S7).

Furthermore, the immobilized double layers of GNPs promoted the proliferation of cells without

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cytotoxicity (Figure 5). Double layers of GNPs also consistently recorded the maximum values in both the in vitro tests and in vivo models (Figures 6–8, S5 and S7). Our study suggest that implants

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with double layers of GNPs can be a useful alternative in osteoporotic patients.

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Figure legends

Figure 1. Schematic Illustration of the coating processes of Gold Nanoparticles (GNPs) on titanium (Ti) surfaces. Experimental design and application of screw implants in rabbits were demonstrated.

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Figure 2. Preparation and characterization of GNPs

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(A) TEM image of GNPs (scale bar = 50 nm). (B) Absorbance values ranging from 400 to 800 nm. (C) Calibration curve using the differences in the absorbance values according to the GNP

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concentrations.

Figure 3. SEM images of TiO2, TiOH, TiSH, TiGNP1, TiGNP2, and TiHA. They were each

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observed at a magnification level of 10 K. Scale bar: 200 µm.

Figure 4. (A) AFM images. (B) Water contact angles of TiO2, TiOH, TiSH, TiGNP1, TiGNP2, and

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TiHA.

Figure 5. (A) Live and dead staining. (B) Cell cytotoxicity of MSCs on the surfaces of cell culture plates for TiO2, TiHA, TiGNP1, and TiGNP2 for 24 or 48 h. The results are expressed as the mean ± SD (n = 4 per group). “*” indicates a significant difference for which p < 0.05. “**” indicates a significant difference for which p < 0.01.

Figure 6. The mRNA expression levels of (A) COL1, (B) Runx2, and (C) OPN on the surfaces of cell culture plates for TiO2, TiHA, TiGNP1, and TiGNP2. OM was used for 3, 7 or 14 d. Plate groups with GM were also added at 3, 7 and 14 d. The results are expressed as the mean ± SD (n = 4 per group) and the experiments was conducted in triplicate. “*” indicates a significant difference for which p < 0.05. “**” indicates a significant difference for which p < 0.01.

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Figure 7. The µCT images and osseointegration parameters. 3D image of each group was provided. (A) Pristine TiO2 screw, (B) screw images in the OVX model, (C) screw images in the SHAM model, (D) BV/TV percentages within the VOI, and (E) BS/TV value within for which VOI. The results are expressed as the mean ± SD (n = 3 per group). “*” indicates a significant difference for which p < 0.05. “**” indicates a significant difference for which p < 0.01.

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Figure 8. Masson-Goldner trichrome staining images

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(A) HA screw in the OVX model, B) GNP2 screw in the OVX model, C) HA screw in the SHAM

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model, and D) GNP2 screw in the SHAM model. Scale bars: 100 μm.

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Graphical Abstract

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The successful titanium (Ti) implant anchorage depends on strong osseointegration between bones and Ti implants. Specifically, osseointegration is important in osteopenia and osteoporosis patients

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due to their low bone densities. Gold nanoparticles (GNPs) are highly beneficial materials as osteogenic agents. In this study, we aimed to evaluate the osseointegration between bones and double

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layers of GNP-immobilized Ti implants. The double layers of GNP-immobilized Ti implants significantly improved the osseointegration than hydroxyapatite coated implants in ovariectomized

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(OVX) and SHAM rabbit models. These findings suggest that implants with double layers of GNPs

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can be a useful alternative in osteoporotic patients.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8