Effect of crystalline phase changes in titania (TiO2) nanotube coatings on platelet adhesion and activation

Accepted Manuscript Effect of crystalline phase changes in titania (TiO2) nanotube coatings on platelet adhesion and activation

Lu Zhang, Xuhui Liao, Alex Fok, Chengyun Ning, Piklam Ng, Yan Wang PII: DOI: Reference:

S0928-4931(17)31042-1 doi: 10.1016/j.msec.2017.08.024 MSC 8229

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

19 March 2017 7 July 2017 9 August 2017

Please cite this article as: Lu Zhang, Xuhui Liao, Alex Fok, Chengyun Ning, Piklam Ng, Yan Wang , Effect of crystalline phase changes in titania (TiO2) nanotube coatings on platelet adhesion and activation, Materials Science & Engineering C (2017), doi: 10.1016/ j.msec.2017.08.024

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ACCEPTED MANUSCRIPT Effect of crystalline phase changes in titania (TiO2) nanotube coatings on platelet adhesion and activation Lu Zhanga#, Xuhui Liaoa#, Alex Fokb, Chengyun Ningc, Piklam Nga, Yan Wanga* a. Department of Prosthodontics, Guanghua School of Stomatology & Hospital of Stomatology, Guangdong Key Laboratory of Stomatology, Sun Yat-Sen University,

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Guangzhou, China, 510055 b. Minnesota Dental Research Center for Biomaterials and Biomechanics (MDRCBB), School of Dentistry, University of Minnesota, MN 55455, USA

c. School of Material Science and Engineering, South China University of Technology,

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Guangzhou, China, 510641

E-mail: [email protected];

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# Co-first author

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* Corresponding author: Yan Wang

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ACCEPTED MANUSCRIPT Abstract Objective: To explore the relationship between various crystalline phases of titania (TiO2) nanotube (TNT) coatings and platelet adhesion and activation. Methods: TNT coatings were fabricated on pure titanium foils by anodization and then randomly divided into four groups. Three groups were annealed at 350 °C, 450 °C and 550 °C in order to obtain different crystalline phases. The remaining group was not annealed

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and served as the control group. X-ray diffraction (XRD) was used to define the crystalline phases of different groups. Surface morphology, elemental composition, surface roughness, and contact angles were measured by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), laser scanning confocal microscopy (LSCM) and contact angle analysis, respectively. Platelets were cultured on the TNT coatings for 30 min and 60 min to assess the number, viability, distribution, and morphology of the adhered platelets. CD62P fluorescence

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expression and the amount of released platelet-derived growth factor (PDGF) were detected to evaluate platelet activation.

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Results: The un-annealed TNT coatings were amorphous and part of TNT converted to anatase after the 350 °C annealing treatment. The quantity of anatase increased upon

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annealing at 450 °C and transformed to rutile at 550 °C. Nanotubes of all four groups maintained a well-ordered structure, but the wall thickness of the nanotubes increased from (11.874±1.660) nm for the un-annealed TNTs to (26.126±2.130) nm for the 550 °C annealed

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TNTs. The surface roughness of the 550 °C annealed TNT coatings was the lowest and the water contact angle was the largest at (28.117±1.182) degree. The number and viability of

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adhered platelets after 30 min and 60 min were the highest on TNT coatings annealed at 450 °C. LSCM and SEM images revealed that the platelets that adhered on the 450 °C

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annealed TNT coatings aggregated, transformed, and spread most obviously. CD62P fluorescence expression results showed that the platelets on the 350 °C and 450 °C annealed TNT coating groups expressed the strongest fluorescence, followed by platelets on the 550 °C annealed group and the un-annealed group. The quantity of released PDGF was highest for the 450 °C annealed group at (4719±86) pg/mL, and lowest for the un-annealed group at (4241±74) pg/mL. Conclusion: Crystalline TNT coatings encourage improved platelet adhesion and activation over amprphous analogues. The TNT coatings annealed at 450 °C resulted in the most

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ACCEPTED MANUSCRIPT improved platelet behavior. The TNT crystalline phase was the predominant influencing factor in platelet adhesion and activation.

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Key Words: Titania nanotubes; Crystalline phase; Platelet adhesion; Platelet activation

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ACCEPTED MANUSCRIPT 1. Introduction Dental titanium implants are widely used to replace missing teeth1. In order to achieve longterm success, it is necessary to obtain and maintain osseointegration between the implant surface and de novo bone2-4. The complex process of osseointegration is initiated upon blood contact during implant placement followed by the adsorption of plasma proteins, clot formation, osteogenic cell migration, and subsequent implant-cell interactions5-8. A variety of material modifications and surface treatments have been used to enhance the osseointegration

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of dental implants4, 9, 10. Similarly, pre-incubation of human whole blood on a titanium (Ti) implant surface enhances the thrombogenicity and the differentiation of human osteoblasts6. Thrombogenicity is considered very important for early implant healing and implant-blood interface integration is a prerequisite for osseointegration11, 12.

In the initial stage of implant healing, blood makes contact with the implant surface

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immediately, followed by the adsorption of plasma proteins and the mediation of platelet adhesion within a matter of a few seconds. Among blood cells, platelets are the first batch of

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cells to adhere on an implant surface and play a significant role during blood clotting, angiogenesis, and osteogenesis around the dental implant6, 12, 13. After adhering to the implant surface, platelets are activated to release bioactive factors (such as platelet-derived growth

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factor (PDGF) and vascular endothelial growth factor (VEGF)) to initiate the coagulation pathway for blood clot formation. The fibrin matrix of the newly formed clot acts as a

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bioengineering framework and facilitates the migration of mesenchymal stem cells (MSCs) from old bone to the implant surface. As such, blood clot formation around the implant

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surface plays an important role in osteoconduction and promotes contact osteogenesis6, 12-14. PDGF and VEGF function as chemotactic factors and facilitate MSC migration, induce

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osteoblast proliferation, and differentiation. These growth factors can also improve early angiogenesis, bone matrix deposition, and bone formation15-21. These factors point to the importance of platelet adhesion and activation on the implant surface in initiating early healing around dental implants and osseointegration. Notably, platelet behavior is greatly influenced by implant surface properties11, 15, 20-26. Nanomaterials are promising biomedical materials with their unique physiochemical and structural characteristics. They have been widely used in biomedical areas, including nanomedicines, nanocarriers for gene delivery and drugs delivery27-31. With the development of nanotechnology, nanostructural modifications have been applied to implant surfaces with a view to improving osseointegration32,33. For instance, Cei et al. reported that titania (TiO2) 4

ACCEPTED MANUSCRIPT nanotube (TNT) coatings enhanced the quality and speed of bone formation7. Another study reported that TNT coatings enhance protein adsorption with faster adsorption of bovine serum albumin (BSA) and slower desorption34. Furthermore, the number of platelets that adhere on TNT coatings is significantly higher than on pure titanium surfaces, thus shortening the blood clot generation time24. Indeed, TNT coatings enhance cell adhesion, proliferation, and differentiation. Oh et al. reported that osteoblast proliferation on TNT coatings was 300-400% higher than on pure titanium after 48 h incubation35. Cells adhered

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on the TNT coatings and connected with each other through pseudopodia and extended to the inner walls of the nanotubes to increase cell-surface anchorage space36. Moreover, alkaline phosphatase (ALP) activity was 50% higher on the TNT coatings than the uncoated pure titanium analogues37. Enhanced expression of ALP, Col-I, and Osx genes was also found on TNT surfaces38 with elevated levels of extra-cellular matrix secreted39. An in vivo study using

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domestic pigs also demonstrated that TNT coatings enhanced bone formation around implants40.

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Generally, TiO2 nanotubes has three forms: amorphous, anatase and rutile. As-fabricated TiO2 nanotubes at room temperature are usually amorphous. Through annealing at different temperatures, amorphous TiO2 nanotubes were found to transform into anatase or mixed

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phase anatase/rutile crystalline forms41,

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Studies have shown that anatase and mixed

anatase/rutile crystalline phases in TNT coatings can improve hydroxyapatite adsorption and 42-45

. However, to date, there is little consensus on the

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the osteogenic cell response16-18,

optimal crystalline phase for the best cell response. Indeed, little attention has been given to

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the influence of TNT crystallinity on platelet behavior. Inspired by the information outlined above, the aim of this study was to explore the influence

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of different TNT coating crystalline phases on platelet adhesion and activation and further understand the inter-relationship between material crystalline phase, platelet behavior and biomaterial bio-activity. To this end, we fabricated TNT coatings on pure titanium surfaces with different crystalline phases through anodization and subsequent heat treatment and evaluated the platelet adhesion and activation on these various surfaces.

2. Materials and Methods 2.1. Fabrication of TNT coatings

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ACCEPTED MANUSCRIPT Titanium discs (n=228; dimensions: 13.0 × 13.0 × 0.2 mm) were prepared from commercially pure titanium foil (99.6% purity, Qichen Material Corporation, Baoji, China). The discs were polished sequentially with SiC grinding papers (Struers, Denmark) from 200 to 1500 grit before being consecutively ultrasonically cleaned with acetone, ethyl alcohol, and deionized water before being dried in air. TNT coatings were fabricated on the surface of the pure titanium discs by anodization with a potentiostat (RNX 605D, Shenzhen Zhaoxin Electronic Instrument Equipment Co., LTD, China) at room temperature and 20 V for 30 min. A

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titanium disc was used as the anode and a copper sheet served as the counter electrode, with 0.15 mol/L NH4F and 0.5 mol/L (NH4)2SO4 in deionized water used as the electrolyte. A total of 228 samples were prepared. In order to obtain TNT coatings with different crystalline phases, the samples were divided into 4 groups (n=57 for each group): un-annealed, annealed at 350 °C, 450 °C and 550 °C. The annealing procedures were conducted in a resistance

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furnace (SGM T40/10, SGM Equipment Company, Luoyang, China), at the three temperatures mentioned, in air, for 6 h, and at a heating rate of 5 °C/min.

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2.2. Surface characterization

The crystalline phases of the samples were identified by X-ray diffraction (XRD, Bruker D8,

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Bruker, Germany) operating at a scan speed of 0.1 s/step in the 2-θ range of 20-60 degree. The surface morphologies of the TNT coatings were observed by scanning electron microscopy (SEM, Navo Nano SEM430, FEI, the Netherlands) at a voltage of 5 kV. The

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dimensions of the nanotubes were measured with Image Pro plus 6.0 software. Three fields from each sample were randomly selected and 20 nanotubes from each field were manually

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measured for the outer, inner diameter and wall thickness of the nanotubes. The chemical compositions of the TNT coatings were assessed through energy dispersive spectroscopy

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(EDS, FEI, the Netherlands), which was incorporated into the SEM system. Surface topography was observed and roughness was measured using laser scanning confocal microscopy (LSCM, LSM 700, Zeiss, Germany) through non-contact method. Contact angles were measured using a contact angle analyzer system (OCA15, Dataphysics, Germany), using a 2 L drop of deionized water. Measurements were taken 10 s after the application of the drop. 2.3. Platelet preparation and incubation This portion of the study protocol was approved by the Sun Yat-sen University Ethics Review Board. Human whole blood was collected from a young healthy volunteer with a 6

ACCEPTED MANUSCRIPT vacuum blood collection tube containing 3.2% sodium citrate as an anticoagulation agent (BD Vacutainer, USA). To prepare platelet-rich plasma (PRP), the collected blood was centrifuged immediately at 200 g for 10 min and the supernatant was collected. The supernatant was centrifuged again at 200 g for 10 min and then the top three-quarters of the supernatant was removed and the rest of the plasma was mixed to yield PRP for immediate incubation. TNT coated samples were placed in 24-well plates (Corning, USA) and 200 μL PRP was added to each well. The 24-well plates were centrifuged at 150 g for 10 min before

2.4. Platelet counting and viability assessment

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being incubated on a horizontal shaker at 37 °C for further investigations.

After incubation with PRP for 30 min and 60 min, the supernatants from each well were collected in separate centrifuge tubes (Corning, USA). In order to measure the number of

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remaining platelets, a 20 μL aliquot of supernatant was added into a cell counter (Z2, Beckman, USA) and counted: then the number of adhered platelets was calculated by subtracting the number of remaining platelets from the number of platelets in the prepared

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PRP11. The samples were then washed three times with 0.01 M 1X phosphate buffered solution (PBS; pH 7.2-7.4) and transferred to new 24-well plates. The viability of the platelets was quantified using commercially available CCK-8 assay kits (Dojindo, Japan)

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according to the manufacturer’s instructions. After 3 h incubation at 37 °C, 100 μL of the test solution from each well was added into wells of a 96-well plate and the absorbance was

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measured using an automatic plate reader (Infinite200, Tecan, Switzerland) at a wavelength

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of 450 nm24.

2.5. Platelet adhesion and distribution

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The distribution of the adhered platelets on the sample surfaces was visualized through fluorescence staining. After incubation with PRP for 30 min, the samples were washed three times in 0.01 M 1X PBS and transferred to a new 24-well plate. A 500 μL aliquot of 10 μg/L rhodamine staining solution (Sigma, USA) was added into each well and the plates were incubated at 37 °C for 15 min in darkness. The staining solutions were then removed and the samples were washed three times in PBS. The adhesion and distribution of the platelets was observed by LSCM (LSM 780, Zeiss, Germany). 2.6. Platelet morphology

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ACCEPTED MANUSCRIPT To observe platelet morphology, adhesion, and activation on the TNT coated surfaces, the samples were incubated with PRP for 30 min and 60 min. The supernatants were then removed and the samples were washed three times in PBS. Then, 500 μL of a 2.5% glutaraldehyde solution was added to each well for 12 h at 4 °C to fix the adhered platelets on the sample surfaces. To dehydrate the platelets, 20%-100% ethyl alcohol solutions were serially added and removed. Sample surfaces were gold coated to observe the platelets using

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SEM. 2.7. CD62P expression

Following 60 min incubation with PRP, the supernatant was removed and the samples were washed three times in PBS. An aliquot of 500 μL 4% paraformaldehyde was added into each well for 15 min to fix the platelets on the sample surfaces. After removal of the

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paraformaldehyde, the samples were blocked with 1% bovine serum albumin for 30 min. Then, PE-conjugated CD62P antibody (Abaca, USA) was added and incubated for 2 h at

were observed by LSCM.

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2.8. Detection of PDGF release

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room temperature in darkness. After removal of the staining solution, the sample surfaces

The amount of PDGF released from the activated platelets was detected using commercially available PDGF-AB ELISA kits (Cloud-Clone, USA), according to the manufacturer’s

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instructions. Briefly, after incubation with PRP for 60 min, 40 μL of the supernatant from each sample was collected in a centrifuge tube and diluted 5-fold with PBS. Then, 100 μL of

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the diluted solutions from each sample was added to wells of a 96-well plate. ELISA reagents were added into the plates according to the manufacturer’s instructions. The plates were

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incubated and absorbance was measured by an automatic plate reader at a wavelength of 450 nm. Curve Expert 1.3 software was used to calculate the amount of released PDGF. 2.9. Statistical analysis Each experiment was repeated at least three times. Results were recorded as the mean ± standard deviation (SD) and analyzed by SPSS software (version 17.0, IBM, USA). One way analysis of variance (ANOVA) was used to compare the difference among various groups and the significant level was set to p< 0.05. 3. Results 8

ACCEPTED MANUSCRIPT 3.1. Surface properties of TNT coatings The crystalline phase patterns of the TNT coatings were obtained by XRD, Figure 1. Unannealed TNT coatings only showed Ti peaks (100) and (101), indicative of the amorphous nature of the un-annealed TNT coatings. The characteristic anatase peak (101) appeared in the 350 °C annealed group. The proportion of anatase increased as the annealing temperature increased from 350 °C to 450 °C, with increasing conversion of amorphous nanotubes to

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anatase. For the TNT coatings annealed at 550 °C, rutile peaks [(110), (101), (111), (211),

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(220)] were also found, representing mixed crystalline phases of anatase and rutile.

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Fig. 1 XRD patterns of TNT coatings annealed at different temperatures

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A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed

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at 450 °C; D: TNT coating annealed at 550 °C.

The surface morphologies of the TNT coatings can be observed in SEM images, Figure 2. Regardless of the annealing temperature, all sample surfaces demonstrated a well-ordered structure composed of nanotubes. The dimensions of the nanotubes were measured by Image Pro software and are detailed in Table 1. Interestingly, while the outer diameters of the nanotubes annealed at different temperatures were similar, the inner diameters of the TNTs annealed at 550 °C were significantly smaller than in the un-annealed TNT coated group (Table 1). Indeed, the nanotube walls thickened significantly as the annealing temperatures increased.

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Fig. 2 Surface morphologies of TNT coatings annealed at different temperatures

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A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed at 450 °C; D: TNT coating annealed at 550 °C. The scan bars in the main images (50000)

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represent 500 nm and in the magnified inserts (100000) represent 50 nm.

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Table 1 Dimensions of TNTs annealed at different temperatures

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Size of TNTs (nm) Group

Outer diameter

Inner diameter

Wall thickness

122.659±10.006

89.817±5.238a

11.874±1.660c

120.301±13.524

84.954±12.502ab

15.163±1.614d

119.547±9.952

80.284±7.640ab

19.061±2.842e

550 °C

114.974±17.300

77.388±10.295b

26.126±2.130f

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0.647

3.426

108.163

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0.590

0.029

0.000

350 °C 450 °C

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Un-annealed

(* different letters refer to significant differences between groups)

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ACCEPTED MANUSCRIPT The elemental compositions of the TNT coatings are listed in Table 2. All nanotube coating surfaces contained titanium (Ti) and oxygen (O). The relative proportions of Ti decreased and O increased with increasing annealing temperature. The un-annealed TNT coatings contained 7.97% of fluorine (F), which decreased upon annealing at 350 °C, and vanished in the 450 °C and 550 °C groups. Table 2 Elemental composition of TNT coatings annealed at different temperatures Element atom proportion (%)

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Group

O

F

Un-annealed

46.11

45.92

7.97

350 °C

45.39

50.92

3.69

450 °C

44.86

55.14

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550 °C

41.93

58.07

-

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Ti

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The surface topographies of the TNT coatings were examined by LSCM, 3D reconstructions of the surfaces are shown in Figure 3. All of the TNT coatings had similarly flat surface profiles, with yellow and green colors in the un-annealed, 350 °C and 450 °C groups, but the

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TNT coatings annealed at 550 °C showed more blue areas, indicative of lower roughness. The surface roughness values of the nanotube coatings are detailed in Table 3. According to

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the surface roughness values, the un-annealed, 350 °C, and 450 °C annealed coatings were similarly rough, while the 550 °C annealed group exhibited significantly lower roughness as

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shown in Table 3.

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Fig. 3 Reconstructed 3D images of TNT coatings annealed at different temperatures A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed

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at 450 °C; D: TNT coating annealed at 550 °C.

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Table 3 Surface roughness of TNT coatings annealed at different temperatures

Ra

Rp

0.731±0.072a

0.918±0.088c

0.718±0.052ab

0.908±0.062cd

0.722±0.091ab

0.907±0.116cd

0.562±0.123b

0.697±0.156d

4.220

4.648

0.022

0.016

Surface roughness (μm）

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Group

450 °C 550 °C F P

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350 °C

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Un-annealed

(*Ra: arithmetical mean deviation of the profile; Rp: average profile peak height. * Different letters refer to significant differences between groups.)

The real-time images of contact angles on TNT coatings are shown in Figure 4. The contact angles recorded for the un-annealed coatings and the 350 °C and 450 °C annealed coatings were (12.117±0.741)°, (13.017±0.845)° and (13.550±0.963)° respectively. However, the 12

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(28.117±1.182)°, Figure 5.

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Fig. 4 Real-time images of the water contact angles captured after droplet application on the TNT coatings annealed at different temperatures

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A: Un-annealed TNT coatings; B: TNT coatings annealed at 350 °C; C: TNT coatings

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annealed at 450 °C; D: TNT coatings annealed at 550 °C.

Fig. 5 Water contact angles of TNT coatings annealed at different temperatures (* different letters refer to significant differences between groups)

3.2. Platelet counting 13

ACCEPTED MANUSCRIPT The numbers of platelets adhered on the TNT coatings after incubation for 30 min and 60 min are presented in Figure 6. At the 30 min time-point, the numbers of adhered platelets were significantly different between different groups (p<0.05). The number of adhered platelets was the lowest for the un-annealed TNT coatings, at (234902±7638) platelets/μL, and the highest for the 450 °C annealed TNT coatings, at (300729±8325) platelets/μL. At the 60 min time-point, the numbers of adhered platelets on all samples increased compared with the values after 30 min incubation. The number of adhered platelets on the 450 °C annealed TNT

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coatings after 60 min was still the highest, at (336836±12618) platelets/μL. Significant differences between the numbers of adhered platelets at 60 min were also found between the

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TNT coatings annealed at different temperatures (p<0.05).

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Fig. 6 The number of platelets adhered on TNT coatings annealed at different temperatures at 30 min and 60 min

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(* different letters refer to significant differences between groups)

3.3. Platelet viability The OD450 value (the absorbance value at 450 nm wavelength) was used to represent the viability of the adhered platelets. After 30 min incubation, the OD450 values for the unannealed TNT coatings, the 350 °C annealed group, and the 450 °C annealed group were (1.342±0.068), (2.078±0.082) and (2.139±0.045), respectively. The platelet viability dropped to (1.865±0.069) for the 550 °C annealed group. Significant differences were found between

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ACCEPTED MANUSCRIPT the un-annealed group and the 350 °C/450 °C groups and the 550 °C group, but not between the middle two groups, i.e. the 350 °C and 450 °C groups (p<0.05). At the 60 min time-point, the viability of the adhered platelets on the TNT coatings annealed at 450 °C was still the highest, at (2.354±0.091), followed by the 350 °C and 550 °C annealed groups, at (2.166±0.095) and (2.042±0.010) respectively. The un-annealed group platelets still exhibited the lowest relative viability, at (1.785±0.097). There were significant differences among all of

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the four groups (p<0.05), Figure 7.

Fig. 7 Platelet viability on TNT coatings annealed at different temperatures after 30 min

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and 60 min incubation

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(* different letters refer to significant differences between groups)

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3.4. Platelet distribution

In order to better understand the interactions between the platelets and the TNT coatings, the adhered platelet distributions on the sample surfaces were visualized after 30 min incubation, Figure 8. The LSCM images confirmed that the number of platelets adhered on the unannealed TNT coatings was considerably lower than on the annealed nanotube coatings. Comparatively, on the annealed TNT coatings, higher numbers of adhered platelets were found with projecting pseudopodia to connect with each other, demonstrating early aggregation. The adhered platelets on the TNT coatings annealed at 450 °C seemed to be distributed most densely, with the highest amount of aggregation and the least number of separate platelets observed. 15

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Fig. 8 LSCM images of platelets adhered on TNT coatings annealed at different temperatures A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed

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at 450 °C; D: TNT coating annealed at 550 °C. The scan bars represent 50 μm.

3.5. Platelet morphology

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The morphologies of the adhered platelets on the TNT coatings are representative of the platelet activation status and were observed through SEM. After 30 min incubation, the platelets on the un-annealed TNT coatings displayed little transformation and maintained their round-shaped, indicative of the adhesion stage. However, the platelets on the annealed TNT coatings all obviously transformed, extending filopodia to form connections with each other, which is indicative of platelet activation. No obvious platelet morphological differences were observed between the 350 °C and 550 °C annealed groups. However, some platelet filopodia on the 450 °C annealed coatings had started to extend, demonstrating a spreading tendency and advanced activation, Figure 9.

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Fig. 9 SEM images of platelets adhered on TNT coatings annealed at different temperatures after 30 min incubation

A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed

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at 450 °C; D: TNT coating annealed at 550 °C. The scan bars represent 2 μm.

After 60 min incubation, the platelets on all of the TNT coatings had transformed and spread

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more obviously compared to those incubated for just 30 min. After 60 min incubation, the platelet morphologies showed more obvious differences among the various TNT coating

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groups. The platelets on the un-annealed TNT coatings did not spread extensively but did protrude filopodia to interconnect. The platelets on the 350 °C annealed TNT coatings spread to form large lamellipodia, indicating a higher degree of activation. On 450 °C annealed TNT coatings, the platelets were almost fully spread, demonstrating the highest degree of activation. Platelets on the 550 °C annealed TNT coatings partially spread and established filopodia connections, Figure 10.

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Fig. 10 SEM images of platelets adhered on TNT coatings annealed at different temperatures after 60 min incubation

A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed

3.6. CD62P expression

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at 450 °C; D: TNT coating annealed at 550 °C. The scan bars represent 2 μm.

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CD62P is a membranous protein that is considered one of the biomarkers of activated platelets and is expressed on platelet surfaces. The expression of CD62P can be measured

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semi-quantitatively by fluorescence measurements. The CD62P expression levels of platelets adhered on TNT coatings after 60 min incubation are shown in Figure 11. The platelets on the un-annealed TNT coatings distributed separately and aggregated the least, and displayed weak fluorescence. The platelets adhered on the 350 °C and 450 °C annealed TNT coatings showed the most aggregation with strong fluorescence expression. Platelets adhered on the 550 °C annealed TNT coatings showed less aggregation and weaker CD62P expression than the 350 °C and 450 °C annealed groups, but stronger expression than that observed in the unannealed TNT group.

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Fig. 11 CD62P expression of active platelets on TNT coatings annealed at different temperatures after 60 min incubation

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A: Un-annealed TNT coating; B: TNT coating annealed at 350 °C; C: TNT coating annealed

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at 450 °C; D: TNT coating annealed at 550 °C. The bars represent 50 μm.

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3.7. Detection of released PDGF Platelet activation was quantified by measuring the concentration of PDGF-AB from activated platelets. The measured PDGF-AB concentrations from the un-annealed TNT coatings and the TNT coatings annealed at 350 °C, 450 °C, and 550 °C were (4241±74), (4488±74), (4719±86) and (4400±82) pg/mL, respectively. The values recorded for the 450 °C annealed group were the highest, while the values recorded for the un-annealed group were the lowest. However, there was no significant difference between the values recorded for the 350 °C and 550 °C annealed groups, Figure 12.

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Fig. 12 PDGF released from platelets on TNT coatings annealed at different temperatures

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(* different letters refer to significant differences between groups)

4. Discussion

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Osseointegration between a dental implant and bone tissue is extremely critical for implant healing and long-term success3, 4. The surface properties of dental implants such as surface

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morphology, surface roughness, chemical composition, crystalline phase, and hydrophilicity can remarkably influence the speed and degree of osteogenesis and the strength of boneimplant contact33,

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. In order to enhance the biological properties of implants (such as

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biocompatibility and bioactivity), researchers have tested a variety of surface treatment techniques. Nanoscale surface modifications have shown promise in dental implant studies

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in-terms of the biological outcomes. However, the thrombogenicity of nanoscale surfaces, thus far, has not been extensively studied and the thrombogenicity of TNT coatings with

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differing crystallinities has not been reported and further research is needed11, 25. In the present study, TNT coatings were fabricated on top of pure Ti surfaces through anodization, and the crystalline phases were tailored through further annealing. According to the XRD results, the as-fabricated TNT coatings were amorphous and partially converted to anatase after annealing at 350 °C. The amount of anatase increased after annealing at 450 °C, and transformed to mixed anatase/rutile crystalline phases upon annealing at 550 °C. This result is consistent with previous studies, which reported that amorphous TNTs started to form anatase at 280 °C, with conversion almost completed at approximately 400 °C, followed by rutile gradually replacing anatase when the annealing temperatures were elevated to 20

ACCEPTED MANUSCRIPT 450 °C and above. If the temperature was raised to above 600 °C, the majority of the anatase converted to rutile, resulting in the collapse of the nanotubes41, 42, 47, 48. Compared to these previous studies, the amount of rutile was higher in the present study, which may be due to the comparatively longer 6 h annealing time used. Previous studies have reported that the anatase size, electron mobility and crystallization were increased by extending the annealing duration from 2 h to 6 h. Longer annealing times can lead to a higher amount of rutile and

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better crystallization41, 49. Changes in the crystalline phase induce changes in the dimensions of the TNTs. The SEM images, shown in Figure 2, show that for all groups, regardless of the annealing temperature used within this study, the nanotube structures were well-ordered on the Ti surfaces. However, the wall thickness of the TNTs increased from (11.9±1.6) nm for the un-annealed TNTs to (26.1±2.1)nm for TNTs annealed at 550 °C. Previous studies have demonstrated that

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the crystalline size of anatase enlarges in response to higher annealing temperatures. Furthermore, the conversion of anatase to rutile, which occurred at the highest temperature

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used in this study, resulted in larger crystals41, 43. Therefore, the TNT walls thickened as the annealing temperature increased and crystalline phases converted from amorphous, anatase to mixed antase/rutile.

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The chemical composition of the nanotubes slightly changed when the annealing temperature increased, as the crystalline phases changed. The TNT coatings were primarily comprised of

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elemental titanium and oxygen. As the annealing temperatures increased, the ratio of Ti/O decreased. In addition, at higher annealing temperatures, the proportion of elemental F

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decreased, before finally vanishing. Rogonini et al. suggested that fluorine present in the nanotubes may originate from the presence of the element in the anodization electrolyte50.

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Transformation of amorphous TNTs to anatase TNTs probably resulted in vacancy defects in the nanotube walls, reducing the proportion of Ti 51. Meanwhile, the F element likely reacts with Ti to form TiF4 and vaporizes out of the TNTs during the annealing process at higher temperatures 42. In this study, the surface roughness of the TNTs with different crystalline phases was similar. Only the TNT coating annealed at 550 °C had a significantly lower surface roughness. Yang et al. reported that rutile firstly formed at the interface between the Ti substrate and the TNT coating and gradually replaced anatase from the bottom of the TNT coating as the annealing temperature increased. Since the crystal size of rutile is larger than the TNT wall thickness,

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ACCEPTED MANUSCRIPT the rutile merges towards the bottom of the TNTs, smoothing the interface between the Ti substrate and the TNT coating, thereby decreasing the surface roughness42. With respect to hydrophilicity, the water contact angles of the un-annealed TNT coatings and those annealed at 350 °C and 450 °C were very similar, close to 10°, indicating excellent hydrophilicity. However, the contact angle of TNT coatings annealed at 550 °C were significantly higher, indicating lower hydrophilicity. Previous studies have suggested that

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rutile growing inside the walls of the nanotubes increased the wall thickness and consequently decreased the inner diameter of the nanotubes, as outlined above. These changes result in a reduction of the specific surface area, surface roughness, and lower the hydrophilicity of the TNT coatings41, 52.

Platelet behavior upon implant contact can be strongly influenced by the surface properties of the Ti implants11, 15, 20-26. In the present study, platelet adhesion and activation were higher on

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the annealed TNT coatings than the un-annealed TNT coatings after 30 min incubation. Among the annealed TNT coatings, 350 °C and 450 °C annealed coatings, comprised of an

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increasing proportion of anatase with increasing annealing temperature, encouraged enhanced platelet adhesion and subsequent activation compared to TNT coatings annealed at 550 °C,

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comprised of mixed anatase and rutile crystalline phases. By extending the incubation time to 60 min, the differences in platelet adhesion and activation between the 350 °C and 450 °C annealed groups had increased. The 450 °C coatings, containing the higher relative amount of

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anatase, resulted in the best platelet adhesion and activation outcomes. Therefore, it can be

behavior.

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deduced that the crystallinity of TNT coatings plays an influential role in-terms of platelet

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To date, the definitive reasons why the crystalline phase changes affects platelet behavior is still unclear and may be explained by band gap theory or vacancy defect theory. Crystalline TiO2 is a semiconductor with an empty energy area in its atom orbits, termed a band gap. The band gap of anatase is 3.2 eV while that of rutile is 3.0 eV. The larger band gap of anatase suppresses electron release, thus more electrons (negative charge) are retained on the material surface, which in-turn may promote protein (positive charge) adsorption on its surface21, 53. Moreover, as anatase transforms to anatase/rutile, the number of oxygen vacancy defects in the TNT coatings decreases43. The oxygen vacancy defects can lead to the dissociation of water to form hydroxyl on the material surfaces, which additionally encourages protein

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ACCEPTED MANUSCRIPT absorption54. Therefore, the anatase TNT coating surface can promote protein adsorption, thus mediating enhanced platelet adhesion. Platelet morphology can be used to evaluate the degree of platelet activation. Morphologically, platelets can be divided into five types: round, dendritic, spread dendritic, spreading and fully spreading. Enhanced activation of platelets is associated with more obvious transformation55. In this study, compared with platelets seeded on un-annealed TNT

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coatings, platelets on annealed TNT coatings showed obvious changes in morphology and extended long filopodia to connect with each other after only 30 min incubation. After 60 min incubation, the platelet spreading was more obvious. In particular, the platelets seeded on TNT coatings annealed at 450 °C showed the best spreading, suggesting the most optimal platelet activation conditions. This indicates that the crystalline structure attained after annealing at 450 °C is most conducive to platelet activation. Possibly this can be explained

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by the fact that crystalline TNTs, especially anatase TNTs, have a more ordered atomic arrangement than the amorphous form, which result in the promotion of cell adhesion and

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spreading56.

In order to further investigate the degree of platelet activation, CD-62P (P-selection) expression was detected. CD62-P is a protein stored in granule form in resting platelets and is

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expressed on the platelet membrane surface upon activation57. In this study, the degree of CD-62P expression on platelets seeded on un-annealed TNT coatings was lower than those

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seeded on the annealed TNT coatings. Although no obvious difference was observed between the expression of platelet CD-62P on TNT coatings annealed at 350 °C and 450 °C, both

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groups showed elevated CD-62P expression compared to the 550 °C annealed group. When platelets are activated, they release a series of growth factors, including PDGF, one of the

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most important growth factors released by platelets, which is associated with subsequent osteoblast response and osteogenesis. Therefore, the amount of PDGF released can be used to quantify the degree of platelet activation11. The results of the present study show that the quantity of PDGF-AB released in the 450 °C annealed group was higher than in the 350 °C annealed analogue, which indicates that the degree of platelet activation was higher on the TNT coatings comprised of higher amounts of anatase. The change in crystalline phase seems to be a dominant factor that directly influences the platelet adhesion and activation behavior on TNT coatings. However, the surface morphology, roughness, elemental composition, and hydrophilicity also affect platelet behavior58. TNTs

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ACCEPTED MANUSCRIPT are nanoscale structures, and thus naturally impart a nanoscale effect, namely significantly greater specific surface area, which enhances protein adsorption and ultimately mediates increased platelet adhesion39, 57, 59-61. Fibronectin (FN) adsorbs on TNT surfaces and mediates platelet adhesion through Arg-Gly-Asp (RGD) sequences and integrins on platelet surfaces36, 62

. However, the surface morphological features of TNT coatings annealed at different

temperatures were highly similar in this study, hence, they were not deemed a critical factor in influencing platelet behavior. As discussed earlier, surface roughness is another important surface roughness increases22,

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surface property. On microscale surfaces, the number of adhered platelets increases as the . The effect of nanoscale surface roughness on platelet

adhesion has not yet been definitively established. In the present study, the surface roughness of TNT coatings annealed at 550 °C was lower than that of the un-annealed coatings and the 350 °C and 450 °C annealed coatings. This may have contributed to the lower platelet

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adhesion observed for the 550 °C annealed group compared to adhesion on the surfaces annealed at lower temperatures. The elemental composition of implant surfaces also may influence platelet behavior. The presence of F can enhance platelet adhesion and activation26.

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There was a small quantity of elemental fluorine on the surface of the TNT coatings which were un-annealed and annealed at 350 °C, which may be a positive factor in-terms of platelet

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behavior. However, this positive effect may not be appreciable in this study due to the stronger influences of other factors such as phase crystallinity. Studies have compared the numbers of platelets adhered on sandblasted and acid-etched and sandblasted and acid-etched

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active surfaces and concluded that increased surface hydrophilicity greatly promoted platelet adhesion21, 56. Therefore, the decreased surface hydrophilicity observed for the TNT coatings

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annealed at 550 °C probably negatively affected the platelet adhesion and activation.

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It is important to note that in this study, all of the findings are based on in vitro experiments in which the complex oral environment could not be exactly simulated. The oral environment and human body response may greatly affect the outcome. Hence, more in vitro and in vivo studies are required to attain definitive conclusions which should contribute to the further refinement of surface techniques for optimal oral implant outcomes.

5. Conclusions Considering the different surface properties of TNT coatings annealed at different temperatures, the change in the crystalline phase seems to be the key factor which influences 24

ACCEPTED MANUSCRIPT platelet adhesion and activation. The results outlined herein point to the superior influence of crystalline TNT coatings on platelet adhesion and activation over un-annealed amorphous counterparts. Among the crystalline groups, TNT coatings annealed at 450 °C contained a higher amount of anatase resulting in improved platelet behavior outcomes, with improved platelet adhesion, better viability, obvious aggregation and activation achieved. Indeed these results indicate that a higher amount of anatase in the TNT coatings encourages better platelet adhesion and activation behavior. As such, 450 °C appears to be the most suitable heat

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treatment temperature to achieve the best TNT coating modifications in-terms of improved platelet response. This investigation comprehensively compared platelet adhesion and activation on TNT coatings with different crystalline phases and explored the relationship between crystalline phase change and subsequent platelet contact behavior. However, the interactions of proteins and whole blood with TNT coatings of different crystallinity merit

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further investigation to better understand the thrombogenicity of the TNT coatings and will

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be the focus of future studies.

Acknowledgments

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This study was financially supported by the National Natural Science Foundation of China

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(No. 81550013).

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ACCEPTED MANUSCRIPT Highlights 1. Crystalline TNT coatings improve platelet adhesion and activation 2. Higher amount of pure anatase in the TNT coatings benefits for better platelet behavior

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3. 450 °C is suitable heat treatment temperature for TNT coating modifications

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