Nano Textured PEEK Surface for Enhanced Osseointegration - ACS

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Bio-interactions and Biocompatibility

Nano Textured PEEK Surface for Enhanced Osseointegration Liping Ouyang, Meiling Chen, Donghui Wang, Tao Lu, Heying Wang, Fanhao Meng, Yan Yang, Jingzhi Ma, Kelvin Wai Kwok Yeung, and Xuanyong Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01425 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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ACS Biomaterials Science & Engineering

Nano Textured PEEK Surface for Enhanced Osseointegration Liping Ouyanga,b,1, Meiling Chenc,1, Donghui Wanga, Tao Lua, Heying Wanga, Fanhao Menga, Yan Yangc, Jingzhi Mac,*, Kelvin W. K. Yeung d,*, Xuanyong Liua,b,* a

State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China b Center

of Materials Science and Optoelectronics Engineering, University of Chinese

Academy of Sciences, Beijing 100049, P. R. China c

Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan, P. R. China d

Department of Orthopaedics and Traumatology, The University of Hong Kong,

Hong Kong, P. R. China Corresponding Author Prof. Xuanyong Liu. E-mail: [email protected]. Tel: +86 2152412409. Fax: +86 21 52412409. Prof. Kelvin W. K. Yeung. E-mail: [email protected] Prof. Jingzhi Ma. Email: [email protected]

Keywords: polyetheretherketone; nanostructure; osseointegration

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Abstract: Polyetheretherketone (PEEK) is widely used in orthopedic and dental applications due to its similar mechanical properties to those of natural bones. However, the inferior osseointegretion and bioinertness hamper the clinic application. Surface texture of biomaterials plays an essential role in controlling cell differentiation through affecting the cell-generated physical forces, thus improving the osseointagration of the substrate. In this work, argon PIII and subsequently hydrogen peroxide treatment are applied to construct nano structure on the PEEK surface. The in vitro results show that the cell adhesion, collagen secretion and extracellular matrix (ECM) deposition can be enhanced both on these two nanostructured surface. The in vivo tests exhibit that the surface fabricated by physical-chemical treatment is more favorable for fibrous tissue filtration inhibition and osseointegration than that fabricated by argon PIII only. This work provides a candidate approach for improving the osseointegration ability of PEEK implant by construing the nanostructure on its surface, which paves the way of applying PEEK in orthopedic and dental applications.

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1. Introduction Polyetheretherketone (PEEK) is the prime candidate for bone implants to replace metallic implants due to its superior properties. Besides, PEEK, a semi crystalline polymer that has an approximate crystallinity of 30-35% and a glass transition temperature of 143 ºC,1,2 bodes well for manufacturing the suitable shape for diverse application. The elastic modulus of PEEK ranges between 3 and 4 GPa, which can be tailored to closely match cortical bone (~18 GPa).1 The degradation of the mechanical properties of metallic implants caused by corrosion can be avoided due to the well-known good chemical resistance of PEEK.3 Though PEEK exhibits many superior properties, the bioinertness and inferior osteoconduction of PEEK hamper its clinical application. Herewith, it is important to improve the bioactive and osseointegration of PEEK for its application in clinic. Modification of surface composition and morphology are two main strategies to obtain implants with superior biological properties, such as antibacterial capacities and osteoconduction.4-15 Considering the stringent safety requirement of biomaterials, materials modified by surface topology construction are more promising in the clinical application than those modified by element doping, which may possess toxicity on cell, blood and organ. The micro/nano-textured implants are more favorable for cell initial fixation and thus exhibit enhanced osteoblast attachment and differentiation

than

smooth-textured

implants.16-18

Nanostructures,

including

nanopores, nanotubes, nanopits, nanosphere, nanorods, nanopalates, play an important role in cell behaviour alternation.16-17, 19-24 The cell response to the ordered structure, 3



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such as TiO2 nanotubes, varies with the diameter of nanotubes, nevertheless the optimal diameter is controversial.17,

19-23

The ideal surface roughness of unordered

structure for optimal osseointegration also has been widely researched.16,

24-33

Developing nanostructures on PEEK surface and investigating their influence on cell behavior will provide PEEK a preferred surface for bone formation. Many efforts have been made to obtain the PEEK with the optimal surface structure that possess well osteoconduction. Yoon BJ et al applied plasma spray processes incorporated grit blasted to alter the surface roughness of PEEK and verified that the increased surface roughness of PEEK can improve cell proliferation.34 Gan K et al used nitrogen plasma immersion ion implantation to construct different roughed surface on PEEK, and the surfaces with the cylindrical structure were more favorable for cell differentiation than the surfaces with granules under the existence of similar nitrogen content.35 Argon plasma immersion ion implantation (PIII) can avoid the introduction of undesirable impurity components, thereby it is a good choice to modify PEEK with the fine structure through disrupting the structure of polymers.5-6, 8-9, 36-38

Except for PIII, concentrated sulfuric acid can etch PEEK, forming 3D

network on its surface, which is verified to have good osseointegration and antibacterial properties.15,

39

Therefore, in this work, argon PIII and followed

hydrogen peroxide treatment are employed to construct nanostructures on the PEEK surface. In vitro osteoblast responses to these two structures are evaluated by rat bone marrow mesenchymal stem cells (rBMSCs). A craniofacial model is utilized to evaluate the osseointegration ability of the samples in vivo. 4


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2. Results

Figure 1. Surface morphology of various samples: (a) PEEK, (b) A-PEEK, (c) AH-PEEK. The surface of PEEK is relative flat, the surface of A-PEEK is convex, and the surface of A-PEEK is concave. The red circles mark the tangent nanostructures, the blue circles mark the separated nanostructures, and the yellow circles mark the intersected nanostructures. The red arrows mark some of the tangent nanostructures, the blue arrows mark some of the separated nanostructures, and the yellow arrows mark some of the intersected nanostructures. Surface characterization. The surface morphologies of the samples are shown in Figure 1. The surface of the pristine PEEK is relative flat (Figure 1a). The samples treated by argon PIII have an upwardly convex structured surface (Figure 1b), and the downwardly concave structure is derived from the upwardly convex structure via the subsequent hydrogen peroxide treatment (Figure 1c). The size of nanostructures ranges from 30 nm to 50 nm. Argon PIII can scissor polymer chain and generate free radicals on the surface of samples, thus providing an active surface for the subsequent modification. Hydrogen peroxide tends to etch the active surface constructed by argon 5



PIII rather than PEEK substrate due to the chemical resistance of pristine PEEK. Hydrogen peroxide treatment is applied only to further study the chemical resistance of pristine PEEK to hydrogen peroxide, the results show that there is not any structure change on the samples (Figure S1), which indicates that PIII treatment before chemical treatment is essential for constructing nanostructure on PEEK surface. The concave nanostructure is derived from the convex nanostructure and the one to one correspondence between the convex and concave nanostructure has been subtly fabricated by utilizing the chemical resistance of pristine PEEK and the active surface generated by argon PIII (hydrogen peroxide cannot etch pristine PEEK, Figure S1). As Figure 1 shows, the positions of the two adjacent convex or concave nanostructures can be concluded in three cases: intersection (the yellow circles), tangent (the red circles), and separation (the blue circles). The arrows in yellow, red and blue mark the intersected, tangent, and separated nanostructures in Figure 1. Surface structure and chemical composition are two main factors that influence the cell response. XPS is employed to characterize the surface chemical states of the samples and the results are displayed in Figure 2a, b and c. Only carbon (C1s) and oxygen (O1s) can be detected on all samples, proving that the surface chemical compositions of PEEK, A-PEEK and AH-PEEK are same. The surface functional groups with same chemical composition can be varied, so it is essential to figure out the surface groups for analyzing the relationship between structures and cells. The Fourier transform infrared spectra was employed to analysis the surface functional groups and the results are displayed in Figure 2d. In the spectra, the diphenylketone 6


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bands at 1650, 1490 and 926 cm-1, and C-O-C stretching vibration of the diaryl groups at 1188 and 1158 cm-1 can be observed on all samples, indicating that there are the same groups existing on the surface of PEEK, A-PEEK and AH-PEEK.39-41 The wavelength and peak intensity of A-PEEK are the same as those of AH-PEEK, indicating that the surface functional groups on A-PEEK and AH-PEE are identical. It can be concluded that the convex and concave nanostructure without undesirable impurity components have been fabricated through argon PIII and hydrogen peroxide treatment. The chemical composition and surface functional groups of the convex nanostructured samples and the concave nanostructured samples are same completely, providing a perfect platform for studying cell response to structure.

Figure 2. XPS full spectra of the samples: (a) PEEK, (b) A-PEEK, (c) AH-PEEK; (d) The FT-IR spectra of the samples; (e) Zeta-potential variation versus pH of the 7



potassium chloride solution acquired from the samples; (f) Water contact angles of the samples, and the insets in the figure are the optical images of water droplet on the corresponding sample surfaces. The surface zeta-potential of the samples is utilized to analysis the electrical state near the surface and the results are displayed in Figure 2e. The zeta-potential of each samples decreases with the ascending pH values. Compared with PEEK, the zeta potential of A-PEEK is more positive whereas the zeta potential of AH-PEEK is more negative. The different zeta potential of A-PEEK and AH-PEEK is sensitive to the surface topology because of the different types of hydroxyl groups on the surfaces with varied geometries.42 The contact angle of the samples is shown in Figure 2f. The pristine PEEK is 81o, whereas the contact angle of A-PEEK increases significantly to 125o on account of the hydrophobic recovery of polymers treated by high energy particles

43.

Because the wettability varies from super hydrophobic to super

hydrophilic through adjusting the surface geometries,44-46 the contact angle decreases dramatically to 51o after treating A-PEEK with hydrogen peroxide. Cell proliferation and cell adhesion. Initial adhesion is the first step that the nanostructure triggers cell, and thus affects cell proliferation and differentiation.23 On the near flat PEEK surface, the cells exhibit round like morphology with pseudopodia (Figure 3a). On the convex A-PEEK surface, the cells extend to fusiform with lamellipodia (Figure 3b). However, the cells on the concave AH-PEEK surface spread to near flat with filopodia (Figure 3c), indicating that the concave surface is more favorable for the cell initial adhesion than the convex and flat surface. The assessment 8


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of cell proliferation activity was detected by alarmarBlueTM assay and the results are shown in Figure 3d. All of the samples with nanostructure can accelerate cell proliferation, whereas the acceleration ability of cell proliferation on concave nanostructure is stronger than that on convex nanostructure, which is contributed to the more favorable cell adhesion of concave nanostructure.

Figure 3. SEM images at different magnifications showing the initial adhesion and spreading of rBMSCs incubated on the samples: (a) PEEK, (b) A-PEEK, (c) AH-PEEK; (d) Reduction percentages of alamarBlueTM showing the proliferation and viabilities of rBMSCs cultured on the sample surfaces (* p < 0.05, *** p < 0.001; All the data are expressed as means ± SD and n=5). The water contact angle of PEEK treated with hydrogen peroxide only was also evaluated, and the results show that the water contact angel is lower than those of 9



A-PEEK and PEEK but higher than that of AH-PEEK (Figure S2). Therefore, the water contact angel may be attributed to both the structure change and surface hydroxyl change. The increased hydroxyl is more favorable for cell adhesion and even proliferation, however, in this work, there is not obvious difference between PEEK and the samples only through H2O2 treatment in cell response ， which have both relative flat surface (as following figure) (Figure S3). The reason may be that the hydroxyl is not enough to affect the cell behavior.

Figure 4. SEM morphologies of the rBMSCs cultured on various sample surfaces for 1 day, 4 days, and 7 days. The insets are the corresponding images at a higher magnification. The morphologies of cells cultured on the samples surface for 1, 4 and 7 days observed by SEM are put in the Figure 4. The cell amount on AH-PEEK is more than A-PEEK and PEEK, which corresponds to the quantitative results detected by 10


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alarmarBlueTM assay. The cell morphology on PEEK is round whereas the cells on A-PEEK extend to fusiform and on AH-PEEK spreads to near flat after culturing for 1 day. When the culture time is prolonged to 4 days, the cells on the PEEK, A-PEEK and A-HPEEK surface extend to star like shape with filopodia, but the height of cells on AH-PEEK is significantly lower than A-PEEK and PEEK, which indicates that the tension of cells on AH-PEEK is stronger than that on PEEK and A-PEEK. Cell differentiation and mineralization. The fluorescence microscopy images of the samples with ALP stain by Mayer’s Hematoxylin Solution stains are presented in Figure 5a-c and the Mayer’s Hematoxylin Solution stains ALP positive areas dark red. The stained area on A-PEEK is slightly larger than that on PEEK, whereas the positive area on AH-PEEK is significantly larger than that on PEEK. The quantitative ALP activity of cells cultured on the samples surface is shown in Figure 5d. There is no significant difference between PEEK and A-PEEK, whereas the ALP activity of cells on AH-PEEK is markedly higher than PEEK and A-PEEK, indicating that the concave nanostructure exhibits more effective ALP enhancement than the flat surface and convex nanostructured surface. ALP activity of the samples treated with hydrogen peroxide only was also evaluated in our work, and the results show that the ALP activity is similar to pristine PEEK but lower than A-PEEK and AH-PEEK, which indicates that it is hard to enhance ALP activity of PEEK through increasing the surface hydroxyl via hydrogen peroxide treatment (Figure S4).

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Figure 5. ALP activities of rBMSCs cultured on the samples for 14 days. Fluorescence microscopy images of rBMSCs: (a) PEEK, (b) A-PEEK, (c) AH-PEEK; and (d) the quantitative activity of rBMSCs cultured on the surface of samples. The micro/nano textured surfaces induce cell to produce a higher collagen secretion and extracellular matrix (ECM) mineralization around the implants and thus trigger an enhanced tissue integration.47-48 The estimate of the collagen secretion of the cells cultured on the samples for 7 and 14 days is shown in Figure 6. The collagen secretion of A-PEEK and AH-PEEK is slightly higher than PEEK after culturing cells on the samples for 7 days, whereas significantly higher than PEEK when the culture time extends to 14 days. It can be concluded that both of the convex and concave nanostructures promote collagen secretion. Alizarin Red stains ECM mineralization red and the ECM of cells cultured on three set of samples for 7 and 14 days is shown 12


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in Figure 7. After culturing for 7 days, the ECM mineralization of cells on PEEK, A-PEEK and AH-PEEK exhibits an up-regulated trend. When the culture time extends to 14 days, the ECM mineralization of cells on AH-PEEK is significantly higher than PEEK and A-PEEK, whereas there are no obviously differences of ECM mineralization between the cells cultured on PEEK and A-PEEK, which indicates that the ECM mineralization ability of cells cultured on the concave nanostructured surface is stronger than the flat and convex nanostructured surfaces.

Figure 6. Collagen secretion of rMSCs cultured on various surfaces for 7 and 14 days. (* p < 0.05, ** p < 0.01. All the data are expressed as means ± SD and n=4)

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Figure 7. Matrix mineralization of rBMSCs cultured on various surfaces for 7 and 14 days. (* p < 0.05, ** p < 0.01. All the data are expressed as means ± SD and n=4) Osteogenesis-related gene expressions. In order to detect the differentiation of rBMSCs on the three set samples, the expressions of four typical osteogenesis-related genes including BMP-2, RUNX-2, ALP and Col-1 were quantified by real-time PCR and the results are shown in Figure 8. At the early stage, the gene expression of BMP-2 of cells cultured on PEEK, A-PEEK and AH-PEEK shows an up-regulated trend, especially cells on AH-PEEK. However, the gene expression of RUNX-2 of the cells cultured for 7 days on AH-PEEK shows a weaker enhancement than A-PEEK. The gene expression of ALP in the cells cultured for 7 days have the same trend with 14


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RUNX-2, but cells on the AH-PEEK and A-PEEK present enhanced gene expressions of ALP. After culturing cells for 14 days, the gene expression of BMP-2 doesn’t show any difference among cells cultured on PEEK, A-PEEK and AH-PEEK, while the gene expression of RUNX-2 in cells on AH-PEEK and A-PEEK is significantly higher than PEEK. COL-1, the latest gene that marks the cell differentiation, shows an up-regulated expression of cells cultured on PEEK, A-PEEK and AH-PEEK at both 7 and 14 days, indicating that the AH-PEEK has a lasting promotion effect on cell differentiation.

Figure 8. Real-time PCR detection of osteogenesis-related gene expressions of the rBMSCs cultured on the samples for 7 and 14 days. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001). 15



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TGFB, BMP and Wnt signaling pathways are the upstream cues of RUNX-2.49-53 AH-PEEK can enhance BMP-2 expression rather than RUNX-2 at the early stage and promote RUNX-2 expression rather than BMP-2 at later stage, which indicates that the signaling pathway that regulates RUNX-2 is not BMP signaling pathway. ALP is one of the downstream genes of RUNX-2, herewith the trend of ALP expression is the same as RUNX-2. Sequential fluorescent labeling. The sequential fluorescent labeling is utilized to mark the post-surgery bone formation. As the Figure 9d shows, the regions of interest are the adjacent area of implants. The slices of the interested regions of the samples are observed by fluorescence microscope and the results are presented in Figure 9a – c. After 2 weeks, the alizarin red deposition can be found on the surfaces of all samples, whereas the area of alizarin red deposition on AH-PEEK and A-PEEK is more than that on PEEK. Calcein (green) is used to label the bone formation after 6 weeks. No obvious green fluorescence can be detected on the PEEK surface, while calcein can be found on the surfaces of A-PEEK and AH-PEEK. The merge of total fluorescence during procedure is put in the third column of Figure 9a – c, and the quantitative fluorescence is presented in Figure 9e. The area of fluorochrome stained bone in mice implanted with different samples increases in the following order:

PEEK < A-PEEK

< AH-PEEK, and the fluorescent area of AH-PEEK is significantly larger than PEEK and A-PEEK. The sequential fluorescent labeling gives a hint that the bone formation abilities around concave nanostructured surface is stronger than flat and convex nanostructured surface, even though the convex nanostructure also has some positive 16


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impacts on bone formation when compared to flat surface.

Figure 9. Characterization of the surrounding bones by sequential fluorescent labeling: (a) PEEK, (b) A-PEEK and (c) AH-PEEK; (d) The schematic of implants; (e) Histogram of percentage for the area of fluorochromes stained bone. *** (p < 0.01) when compared with PEEK. Micro-CT evaluation of bone formation. The 2-D reconstructed micro-CT images of the three groups are displayed in Figure 10a -c. Around PEEK surface, the wound manufactured by craniofacial model cannot be sealed, whereas the wound of AH-PEEK can be completely sealed. The bone-bonding ability is essential for osseointegration. The obvious distance between newly formed bone and PEEK surface can be observed, whereas the distance between newly formed bone and A-PEEK is minished. Around AH-PEEK, the newly formed bone is tightly anchored to the surface, indicating that the concave nanostructured surface is more favorable for osseointegration than flat and convex nanostructured surfaces.

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Figure 10. Characterization of implants and the surrounding bones by Micro-CT: (a) PEEK, (b) A-PEEK and (c) AH-PEEK; Methylene-fuchsin staining of samples at low and high magnifications: (d1) (e1) PEEK, (d2) (e2) A-PEEK and (d3) (e3) AH-PEEK. Histological evaluation. The histological sections post 8-week implantation are processed by methylene-fuchsin staining and the results are shown in Figure 10d and e. At a defect site, once the bone defect is occupied by fibrous tissue, it is hard to have further bone formation

54-56.

The fibrous tissue, in addition, contributes to the weak

bone-bonding between the samples and tissue. A thick covering of fibrous tissue encompassing the PEEK samples can be observed and the tissue is easy to peel off from the PEEK surface, which indicates a weak osseointegration of flat surface. Around A-PEEK, the fibrous tissue infiltration is milder than PEEK, indicating that the convex nanostructure has a positive effect on inhibiting fibrous tissue filtration. Very few fibrous tissue can filtrate around AH-PEEK and the tissue is tightly anchored to the surface, which imparts that the concave nanostructure is more favorable for fibrous tissue filtration inhibition and bone formation. 18


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3. Discussion Surface texture of biomaterials plays an essential role in controlling cell differentiation through affecting the cell-generated physical forces. Consequently, vast researches focused on the surface topology modification of numerous materials substrates and aimed to figure out the optimal surface roughness and nanostructure size. However, almost all the researches attempted to investigate the cell response to the shape, density, size and depth/ height of either convex nanostructure

25-29

or

concave nanostructure 30-33 separately, whereas, few studies discussed the influence of the convex and concave nanostructure on bone formation. In this work, the PEEK surface with convex nanostructure is produced by argon PIII, and the concave nanostructure is derived from convex nanostructure by subsequent hydrogen peroxide immersion. The one to one correspondence between the convex nanostructure and concave nanostructure can be subtly obtained due to the chemical resistance of PEEK and the active surface of A-PEEK. The cell physical forces affected by surface texturing are produced by myosin bundles sliding along actin filaments and then are transported to the ECM. ECM is a three-dimensional fibrillary protein scaffold surrounding and anchoring cells 47-48, 57-60, which can specifically bond to the integrin

61-62.

Consequently, the surface texturing

plays essential role in cell adhesion. Because the diameter of integrin is 15 nm

21, 63,

the integrin cannot be anchored onto the internal surfaces of the gap when the gap between two upwardly convex surfaces on A-PEEK is smaller than 15 nm. However, the downwardly concave nanostructure is free of gap, because there is not a well 19



between two concave nanostructures. Consequently, this phenomenon endows downwardly concave nanostructure with favorable cell adhesion. Besides, the hydrophilic downwardly concave surface are more favorable for cell adhesion than the hydrophobic upwardly convex surface 64. It can be concluded that the downwardly concave surface enhances cell initial adhesion and thus affects cell response. The cells adhere onto the substrate and simultaneously the plasma membrane migrates into the nano caves or the gap of the convexes. Figure 11 illustrated the scheme of the effect of substrate on the cell migration. The space for plasma membrane migration is marked by blue, and it can be observed that the intersectant and tangent nano-caves in concave structured samples provide more space for cell membrane migration than the corresponding convexes in convex structured samples (Figure 11a). However, the space for cell membrane migration of the separated caves in concave textured samples and convexes in convex textured samples depends on the gap between two nanostructures. When the plasma membrane migrates into the nano caves or the gap of the convexes, various proteins in the plasma membrane sense and response to the textured surface, thus triggering cell shape change and cell motility. The plasma membrane tension is sensed and regulated by the curvature-sensing proteins. As Figure 11b shows, the curvature-sensing proteins associate with the curved membrane, thus promoting cell migration and spreading. However, the curvature-sensing proteins would not bond to the flat plasma membrane.65 Therefore, it is the curved region instead of the flat region of two separated caves/convexes that influences plasma membrane tension. The curvature-sensing proteins stimulate cell to 20


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lower the membrane tension through decreasing the actin polymerization in the curved region.66 The curved interfaces of the substrate could keep the membrane curved all the time, stimulating the cell to keep releasing the membrane tension through migrating itself onto the adjacent region. And the feedback of the adjacent curved interfaces further promotes the cell to migrate onto the adjacent region. The space that can stimulate cell migration is marked in Figure 11c. It can be observed that the space that can stimulate cell migration of concave textured samples is more than convex textured samples. In summary, the concave textured samples are more favorable for cell adhesion and migration. As the Figure 3 shows, the area of the cells on the AH-PEEK is more than that on the A-PEEK. Besides, the cells on the AH-PEEK surfaces are relative flat than that of A-PEEK, indicating that the downwardly concave surface provides more advantages than upwardly convex surface, inducing more tension in cells. Cell tension regulates cell proliferation and differentiation through stimulating the osteogenic genes.67-69 At early stage, the BMP-2 is upregulated of cells on AH-PEEK whereas downregulated at later stage. RUNX-2 expression exhibits the opposite trend with BMP-2, which indicates that the signal pathway at early stage is BMP related signal pathway. However, the downstream cues of BMP-2 are not clear and need to be further explored. At late stage, the referred gene of the signal pathway is RUNX-2. Nevertheless, the upstream cues of RUNX-2 need to be further discussed.

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Figure 11. The scheme of the effect of substrate on the cell migration. The space for plasma membrane migration is marked by blue, and it can be observed that the intersectant and tangent nano-caves in concave structured samples provide more space for cell membrane migration than the corresponding convexes in convex structured samples. Cell adhesion, collagen secretion and ECM deposition are affected by the surface structure, thus paving the way of the osseointegration enhancement in vivo. The fibrous tissue infiltration is inhibited on the AH-PEEK surface due to the enhanced cell initial adhesion. The upregulated collagen secretion and ECM deposition of AH-PEEK contribute to the superior osseointegration ability. The preferable ability of

22


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fibrous tissue infiltration inhibition and osseointegration of downwardly concave surface than upwardly convex surface provide better repair of the bone defection. 4. Conclusions The nanotextured PEEK surface is fabricated by argon PIII and subsequent hydrogen peroxide treatment. The in vitro cell response results show that cell adhesion, collagen secretion and ECM deposition can be enhanced both on the PEEK surface fabricated by argon PIII and physical-chemical treatment, especially on the surface fabricated by physical-chemical treatment. The in vivo tests exhibit that the PEEK surface fabricated by physical-chemical treatment is more favorable for fibrous tissue filtration inhibition and osseointegration than that fabricated by argon PIII. In summary, the nanostructure on PEEK fabricated by argon PIII followed by hydrogen peroxide treatment provides a preferable surface for bone regeneration than that fabricated by argon PIII only, which provides a potential candidate in orthopedic and dental applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: http://pubs.acs.org. Detailed experimental materials and methods ， morphology of the sample only through H2O2 treatment, contact angel of the samples，cell adhesion on the samples

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treating with hydrogen peroxide, ALP activities of rBMSCs cultured on the samples treating with hydrogen peroxide. AUTHOR INFORMATION Author Contributions 1

These authors contributed equally to this work

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Financial support from the International Partnership Program of Chinese Academy of Sciences Grant No.GJHZ1850, National Science Foundation for Distinguished Young Scholars of China (51525207), Shanghai Committee of Science and Technology, China (18410760600), National Natural Science Foundation of China (81641035, 81401524), Hubei provincial Natural Science Foundation of China No. 2014CFB978 are

acknowledged.

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For Table of Contents Use Only

Nano Textured PEEK Surface for Enhanced Osseointegration Liping Ouyanga,b,1, Meiling Chenc,1, Donghui Wanga, Tao Lua, Heying Wanga, Fanhao Menga, Yan Yangc, Jingzhi Mac,*, Kelvin W. K. Yeung d,*, Xuanyong Liua,*

The downwardly concave surface that constructed by argon PIII and hydrogen peroxide treatment is more favorable for fibrous tissue filtration inhibition and osseointegration than upwardly convex surface.

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Figure 1. Surface morphology of various samples: (a) PEEK, (b) A-PEEK, (c) AH-PEEK. The surface of PEEK is relative flat, the surface of A-PEEK is convex, and the surface of A-PEEK is concave. The red circles mark the tangent nanostructures, the blue circles mark the separated nanostructures, and the yellow circles mark the intersected nanostructures. The red arrows mark some of the tangent nanostructures, the blue arrows mark some of the separated nanostructures, and the yellow arrows mark some of the intersected nanostructures.


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Fgiure 2. XPS full spectra of the samples: (a) PEEK, (b) A-PEEK, (c) AH-PEEK; (d) The FT-IR spectra of the samples; (e) Zeta-potential variation versus pH of the potassium chloride solution acquired from the samples; (f) Water contact angles of the samples, and the insets in the figure are the optical images of water droplet on the corresponding sample surfaces.



Figure 3. SEM images at different magnifications showing the initial adhesion and spreading of rBMSCs incubated on the samples: (a) PEEK, (b) A-PEEK, (c) AH-PEEK; (d) Reduction percentages of alamarBlueTM showing the proliferation and viabilities of rBMSCs cultured on the sample surfaces (* p < 0.05, *** p < 0.001; All the data are expressed as means ± SD and n=5).


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Figure 4. SEM morphologies of the rBMSCs cultured on various sample surfaces for 1 day, 4 days, and 7 days. The insets are the corresponding images at a higher magnification.



Figure 5. ALP activities of rBMSCs cultured on the samples for 14 days. Fluorescence microscopy images of rBMSCs: (a) PEEK, (b) A-PEEK, (c) AH-PEEK; and (d) the quantitative activity of rBMSCs cultured on the surface of samples.


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Figure 6. Collagen secretion of rMSCs cultured on various surfaces for 7 and 14 days. (* p < 0.05, ** p < 0.01. All the data are expressed as means ± SD and n=4)



Figure 7. Matrix mineralization of rBMSCs cultured on various surfaces for 7 and 14 days. (* p < 0.05, ** p < 0.01. All the data are expressed as means ± SD and n=4)


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Figure 8. Real-time PCR detection of osteogenesis-related gene expressions of the rBMSCs cultured on the samples for 7 and 14 days. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).



Figure 9. Characterization of the surrounding bones by sequential fluorescent labeling: (a) PEEK, (b) A-PEEK and (c) AH-PEEK; (d) The schematic of implants; (e) Histogram of percentage for the area of fluorochromes stained bone. *** (p < 0.01) when compared with PEEK.


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Figure 10. Characterization of implants and the surrounding bones by Micro-CT: (a) PEEK, (b) A-PEEK and (c) AH-PEEK; Methylene-fuchsin staining of samples at low and high magnifications: (d1) (e1) PEEK, (d2) (e2) A-PEEK and (d3) (e3) AH-PEEK.



Figure 11. The scheme of the effect of substrate on the cell migration. The space for plasma membrane migration is marked by blue, and it can be observed that the intersectant and tangent nano-caves in concave structured samples provide more space for cell membrane migration than the corresponding convexes in convex structured samples.


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Nano Textured PEEK Surface for Enhanced Osseointegration - ACS

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